Dissecting the Role of Presynaptic GABA a Receptors in Nerve Terminal Function

by Philip Long

Department of Pharmacology, The School of Pharmacy, 29 - 39 Brunswick Square, London, WCIN lAX

A thesis submitted for the degree of Doctor of Philosophy in Pharmacology for the University of London 2007

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 This thesis describes research conducted in the School of Pharmacy, University of London between 2003 and 2006 under the supervision of Dr Jasmina Jovanovic. I certify that the research described is original and that any parts of the work that have been conducted by collaboration are clearly indicated. I also certify that I have written all the text herein and have clearly indicated by suitable citation any part of this dissertation that has already appeared in publication.

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_ 9 - Abstract

Fast synaptic inhibition in the mammalian brain is mediated principally by GABAa receptors, a large and diverse family of Cl permeable ion channels. Emerging evidence points to the presynaptic localisation of these receptors and their role in regulating intraterminal Ca^"^ levels and neurotransmitter release in some areas of the CNS. We used purified nerve terminals (synaptosomes) to establish the presence of functional presynaptic

GABAa receptors in the rat neocortex. As a biochemical read-out of presynaptic GABAa receptor activity, we measured Ca^^-dependent changes in the phosphorylation state of synapsin I. A dose-dependent decrease in synapsin I phosphorylation was detected in response to , and GABase, while and caused a dose-dependent increase in synapsin I phosphorylation. Immunohistochemical analysis revealed this phospho-form of synapsin I to be localised to glutamatergic nerve terminals.

In functional studies, GABAa receptor activation by muscimol caused a dose-dependent inhibition of glutamate release, which was abolished by picrotoxin. In order to investigate the mechanism(s) by which presynaptic GABAa receptor activity leads to inhibition of glutamate release, we measured release in the presence of the NKCCl antagonist , which abolished the muscimol-induced inhibition of glutamate release. When we subsequently investigated the role of voltage-gated Ca^^ channels on muscimol- dependent inhibition of glutamate release, we found that muscimol inhibited glutamate release in the presence of co-conotoxin GVIA, co-agatoxin IVA and NiCb. However, the inhibition of release by muscimol was abolished by SNX-482 and nifedipine. Muscimol- dependent decrease in release was also abolished in the presence of W7. Our results indicate that the activation of presynaptic GABAa receptors in the rat neocortex is coupled to depolarisation of the nerve terminal membrane, leading to inhibition of glutamate release via Ca^'^-dependent Ca^'^ channel inhibition linked to L- and R-type VDCCs.

-3 Acknowledgements

I would like to begin by thanking my supervisor Dr Jasmina Jovanovic for all her assistance and guidance throughout the course of my PhD, especially for her constructive comments while writing this thesis.

I would also like to express my gratitude to Dr Talvinder Sihra for integrating me into his lab and providing creative input and technical help.

Thanks to Dr Audrey Mercer and Rahima Begum for their help carrying out the immunohistochemistry.

Finally, thanks to my friends and family for their help and support during this time, especially Rosa Sancho for ensuring I didn’t have to live in a box and to Chris Moore and Sabina Muneton for proof-reading my thesis.

-4 Contents

Abstract ...... 3

Acknowledgements ...... 4

Abbreviations ...... 11

1. Introduction ...... 16 1.1 GAB A as a neurotransmitter ...... 17 1.2 GAB A Receptors ...... 19

1.2.1 GABAa Receptors ...... 19

1.2.2 GABAb Receptors ...... 19 1.2.3 GAB Ac Receptors ...... 19

1.3 GABAa receptor structure ...... 20

1.4 Regulation of GABAa receptor function by allosteric modulators ...... 23

1.5 Synthesis, expression and trafficking of GABAa receptors ...... 27

1.6 Subcellular localisation of GABAa Receptors ...... 33

1.7 Modulation of GABAa receptors by protein phosphorylation and association with signalling molecules ...... 34

1.8 Presynaptic GABAa Receptors ...... 38

1.9 Cation co-transporters determine the functional outcome of GABAa receptor activation ...... 42 1.10 Neurotransmitter release ...... 44 1.11 Synapsins ...... 48 1.12 Voltage-gated Ca^^ Chaimels ...... 51 1.13 Modulation of neurotransmitter release by presynaptic neurotransmitter receptors ...... 54 1.13.1 Presynaptic G-Protein Coupled Receptors ...... 54 1.13.1.1 Metabotropic Glutamate Receptors ...... 54 1.13.1.2 GAB As receptors ...... 55 1.13.1.3 Histamine receptors ...... 55 1.12.1.4 Adenosine receptors ...... 56 1.13.1.5 Muscarinic acetylcholine receptors ...... 56 1.13.2 Presynaptic ligand-gated ion channels ...... 57

- 5 - 1.13.2.1 receptors ...... 57 1.13.2.2 Nicotinic acetylcholine receptors ...... 57 1.13.2.3 5HT) receptors ...... 58 1.13.2.4 NMDA receptors ...... 59 1.13.2.5 AMPA receptors ...... 59 1.13.2.6 Kainate receptors ...... 60 1.13.2.7 P2X receptors ...... 61 1.13.2.8 V anilloid receptors ...... 61 1.14 Isolated Nerve Terminals (Synaptosomes) ...... 61 1.15 Aims...... 62

2. Materials and Methods ...... 65

3. Regulation of presynaptic Ca^^-dependent signalling pathways and synapsin I phosphorylation by GABAa receptors ...... 80

4. Modulation of glutamate release by presynaptic GABAa receptors ...... 115

5. Modulation of Plasma Membrane Potential and Intraterminal Ca^"^ Concentration by

Presynaptic GABAa Receptors ...... 145

6. Functional coupling of the presynaptic GABAa receptors to the NKCCl cation co­ transporter and voltage-dependent Ca^^ channels ...... 169

7. Discussion ...... 197

List of References ...... 214

- 6 - List of Figures

Figure 1. Structure of GABAa receptors ...... 21 Figure 2. Domain model of rat synapsin la and Ib showing location of phosphorylation sites...... 50 Figure 3. Glutamate is metabolised to a-ketoglutarate and ammonia ...... 75 Figure 4. Presynaptic activity of CaM kinases and Ca^^-dependent phosphorylation of synapsin la and Ib are reduced in the absence of extrasynaptosomal GAB A ...... 87 Figure 5. Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is decreased in the presence of GABase ...... 88 Figure 6. Presynaptic activity of CaM kinases and Ca^^-dependent phosphorylation of

synapsin I are reduced in the presence of GABAa receptor antagonist bicuculline.... 91 Figure 7. Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is decreased in the presence of bicuculline ...... 92 Figure 8. Presynaptic activity of CaM kinases and Ca^^-dependent phosphorylation of

synapsin I are reduced in the presence of GABAa receptor antagonist picrotoxin 95 Figure 9. Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is decreased in the presence of picrotoxin ...... 96 Figure 10. Presynaptic activity of CaM kinases and Ca^^-dependent phosphorylation of

synapsin I are increased in the presence of GABAa receptor agonist isoguvacine 98 Figure 11. Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is increased in the presence of isoguvacine ...... 99 Figure 12. Presynaptic activity of CaM kinases and Ca^^-dependent phosphorylation of

synapsin I are increased in the presence of GABAa receptor agonist muscimol 102 Figure 13. Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is increased in the presence of muscimol ...... 103

Figure 14. GABAa receptor subunits, synapsin and GAD are abundantly expressed in rat cerebrocortical synaptosomes ...... 105 Figure 15. Ca^^-dependent phosphorylation of synapsin I at P-site 3 occurs in glutamatergic nerve terminals in neocortical layers I - III ...... 107 Figure 16. Ca^^-dependent phosphorylation of synapsin I at P-site 3 occurs in glutamatergic nerve terminals in neocortical layer IV ...... 108

7 - Figure 17. Ca^^-dependent phosphorylation of synapsin I at P-site 3 occurs in glutamatergic nerve terminals in neocortical layers V and VI ...... 109 Figure 18. Muscimol causes a dose-dependent inhibition of 4AP-evoked glutamate release 121 Figure 19. Muscimol has no effect on Ca^^-independent release of glutamate in the presence of 4AP ...... 122 Figure 20. Isoguvacine causes a dose-dependent inhibition of 4AP-evoked glutamate release...... 123 Figure 21. Muscimol-induced decrease of 4AP-evoked glutamate release is inhibited by picrotoxin ...... 125 Figure 22. Picrotoxin has no effect on 4AP-evoked glutamate release ...... 126 Figure 23. Muscimol-induced decrease in 4AP-evoked glutamate release from hippocampal synaptosomes, inhibited by picrotoxin ...... 127 Figure 24. Muscimol has no effect on 4AP-evoked glutamate release from cerebellar synaptosomes ...... 128 Figure 25. Muscimol causes an inhibition of glutamate release triggered by 10 mM KCl 130 Figure 26. Muscimol has no effect on glutamate release triggered by 30 mM KCl 131 Figure 27. Muscimol has no effect on glutamate release triggered by ionomycin 132 Figure 28. Muscimol-induced inhibition of 4AP-evoked glutamate release in the presence of ...... 134 Figure 29. Muscimol-induced inhibition of 4AP-evoked glutamate release in the presence of ...... 135 Figure 30. Muscimol-induced inhibition of 4AP-evoked glutamate release in the presence of ...... 136 Figure 31. Picrotoxin has no effect on KCl-triggered depolarisation of synaptosomes .. 153 Figure 32. Muscimol has no effect on KCl-triggered depolarisation of synaptosomes... 154 Figure 33. Muscimol has no effect on KCl-triggered depolarisation of synaptosomes... 155 Figure 34. Isoguvacine has no effect on KCl-triggered depolarisation of synaptosomes 157 Figure 35. Isoguvacine has no effect on KCl-triggered depolarisation of synaptosomes 158 Figure 36. Muscimol has no effect on 4AP-triggered depolarisation of synaptosomes .. 159

Figure 37. Intrasynaptosomal Ca^"^ concentration in the presence of GABAa receptor agonist muscimol ...... 161 Figure 38. Regulation of intrasynaptosomal Ca^^ levels by the GABAa receptor agonist muscimol ...... 162 Figure 39. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of bumetanide ...... 177 Figure 40. Muscimol has no effect on 4AP-evoked glutamate release in the absence of extrasynaptosomal Ca^"^ ...... 178 Figure 41. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of BaCli ...... 179 Figure 42. Muscimol-induced inhibition of 4AP-evoked glutamate release is independent of the activity of P/Q type VGCCs ...... 182 Figure 43. Muscimol-induced inhibition of 4AP-evoked glutamate release is independent of the activity of N-type VGCCs ...... 183 Figure 44. Muscimol-induced inhibition of 4AP-evoked glutamate release is independent of the activity of T-type VGCCs ...... 184 Figure 45. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of L-type VGCC blocker nifedipine ...... 186 Figure 46. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of R-type VGCC blocker SNX-482 ...... 187 Figure 47. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the R-type Ca^^ channel knock-out mice ...... 188 Figure 48. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of W 7 ...... 191

Figure 49. Molecular mechanism underlying the presynaptic GABAa receptor-mediated inhibition of glutamate release ...... 212

- 9 - List of Schematics

Schematic 1 incubation protocols used for phosphorylation experiments in Chapter 3...... 85 Schematic 2 Drug incubation protocols used for glutamate release experiments in Chapter 4 ...... 119 Schematic 3 Drug incubation protocols used to measure changes in synaptosomal membrane potential in Chapter 5 ...... 150 Shcematic 4. Drug incubation protocols used to measure changes in intraterminal Ca^"^ concentration in Chapter 5 ...... 151 Schematic 5 Drug incubation protocols used for glutamate release experiments in Chapter 6...... 175

10 - Publications

Society for Neuroscience Annual Meeting, Atlanta, GA, USA. October 2006.

Title: GABAa receptor-mediated presynaptic modulation

Authors: P. LONG, A. Mercer, R. Begum, T. S. Sihra, J. N. Jovanovic.

Fast synaptic inhibition in the brain is mediated principally by GABAa receptors, a large and diverse family of Cl' permeable ion channels. GABAa receptors are highly enriched in domains of the postsynaptic membrane which precisely appose GABA-releasing presynaptic terminals. Emerging experimental evidence points to the presynaptic localization of these receptors in some areas of the CNS where they are proposed to regulate intraterminal Ca^^ levels and neurotransmitter release. To establish the presence of functional GABAa receptors at presynaptic sites in the rat neocortex, we used isolated nerve terminal preparation (synaptosomes) to investigate the regulation of presynaptic signaling cascades and neurotransmitter release in the presence of specific agonists and antagonists of these receptors. As a biochemical read-out of the activity of presynaptic GAB A A receptors, we measured Ca^^-dependent changes in the phosphorylation state of a presynaptic-specific protein, synapsin I, using a phosphorylation state-specific antibody specifically recognizing this protein when targeted by CaM kinase II. A dose-dependent decrease in synapsin I phosphorylation reflecting a decrease in intraterminal [Ca^^Ji was detected in response to bicuculline, a competitive antagonist, and picrotoxin, a channel blocker, of GABAa receptors. Conversely, the GABAa receptor specific agonist muscimol caused a dose-dependent increase in synapsin I phosphorylation by CaM kinase II.

Immunoblotting with GABAa receptor subunit-specific antibodies confirmed the presence of al, a2, (32, p3, and a specific enrichment of y2 subunit in the fraction of highly purified terminals. The effects of GABAa receptor active compounds on plasma membrane potential, intraterminal [Ca^^] and glutamate and G ABA release were also investigated using on-line fluorescence assays. Together, our result indicate that the activation of presynaptic GABAa receptors in the rat neocortex is coupled to the depolarization of nerve terminal membrane leading to an increase in intraterminal Ca^"^ concentration as opposed to their classically described hyperpolarizing postsynaptic effects.

11 - Abbreviations

ACh acetylcholine (û-Aga IV A co-Agatoxin IV A Allopregnanolone 5a-Pregnan-3p-ol-20-one 3 (3-acetate AMPA a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid 4AP 4-aminopyridine APS ammonium peroxodisulphate ATP adenosine triphosphate BDNF brain-derived neurotrophic factor Bicuculline bicuculline methiodide BIG2 brefeldin A-inhibited GDP/GTP exchange factor 2 BSA bovine serum albumin intraterminal Ca^"^ concentration CaMK Ca^'^/calmodulin-dependent protein kinase cAMP adenosine 3’,5’-cyclic adenosine monophosphate GDI Ca^^-dependent inhibition CNS central nervous system (O-CTx GVIA co-Conotoxin GVIA DAG diacylglycerol DiSCsCS) 3,3 '-dipropylthiadicarbocyanine iodide

Ec\ chloride reversal potential

^GABA GABA reversal potential EGTA ethylene glycol-bis ((3-amino-ethyl ether)N,N,N’,tetra acetic acid EPSC excitatory postsynaptic current EPSP excitatory postsynaptic potential ERK extracellular signal-regulated protein kinase Fura-2 AM fura-2 acetoxymethyl ester GABA y-aminobutyric acid GABARAP GAB A A receptor-associated protein GDH glutamate dehydrogenase GEF guanine nucleotide exchange factor

- 12 - GODZ Golgi-specific protein with a DHHC finger domain GPCR G-protein-coupled receptor

GRIP GABAa receptor interacting factor GTP guanosine-5'-triphosphate HBM hepes buffered medium Hepes N-2-hydroxyethylpiperazine-N’-2-ethansulfonic acid HPLC high performance liquid chromatography 5-HT 5 -hydroxytryptamine HVA high-voltage activated Ca^'^ channels

IP3 inositol 1,4,5-trisphosphate IPSC inhibitory postsynaptic current IPSP inhibitory postsynaptic potential Isoguvacine isoguvacine hydrochloride KCC potassium chloride co-transporter KCl potassium chloride Kd dissociation constant kDa kilodalton LDCV large dense core vesicle LGIC ligand-gated ion channel LTP long-term potentiation LVA high-voltage activated Ca^'^ channels MAPK mitogen-activated protein kinase mGluR metabotropic glutamate receptors Muscimol muscimol hydrochloride NADP+ adenine dinucleotide phosphate NKCC sodium potassium chloride co-transporter NMDA N-methyl-d-aspartate NSF N-ethylmaleimide-sensitive fusion protein PKA protein kinase A PKC protein kinase C PLC phospholipase C PPl protein phosphatase 1 PP2A protein phosphatase 2A PP2B protein phosphatase 2B (calcineurin) RIM rab 3 interacting molecule SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate -polyacrylamide gel electrophoresis S.E.M. standard error of the mean SNAP-25 synaptosome-associated protein 25 SNAPs soluble NSF attachment proteins SNARE SNAP receptor SSV small synaptic vesicle TBS tris-buffered saline TEMED N, N, N’, N'-tetramethylethylethylenediamine t-SNARE target-associated SNARE Tris tris(hydroxymethyl)methylamine TTX tetrodotoxin VAMP vesicle-associated membrane protein VGCC voltage-gated Ca^^ channel v-SNARE vesicle-associated SNARE W7 N-(6-aminohexyl)-5-chloro-I-naphthalenesulfonamide hydrochloride

- 14 - CHAPTER ONE

- 15 - 1. Introduction

The mammalian brain contains approximately 10^^ neurones, which are able to communicate in a highly regulated manner at specialised contacts known as synapses. It has been estimated that the number of synapses can reach up to 10^ - 10"^ per neurone, equating to around 10^^ synaptic contacts in the human brain (Williams and Herrup, 1988). Synapses are morphologically and functionally specialised cell-cell contacts, which can be divided into two general classes: electrical synapses and chemical synapses. Electrical synapses (gap junctions) permit direct, passive flow of electrical current from one neurone to another, whereas chemical synapses are points at which one neurone communicates with another via the conversion of an electrical signal into a chemical signal (Eccles, 1982). Chemical synapses are composed of presynaptic terminals, specialised enlargements of the axon from which transmitter release occurs, the postsynaptic terminal, which receives the signal, and the synaptic cleft, a small gap separating presynaptic and postsynaptic compartments. Neurotransmitters are stored in synaptic vesicles, specialised exocytotic organelles, uniform in size, which fuse with the presynaptic membrane and release the neurotransmitter. Two types of vesicle store neurotransmitters, small synaptic vesicles (SSVs) and large dense-core vesicles (LDCVs). SSVs are approximately 50 nm in diameter and store neurotransmitters that mediate ‘fast’ synaptic transmission, such as GABA, glycine, glutamate and ACh. LDCVs are larger than 75 nm in diameter and store neurotransmitters which mediate ‘slow’ synaptic transmission, such as catecholamines (eg. noradrenaline, dopamine) and neuropeptides. Each SSV stores several thousand molecules of neurotransmitter.

In response to an action potential, Ca^^ enters the presynaptic terminal through voltage- gated Ca^"^ channels (VGCCs) and results in fusion of SSVs with the plasma membrane. The stored neurotransmitter is then released into the synaptic cleft as a discrete amount known as quanta, where they diffuse and bind to receptor molecules in the postsynaptic neurone. Synaptic transmission can be either excitatory or inhibitory. The majority of excitatory synapses store glutamate as their neurotransmitter, which binds to postsynaptic glutamate receptors following release. Activation of these receptors results in plasma membrane depolarisation and thereby neuronal excitation due to opening of ion channels

- 16 - permeable to Na^, and/ or Ca^^. The majority of inhibitory synapses in the brain are GABAergic. The inhibitory effects of GABA are due to activation of GABAa receptors, which are GABA-gated CI/HCO 3 channels, resulting in hyperpolarisation of the plasma membrane and inhibition of neuronal activity.

1.1 GABA as a neurotransmitter y-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain, functioning as a transmitter at 30 - 40% of synapses (Hendry et ah, 1987; Beaulieu, 1993), where it plays an essential role in regulating neuronal excitability, neuronal plasticity and network synchronisation. The GABAergic system is implicated in a variety of disorders, such as anxiety (Lydiard, 2003), epilepsy (Scheffer and Berkovic, 2003), substance abuse, sleep disorders (Korpi et al., 2002) schizophrenia (Wassef et al., 2003), pain (Enna and McCarson, 2006) and withdrawal (Aguayo et al., 2002). Some anaesthetics also act on this system, along with such as , and (Korpi et al., 1997). GABA was first characterised as a neurotransmitter in crustaceans in the 1950’s by Katz, Kuffler and others, although it was not until the late 1960’s that it was demonstrated to display the functional characteristics of a classical neurotransmitter in the mammalian brain (Kmjevic and Schwartz, 1966). GABA is formed when (glutamate) is decarboxylated by glutamic acid decarboxylase (GAD), which is found almost exclusively in GABA producing (GABAergic) neurones (Kuriyama et al., 1966; Collins, 1972). In this reaction, GAD requires a cofactor, pyridoxal phosphate (PLP), which is derived from vitamin Bg (Roberts and Frankel, 1950; Miller and Martin, 1973). The activity of GAD regulates the steady-state concentration of GABA and is stimulated by inorganic phosphate and inhibited by ATP, GABA and aspartate (Grossfeld et al., 1984). In nerve terminals, approximately 30 % of GABA that is synthesised is stored in SSVs following loading by a vesicular neurotransmitter transporter (Eon and Edwards, 2001), with the remaining 70 % distributed in the cytosol of axons, dendrites and cell bodies (Martin and Tobin, 2000).

The arrival of an action potential within the neurone leads to a cascade of Ca^^-regulated protein-protein interactions between synaptic vesicles and plasma membrane, resulting in the release of GABA from the presynaptic terminal (Sudhof and Jahn, 1991). Different

- 17 - mechanisms may be involved in SSV exocytosis in glutamatergic and GABAergic nerve terminals. Evidence suggests that the presynaptic proteins Muncl3-1, Rab3-interacting molecule lot (RIMlot), and synapsin exert different modulatory effects on the release of GABA in comparison with the release of glutamate (Augustin et al., 1999; Gitler et al., 2004). Additionally, there is conflicting evidence for the role of SNAP-25, one of the proteins required for SSV exocytosis, in GABAergic terminals. Although some studies have demonstrated the absence of this protein in hippocampal GABAergic terminals (Verderio et al., 2004; Frassoni et al., 2005), other studies have clearly indicated the critical role of this protein in GABAergic transmission (Tafoya et al., 2006). Following exocytosis, released GABA subsequently binds to specific receptors (GABA a, GABAb and GAB Ac) located in the postsynaptic plasma membrane, eliciting a response within the postsynaptic neurone.

Numerous studies have shown that uptake of GABA released into the extracellular space occurs mainly by neurones but also by astrocytes (Schousboe, 2003) via an active transport mechanism, thereby terminating its synaptic actions. Currently, three GABA transporters (GATs) have been shown to be expressed in the cerebral cortex, GAT-1, GAT-2 and GAT- 3, which are 70 to 80 kDa proteins containing 12 hydrophobic membrane-spanning domains. GABA is transported along with two Na"^ ions and one Cl" ion via an electrogenic process (Keynan and Kanner, 1988; Kavanaugh et al., 1992), although the involvement of C r is disputed (Loo et al., 2000). When GABA is taken up by GABAergic neurones, it can be transported back into synaptic vesicles and made available for synaptic transmission (Roettger and Amara, 1999). GABA is metabolised by the actions of GABA-transaminase, the transfer of the amino group from GABA to a-ketoglutarate requiring pyridoxal-5’- phosphate (PLP) as a carrier to give succinic semialdehyde (SSA). SSA is subsequently oxidised by succinic semialdehyde dehydrogenase (Bessman et al., 1953; Hall and Kravitz, 1967).

In addition to its role in synaptic transmission, GABA also plays a trophic role in neuronal development. GABA has been shown to modulate cell proliferation and survival (Nguyen et al., 2003; Haydar et al., 2000), affect neuronal migration (Behar et al., 1998) and increase the length and branching of neurites (Spoerri, 1988; Borodinsky et al., 2003), following

- 18 - initial reports that it could promote dendritic growth and alter development of presynaptic specialisations (Wolff et al., 1978). Although it was originally thought that these trophic actions were a result of GABA-mediated hyperpolarisation (Meier et al., 1991), it now appears that the depolarising actions of GABAa receptors are instead responsible for these processes (Ben-Ari, 2002).

1.2 GABA Receptors

1.2.1 GABAa Receptors

GABAa receptors are GABA-gated Cl' channels that mediate the majority of fast synaptic inhibition in the brain and spinal cord (Macdonald and Olsen, 1994). It has become more apparent recently that in addition to phasic, action potential-dependent effects, GABA can also produce a tonic inhibition due to the continuous activation of extrasynaptic GABAa receptors (Semyanov et al., 2004). In order to maintain normal brain function, synaptic excitation has to be tightly balanced with the synaptic inhibition mediated by the activation of GAB A A receptors.

1.2.2 GAB As Receptors

GABAb receptors are located both postsynaptically, where they modulate the activity of ion channels and neurotransmitter receptors, and presynaptically where they modulate neurotransmitter release (Bowery et al., 1980; Hill and Bowery, 1981). GABAb receptors belong to a family of G-protein-coupled metabotropic receptors containing seven membrane spanning domains (Kaupmann et al., 1997). These receptors are heterodimers of two subunits, GABAb i and GABAb 2, which are essential for expression as well as receptor activation and ligand specificity (Bowery and Enna, 2000; Vacher and Bettler, 2003; Schuler et al., 2001). The GABAbi subunit contains the ligand binding site (Galvez et al.,

1999), with the GABAb 2 subunit linked to the pertussis toxin-sensitive G/Go protein (Margeta-Mitrovic et al., 2001).

1.2.3 GAB Ac Receptors

GAB Ac receptors are GABA-gated Cl' channels composed exclusively of p subunits (Chebib and Johnston, 1999; Feigenspan and Bormann, 1998) which were defined

- 19 - pharmacologically as being insensitive to bicuculline and baclofen (Bormann, 2000). They are highly expressed in the vertebrate retina (Enz et al., 1996) and localised in the axon terminals and dendrites of bipolar cells (Koulen et al., 1998). Evidence has recently emerged that GABAa and GAB Ac receptor subunits may be able to form heteromeric complexes in some central neurones (Milligan et al., 2004).

1.3 GABAa receptor structure

The GABAa receptors belong to the ligand-gated ion channel superfamily which also includes nicotinic acetylcholine receptors, 5-HTs receptors and glycine receptors (Macdonald and Olsen, 1994). Structurally they represent heteropentameric assemblies of subunits classified into 7 different classes: a l - 6 , p i - 3, yl - 3, Ô, e, n, 0 (Whiting, 1999). Each subunit, which is approximately 50 - 60 kDa, has a conserved structure consisting of a large extracellular amino (N)-terminal, four a-helical membrane spanning domains (TM), a large intracellular loop between TM3 and TM4 and a short extracellular carboxyl (C)- terminal. The 5 subunits arrange to form a channel which allows the passage of Cl and

HCO3 ions, with the five TM2 domains forming the channel lining (Fig. 1). The agonist binding sites are thought to be formed by two cysteine binding loops on the N-terminal domains of a and p subunits (Connolly and Wafford, 2004). The majority of GABAa receptors contain a stoichiometry of 2 a, 2 p and ly subunits, with the largest population being al P2y2, followed by a2p3y2 and a3p3y2 (Fritschy and Brunig, 2003). Two different forms of y2 subunit exist, y2S and y2L, differing by the presence or absence of a

24-base-pair ( 8 -amino acid) insertion in the cytoplasmic domain, between TM3 and TM4. (Whiting et al., 1990). Many neurones have shown a large heterogeneity of subunit expression (Laurie et al., 1992; Fritschy and Mohler, 1995), although a better understanding of receptor assembly mechanisms is required to elucidate the level of functional receptor heterogeneity in the brain. The exact role of ô, 8 and 0 subunits remains to be determined, but it is likely that they form various combinations with a and p subimits in place of y subunits. These subunits show restricted patterns of expression throughout the brain (Shivers et al., 1989; Bonnert et al., 1999; Whiting et al., 1997).

-20 2a: 1 - 6 2p: 1 - 3 1 r 1 - 3

N Q

'Î--

Figure 1. Structure of GABAa receptors. a. GABAa receptors are schematically depicted as heteropentamers comprised of 2a, 2(3 and ly subunit. b. Each subunit is composed of a large extracellular N-terminal domain, 4 transmembrane spanning domains (TMl - 4) and a short extracellular C-terminal domain. TM2 is thought to form the lining of the ion channel. A large intracellular loop between TM3 and 4 contains binding motifs for the majority of GABAa receptor interacting proteins and a number of phosphorylation sites. c. Schematic diagram of 5 subunits forming a functional receptor with a centrally located ion channel (Reproduced from Moss and Smart, 2001).

-21 - It has been demonstrated that a and (3 subunits are required for GABAa receptor activation (Schofield et al., 1987), whilst the y subunit has little effect on agonist activation, although it is required for sensitivity to benzodiazepines (Pritchett et al, 1988). Binding of GABA occurs at the extracellular N-terminal domain of the protein at the interface of two adjacent subunits, with residues from each subunit contributing to the binding site (Sigel et al., 1992; Boileau et al., 1999; Boileau et al., 2002; Holden and Czajkowski, 2002; Newell et al., 2004). Residues from at least six polypeptide regions, named loops A to F, are involved (Akabas, 2004). A prototypical GABAa receptor appears to have two similar GABA binding sites which are located in a region between a and (3 subunits (Kash et al., 2004). The residues Thr-202, Ser-204, Tyr-205, Arg-207 and Ser-209 were found to be necessary for agonist and antagonist binding in the p2 subunit (Wagner and Czajkowski, 2001), while in the a l subunit, Phe-65, Arg-67 and Ser-69 are thought to be required (Boileau et al., 1999). The binding site is also located on the extracellular N-terminal domain of the receptor and is situated at the interface between the a and y subunits (Sigel, 2002). Studies using mutational analysis of the GABA binding site in the a l subunit show that different local movements within the site are elicited following binding of different ligands. This demonstrates that individual structural changes are produced by agonist induced channel gating, antagonist binding and allosteric modulation (Kloda and Czajkowski, 2007).

A high degree of sequence identity is observed in the TM2 region of GABAa receptor subunits, showing several highly conserved hydrophilic residues. It has been suggested that interaction between -OH groups on these residues and water molecules forming a hydration shell around the chloride ion, provides energetic stabilisation as they enter the pore domain (Kash et al., 2004). This region of the TM2 domain may enhance channel conductance, though is probably not involved in its selectivity. Instead, the extracellular ends of the TMl and TM2 segments contain a number of positively charged arginine and lysine residues that have been suggested to play a role in the selectivity filter of the channel. Recently, however, it has been demonstrated that the TM1-TM2 loop of the p subunit may have a prominent role in ion selectivity (Jensen et al., 2002). Experiments have shown that following the removal of GABA, picrotoxin can be trapped in the closed channel, suggesting that a closed channel gate is present between the picrotoxin binding site

- 2 2 - and extracellular end of the channel (Bali and Akabas, 2007). It has also been proposed that certain p subunit cys-residues dimerise in the closed state, forming disulfide bonds which lock the channel in its closed state (Yang et al., 2007).

The affinity of GABAa receptors for GABA and other ligands which bind to the same site, such as muscimol and isoguvacine, varies according to the subunit composition of the receptor (Macdonald and Olsen, 1994). At equilibrium, the binding of GABA agonists is heterogeneous with a high affinity component (Kd =10-20 nM) and one or more low affinity sites (Kd =1-10 pM to 50 - lOOpM) (Fisher and Olsen, 1986).

1.4 Regulation ofGABA a receptor function by allosteric modulators

GABAa receptors contain several modulatory sites for a large variety of pharmacologically important agents. Depending on the kinetic actions, allosteric modulators influence the equilibrium binding of the ligand: it can be reduced, left unaltered or increased, which defines them as negative, neutral and positive modulators respectively. Agents which are known to act as allosteric modulators on GABAa receptors include benzodiazepines, barbiturates, neurosteroids, general anaesthetics and Zn^"^.

Benzodiazepines are used clinically and exert their anticonvulsive, muscle relaxant, sedative and anxiolytic effects via positive allosteric modulation of GABAa receptors. They do not act to directly activate the receptors. It has been shown that a and y subunits of GABAa receptors are necessary in order for benzodiazepines to functionally modulate the receptor channel. The highest degree of sensitivity is conferred by the y2 subunit, while the less abundant yl and y3 subunits show significantly lower sensitivity (Sigel et al., 1990a). Several reports confirm the view that ligands of the benzodiazepine site exert their effects by modulating the EC 50 of the full agonists GABA and muscimol (Sigel and Baur, 1988; Wafford et al., 1992). An antagonist of this binding site is also in clinical use and negative allosteric modulators are used as investigative tools (Sieghart, 1995).

The binding pocket for benzodiazepines is thought to be dependent on amino acid residues His-101, Tyr-159, Gly-200, Thr-206 and Tyr-209 on the a l subunit, and Phe-77, Ala-79, Thr81 and Met-130 on the y2 subunit (Sigel and Buhr, 1997; Schaerer et al., 1998; Teissere and Czajkowski, 2001). The amino acid residues on al and p2 subunits which are

- 2 3 - involved in the formation of the GABA binding site are homologous to many of these amino acid residues (Sigel et al., 1992; Smith and Olsen, 1994; Newell and Czajkowski, 2003).

Functional analysis of benzodiazepine action in vivo has been carried out by creating transgenic mice bearing point-mutations of residues that are essential for benzodiazepine binding. Subunits which contain a conserved histidine residue (al-HlOl, a2-H101, a3- H126 and a5-H105) are able to bind diazepam, unlike those containing an arginine in equivalent positions (a4-R99 and a6-R100). In these mice the physiological function of the receptor remained unchanged, whist their modulation by benzodiazepines was prevented. Various behavioural tests have been carried out with these mice, which led to the conclusion that specific a subunit containing GABAa receptor subtypes are involved in specific types of behaviour regulated by benzodiazepines. For example, a 1-containing receptors are responsible for mediating sedative, anaesthetic, and anticonvulsant effects (Rudolph et al., 1999; McKeman et al., 2000), while a2- and a3-containing receptors mediate anxiolytic effects (Low et al., 2000). Receptors containing the a2 subunit also mediate muscle relaxant activity, which in addition requires a3 and oc5 subunits (Crestani et al., 2001; Crestani et al., 2002). Chronic activation of o5-containing receptors has been demonstrated to be necessary for the development of sedative tolerance to diazepam (van Rijnsoever et al., 2004).

GABA a receptor function can be strongly and specifically enhanced by low nanomolar concentrations of (Barbaccia et al., 1997), naturally occurring which are synthesised in the brain. These neurosteroids are also capable of acting as GABA-mimetics, directly activating GABAa receptors at submicromolar to micromolar concentrations (Stoffel-Wagner, 2003). It was originally thought that these neurosteroids originate from endocrine glands and therefore would have to cross the blood- brain barrier. However, it has subsequently arisen that some neurones and glia express the enzymes required for synthesis (Mellon et al., 2001), and that the brain is a steroidogenic organ capable of synthesizing these steroids de novo (Stoffel-Wagner, 2003). It appears that neurosteroids act in a paracrine fashion to modulate local inhibitory transmission, in addition to acting as remote endocrine messengers. Immunohistochemical

-24 studies of the distribution of pregnane steroids synthesising enzymes in the brain have shown a widespread distribution in the adult rat, especially in the olfactory bulb, striatum and cerebral cortex. Immunoreactivity appears to be limited to cell bodies and thick dendrites of mainly excitatory neurones (Saalmann et al., 2007).

The interaction of neurosteroids with GABAa receptors is dependent on their structure and is enantioselective, pointing to the presence of a specific GABAa receptor steroid binding site (Lambert et al., 2001). It has recently been shown that activation and potentiation of

GABAa receptors by neurosteroids are mediated by two different groups of amino acid residues in the TMl and TM2 domains. When apy receptors were heterologously expressed in HEK cells, potentiation of GABAa function was shown to be mediated by aGln-241 and ocAsn-407, while the GAB A mimetic effects of neurosteroids were caused by aThr-236 and PTyr-284 (Hosie et al., 2006).

The subunit composition of GABAa receptors can have a significant effect on neurosteroid- dependent regulation of receptors in some instances. However, studies have shown that varying the type of a subunit, when expressed with P2 and y2L subunits in Xenopus oocytes, has only small effects on the actions of allopregnanolone. However, these effects may be physiologically significant in vivo (Belelli et al., 2002). The modulatory effect of neurosteroids on GABAa receptor function are also largely unaffected by the type of p subunit incorporated in those receptors (Sanna et al., 1997; Belelli et al., 2002). Conversely, allopregnanolone enhances the effects of GAB A to a greater degree at alpl receptors in comparison to alplyl receptors. It appears that the y subunit isoform has no effect on the maximum effect of allopregnanolone but can alter its potency (Maitra and Reynolds, 1999). The presence of Ô subunits in place of y subunits has been shown to increase the modulatory effects of neurosteroids on GABAa receptors (Brown et al., 2002; Wohlfarth et al., 2002). This is in agreement with studies in mice lacking the ô subunit, which showed that the anaesthetic and anticonvulscant effects of neurosteroids are diminished in these animals (Mihalek et al., 1999). In addition, neurosteroids increase the extrasynaptically mediated “tonic” currents in dentate and cerebellar granule cells (Stell et al., 2003). Further evidence to suggest that the composition of GABAa receptors may be important for determining the effects of neurosteroids is provided by the observation that

- 2 5 - receptors incorporating the 8 subunit are insensitive to the modulatory effect of a number of neurosteroids, with allopregnanolone being an exception (Whiting et al., 1997). However,

8 -containing GABA a receptors can still be directly activated by a number of neurosteroids (Davies et al., 1997). Therefore, to date it remains unclear how important the composition of GABAa receptors is in determining neurosteroid selectivity.

Barbiturates, such as and phénobarbital, like benzodiazepines, increase the potency of GAB A, shifting its dose-response curve to the left and also increasing the size of the maximum G ABA response. They have been found to enhance GABA-mediated currents by increasing the channel open time, while having little effect on channel opening frequency (Twyman et al., 1989). Unlike benzodiazepines, barbiturates do not require y subunits to produce their effects, and at higher concentrations they can act as GABA- mimetics to directly activate GABAa receptors in the absence of GAB A (Simmonds, 1981).

Ethanol is a highly potent anxiolytic, sedative-hypnotic, muscle relaxant, amnesic and anticonvulsant drug which affects GABAa receptor function. However, direct evidence to demonstrate an interaction between ethanol and synaptic GABAa receptors is inconclusive (Kumar et al., 2004). In cultured cerebellar granule cells, only very high ethanol concentrations ( 1 0 0 - 300 mM) were found to have a stimulatory effect on GABAa receptor activity (Casagrande et al., 2007). Other reports suggest that ethanol enhances the tonic currents mediated by those receptors containing Ô subunits (Wallner et al., 2003; Wei et al., 2004). Ethanol administration may also dramatically increase the concentrations of neurosteroids in the cerebral cortex and hippocampus to pharmacologically active levels that affect GABAa receptor function (Barbaccia et al., 1999).

The anticonvulsant is selective for receptors containing p2 or P3 subunits, decreasing the EC 50 and the apparent maximal response for GAB A (Wafford et al., 1994), and increasing the rate of receptor desensitisation (Donnelly and Macdonald, 1996).

GABAa receptors are also modulated by Zn^"^ ions. They inhibit the receptor via an allosteric mechanism which alters the sensitivity of the GABAa receptor to GAB A. The degree to which this inhibition occurs is dependent on the subunit composition of the

- 2 6 - receptor, with ap-containing receptors being the most sensitive and apy receptors the least sensitive to Zn^ (Hosie et ah, 2003).

General anaesthetics represent another group of drug which interact with GABAa receptors and produce concentration-dependent effects on GABAa receptor function. At low concentrations they potentiate submaximal GABA-induced currents, while at higher concentrations they cause direct activation of these receptors. Although their exact mechanisms remain unclear, one hypothesis is that the polar anaesthetics enhance the function of GABAa (as well as glycine) receptors by causing slight rotations of the subunits resulting in a conformational change of the receptor (Horenstein et al., 2001). Recently, it has been suggested that this conformational change occurs at the TM2 segment in a similar way to when receptors are activated by G ABA (Rosen et al., 2007). Evidence has been presented for the role of p3-containing GABAa receptors in mediating some of the actions of general anaesthetics (Zeller et al., 2007). Studies utilising [^H]azietomidate, a radiolabeled analogue of , have identified amino acids that are part of the binding site: in the a l subunit, Met-236 (and the homologous methionines in a2, a3 and oc5) in the TMl transmembrane helix, and in the p3 subunit, Met-286 (and the homologous methionines in pi and P2) in the TM3 transmembrane helix. These methionines are located at the interface between a and P subunits as part of a single binding pocket within the transmembrane domain (Li et al., 2006). Additionally, some effects caused by the anaesthetic etomidate appear to be at least partially dependent on which p subunit is contained within the GABAa receptor complex, as behavioural responses differ in mice expressing point mutations (N265M) in the P2 and p3 subunits when etomidate is administered (Jurd et al., 2003).

1.5 Synthesis, expression and trafficking of GABAa receptors

The synthesis and assembly of GABAa receptors occurs in the endoplasmic reticulum. It has been proposed that trafficking of these receptors to various functional domains at the plasma membrane involves sorting of the receptors within the Golgi apparatus and transporting them in some form of secretory vesicles (Barnes, Jr., 2001). Proteins that are known to participate in these processes include GABAa receptor-associated protein (GABARAP), A-ethylmaleimide-sensitive factor (NSF) and Plic-1/2. Following this,

- 2 7 - accumulation of GABAa receptors at postsynaptic sites, which is essential for efficient synaptic transmission, appears to require the involvement of various proteins such as gephyrin, rapsyn, dystrophin, actin and microtubules, although the specific roles of these proteins are still poorly understood.

The stability of GABAa receptors at inhibitory synapses is essential for the control of neuronal excitability, as the efficiency of neurotransmission is dependent on the clustering of receptors at sites directly opposed to transmitter release sites. Although little is known about the mechanisms that anchor and cluster inhibitory neurotransmitter receptors in neurones, a number of proteins that directly associate with GABAa receptors have been suggested to play a role.

GABARAP is a microtubule binding protein that has been found to co-localise with GABAa receptors in cultured cortical neurones (Wang et al., 1999; Leil et al., 2004), due to a direct interaction with the y2 subunit (Nymann-Andersen et al., 2002a). GABARAP was proposed to play a role in sorting and transporting of intracellular vesicles containing GAB A A receptors to the plasma membrane by a microtubule-dependent mechanism (Kittler et al., 2001). Further evidence to indicate that the effects of GABARAP are due to interaction with microtubules was provided when co-expression of GABARAP and (Xlp2y2S-containing GABAa receptors in Xenopus oocytes was demonstrated to increase the amount of protein expressed at the cell surface and enhance GABA-mediated currents (Chen et al., 2005). This facilitation was shown to require the receptor y2 subunit and the microtubule-binding domain of GABARAP, as ablation of this domain or disruption of microtubule polymerisation abolished the stimulation of GABA-mediated currents (Chen et al., 2005). GABARAP has been shown to alter inactivation and desensitisation of GABAa mediated currents in HEK293 cells. Rather than altering receptor kinetics directly, this was postulated to occur by increasing the surface expression of y2 containing receptors instead of ap pentamers lacking y2 (Boileau et al., 2005). When apy receptors are co-expressed with GABARAP in L929 cells, high G ABA concentrations were found to increase both the maximum single channel conductance and mean open time of GABAa channels, indicating that ion channel properties may be shaped by trafficking processes (Luu et al., 2006). Once GABAa receptors are inserted into the plasma membrane, dissociation of GABARAP may allow a direct binding of the y2 subunit to the y or p subunits of neighbouring receptor

- 2 8 - molecules, partaking in synaptic clustering (Nymann-Andersen et al., 2002b). However, GABARAP knock-out mice display no loss of synaptic localisation of GABAa receptors, implying that GABARAP may not be essential for the trafficking and clustering of these receptors (O'Sullivan et al., 2005). Alternatively, the role of GABARAP in synaptic

localisation of y 2 -containing GABAa receptors may have been taken on by another protein in these knock-out mice as part of the survival mechanisms. It is also possible that GABARAP plays a less prominent role, such as accelerating or promoting processes leading to GABAa receptor clustering (O'Sullivan et al., 2005). GABARAP also binds directly to NSF, a protein that plays an essential role in intracellular membrane trafficking (Rothman, 1994; Leil et al., 2004). GABARAP and NSF complexes have been detected in neurones and are shown to co-localise in intracellular membrane compartments (Kittler et al., 2001). This would suggest a role for GABARAP in transport of GABAa receptors between intracellular storage compartments and the cell surface, and hence formation of GABAergic synapses, rather than participating in synaptic anchoring.

The thioacyl transferase Golgi-specific protein with a DHHC zinc finger domain (GODZ) has been demonstrated to palmitoylate the GABAa receptor y 2 subunit on multiple cysteine residues of the major intracellular loop in hippocampal neurones (Rathenberg et al., 2004; Keller et al., 2004). GODZ-mediated palmitoylation of GABAa receptors has been suggested to play a role in trafficking of GABAa receptors, as well as the regulation of GABAa receptor clustering and cell surface stability. Studies in neurones using GODZ- specific RNAi have shown that GODZ is needed for accumulation of GABAa receptors at synapses and normal GABAergic function. GODZ is also indirectly required for GABAergic innervation (Fang et al., 2006). GODZ-mediated palmitoylation of GABAa receptors therefore contributes to the formation and normal function of GABAergic inhibitory synapses.

GABAa receptor associating protein, GRIF - 1 (GABAa receptor interacting factor) was discovered using the yeast two-hybrid assay. GRIF-1 is a 913 amino acid protein which interacts with the large intracellular loop of the |32 subunit of GABAa receptors (Beck et al., 2002). GRIF-1 also directly interacts with kinesin (Brickley et al., 2005) and belongs to the same family as HAPl, another GABAa receptor binding potein. It has been

2 9 - hypothesised that GRIF - 1 plays an important role in intracellular trafficking of GABAa receptors.

Another protein shown to associate with GABAa receptors following yeast two-hybrid studies is brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) (Charych et al., 2004), which has a high binding affinity for the second intracellular loop of all the P subunits. Following co-expression of BIG2 and the p3 subunit in HEK-293 cells, a loss of P3- containing receptors from the ER and/or Golgi apparatus is observed. BIG2 was shown to be associated with microtubules, as well as co-localising with GABAa receptors at the neuronal plasma membrane. These results suggest that BIG2 plays a role in transport of newly assembled GABAa receptors from the trans-Go\g\ network to the cell surface (Charych et al., 2004).

The ubiquitin-like protein Plic-1 is another protein that has been shown to interact with GABAa receptors through its ubiquitin-associated (UBA) domain and the intracellular domains of a and P subunits. It is thought that Plic - 1 may facilitate the insertion of GABAa receptors into the plasma membrane, and may also act as a negative modulator of proteasome activity, resulting in the increased half-life of GABAa receptor subunits (Bedford et al., 2001).

It has been established that cytoskeletal proteins such as tubulin and tubulin-associated proteins are involved in membrane transport and clustering of GABAa receptors. One such protein is gephyrin, which is expressed along with GABAa receptors in various brain regions (Sassoe-Pognetto and Fritschy, 2000). Although both native and recombinant receptors which contain only a and p subunits can form receptors which insert into the plasma membrane, synaptic clustering requires either the y2 or y3 subunit (Essrich et al., 1998). Surprisingly, the use of chimeric y2 subunits suggests that the fourth transmembrane domain of the y 2 subunit is required for postsynaptic clustering of GABAa receptors, while the major intracellular loop is unnecessary. However, both of these domains appear to contribute to the association of GABAa receptors with gephyrin (Alldred et al., 2005). Interestingly, the lack of clustering of GABAa receptors and gephyrin in y2-deficient mice can be overcome by overexpression of the y3 subunit (Baer et

3 0 - al., 1999). However, in the retina and spinal cord of gephyrin deficient mice, and in rat and mouse Purkinje cells, gephyrin independent clustering of GABA a receptors has been noted

(Kneussel et al., 1999; Kneussel et al., 2001). This would indicate that for certain GABAa receptor subtypes or certain types of synapses, other mechanisms of clustering must exist. It has also been suggested that gephyrin may increase the accumulation of GABAa receptors by inhibiting their diffusion. The use of RNA interference (RNAi) to inhibit the expression of gephyrin was shown to reduce the amount of GABAa receptor clusters at the cell surface without affecting the total number of GABA a receptors expressed. These clusters were shown to be more mobile in the absence of gephyrin (Jacob et al., 2005). A potential role for gephyrin in intracellular trafficking has also been reported, following the observation that internalised GABA a receptors are relocated to a subsynaptic pool of receptors associated with gephyrin (van Rijnsoever et al., 2005).

Dystrophin is selectively clustered with GABA a receptors in some GABAergic synapses, where it has been proposed to regulate their stability, based on a reduced number of these clusters found in dystrophin knock-out mice (Kneussel et al., 1999). In cultured hippocampal neurones, a-dystroglan, |3-dystroglan and syntrophin, members of the dystrophin-associated protein complex (DPC), are co-localised with GABAa receptors and dystrophin (Brunig et al., 2002). Experiments with knock-out mice also revealed that the expression of GABA a receptor subunits was decreased in mice lacking the dystrophin gene (Wallis et al., 2004). It has been suggested that these proteins may provide a scaffold stabilising GABAergic postsynaptic apparatus during synaptogenesis, or that they stabilise GABAergic postsynaptic densities following neuronal circuit formation and synaptic remodeling (Fritschy et al., 2003).

Another cytoskeleton-related molecule that has been put forward to play a role in GABA a receptor clustering is the acetylcholine receptor clustering molecule, rapsyn (Ebert et al.,

1999). Although there is evidence for interaction between rapsyn and GABAa receptors, there is only a very low level of rapsyn in the brain, which may discount this protein from playing a major role in GABAa receptor clustering (Yang et al., 1997).

The most recent protein linked to GABAa receptor clustering is the actin-binding protein radixin, a member of the ERM (Ezrin, Radixin and Moesin) family. Radixin is proposed to

-31 - be the first directly interacting molecule that anchors GABA a receptors to cytoskeletal elements, following studies which demonstrate that intramolecular activation of radixin is a

functional requirement for GABAa receptor oc5 subunit binding. Additionally, depletion of radixin expression, as well as replacement of the radixin F-actin binding motif interferes

with GABAa receptor a5 cluster formation (Loebrich et al., 2006).

GABAa receptors cycle between the internal endosomal system and synaptic sites following their internalisation by clathrin- and dynamin-mediated endocytosis. Internalisation is mediated by a direct interaction between a and p adaptins within the AP2 complex, which are necessary for recruiting cell surface proteins into clathrin coated pits, and GAB A A receptor p and possibly y subunits (Kittler et al., 2000). A dileucine-binding motif on the p2 subunits which mediates AP2 binding is critical for dynamin-dependent

endocytosis. Disruption of this interaction leads to an increase in surface levels of GABAa receptors and thereby an increased whole-cell response to GAB A (Herring et al., 2003).

After entering the endosomal system, GABA a receptors can either be degraded or returned to the cell surface (Kittler and Moss, 2003). While it has been established that GABAa receptors cycle between synaptic sites and internal endocytic structures, it is unclear exactly how neurones regulate this process. Some evidence has been provided for insulin-mediated

translocation of GABA a receptors from the intracellular compartment to the plasma membrane (Wan et al., 1997; Wang et al., 2003; Vetiska et al., 2007). Changes in cytoskeletal structure mediated by the small GTPase R ad has also been postulated to play a role in these processes (Meyer et al., 2000).

Internalised GABAa receptors are either rapidly recycled to the cell surface membrane or they are targeted for lysosomal degradation (Kittler et al., 2004), depending on their

association with Huntingtin-associated protein 1 (HAPl), which directly binds to GABAa receptors and prevents their degradation. This suggests that HAPl plays a key role in the regulation of intracellular sorting of internalised GABAa receptors (Kittler et al., 2004).

Evidence indicates that one important mechanism of regulating GABAa receptors at the cell surface includes diffusion of receptors within the plane of the membrane (Thomas et al., 2005; Bogdanov et al., 2006). This lateral transport between distinct membrane pools is

-32 rapid in comparison with insertion from intracellular pools and appears to be an important mechanism for modulating the efficacy of GABAa receptors. Studies show that trafficking pathways that promote rapid importation of receptors into the synapse occur via diffusion from extrasynaptic domains as opposed to insertion from intracellular pools (Thomas et al., 2005). Newly inserted receptors at synaptic sites have been shown to access synaptic sites within 15 minutes via this diffusion (Bogdanov et al., 2006).

GABAa receptors have also been shown to play a role in regulation of GABAb receptor trafficking. In HEK 293 cells, association of the GABAbi subunit with the GABAa receptor 7 2 subunit promotes the cell surface expression of GABAbi in the absence of

GABAb 2, which is usually required for receptor trafficking to the cell surface (Balasubramanian et al., 2004).

In conclusion, a wide variety of proteins interact with GABAa receptors to modulate their synthesis, expression and trafficking. Concentration of GABAa receptors at synaptic sites is a multi-faceted dynamic process which greatly influences the synaptic efficacy of GABAergic transmission.

1.6 Subcellular localisation of GABAa Receptors

Structural diversity of GABAa receptors underlies significant functional diversity of GABAa receptors and affects their subcellular localisation profoundly. In hippocampal pyramidal neurones, receptors which contain al subunits are distributed evenly at all inhibitory synapses on the neuronal soma, dendrites, spines and axon initial segment. In comparison, receptors containing the a 2 subunit appear to be localised at axo-axonic synapses on the axon initial segment (Nusser et al., 1996). Cerebellar granule cells express a variety of subunits, which include al, a 6 , P2 , ps, 7 2 and 5, and can form receptors containing a/p2(p3)/72 or a/p2(p3)/6 (Laurie et al., 1992). The receptors containing the 7 2 subunit are found at synaptic sites on granule cells (Brickley et al., 1999), whereas those containing the Ô subunit are found extrasynaptically (Nusser and Mody, 2002). Receptors containing a4, a5 and a 6 are also found in some poulations of extrasynaptic GABAa receptors (Semyanov et al., 2004; Caraiscos et al., 2004; Farrant and Nusser, 2005), with receptors containing the a 6 subunit being expressed exclusively in the dorsal cochlear

- 3 3 - nucleus (Sieghart and Sperk, 2002) and cerebellar granule cells (Fritschy and Brunig, 2003).

1.7 Modulation ofGABA a receptors by protein phosphorylation and association with signalling molecules

It has become evident that direct phosphorylation of GABAa receptors is of major importance in their dynamic modulation, affecting channel gating, functional regulation and possibly even cell surface stability. In vitro phosphorylation studies in combination with

studies using heterologously expressed receptor subunits have established that GABA a receptor pi-3 subunits are phosphorylated at a conserved serine residue, Ser-408 in p3, Ser- 409 in pi and p3, and Ser-410 in P2, by protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG) and calcium/calmodulin-dependent kinase type 2 (CaMK II) (Moss et al., 1992a; McDonald and Moss, 1994; McDonald and Moss, 1997). The y2S subunit is phosphorylated by PKC and CaMK II on Ser 327. The y2L subunit, which contains an additional 8 amino acids in its intracellular domain, is also phosphorylated by PKC and CaMK II at Ser-343 (Moss et al., 1992a). PKA-dependent phosphorylation of heterologously expressed GABAa receptors containing pi subunits causes a decrease of

GABA-gated currents in HEK293 cells (Moss et al., 1992b). In contrast, GABAa receptors containing P3 subunits show an enhanced GABA-activated response when phosphorylated by PKA in the same cell type. No functional effects have been noted in cells expressing

GAB A A receptors containing the p2 subunit (McDonald et al., 1998). PKC-dependent modulation of GABAa receptors composed of ap and aPy subunits has also been investigated. Phosphorylation of Ser-327/343 in y2S/L subunits, as well as phosphorylation of Ser-409/410 in pi and P 2 subunits, was shown to inhibit GABAa receptor function (Moss and Smart, 1996). The PKC-pII isoform has been shown to bind to the intracellular loop of pi and p3 subunits directly, specifically phosphorylating Ser-409 in pi and Ser- 408/409 in p3 subunits. Additionally, it has been demonstrated that the tyrosine kinase Src phosphorylates Tyr-365/367 in the ^ 2 subunit, thereby enhancing the function of GABAa receptors composed of a ip iy 2 and aip2y2 subunits (Moss et al., 1995).

- 3 4 - Studies utilising phosphorylation state-specific antibodies have begun to demonstrate which of the signalling pathways are involved in controlling the phospho-dependent modulation of GABAa receptors in neuronal systems. It has been shown that the P3 subunit is basally phosphorylated on residues Ser408/409 by PKC (Brandon et al., 2000), whilst the y2 subunit is basally phosphorylated on residues Tyr365/367 (Brandon et al., 2001). BDNF, acting via TrkB receptors, causes an enhancement in miniature IPSC amplitude followed by a prolonged depression of miniature IPSC amplitude in cultured hippocampal neurones.

This occurs simultaneously with increased phosphorylation of the GABAa P3 subunit receptor by PKC, followed by dephosphorylation by protein phosphatase 2A (PP2A)

(Jovanovic et al., 2004). Therefore, it appears that the function of GABA a receptors can be bidirectionally modulated by BDNF regulating GABAa receptor phosphorylation and cell- surface stability (Tanaka et al., 1997; Henneberger et al., 2002; Jovanovic et al., 2004). Pharmacological studies with drugs that activate or inhibit PKC show that PKC modulates GAB A A receptor surface density, chloride conductance and sensitivity to positive allosteric modulators such as neurosteroids, ethanol, benzodiazepines and barbiturates (Song and Messing, 2005).

The receptor for activated C-kinase (RACK-1) is a scaffolding protein involved in the subcellular targeting of both a and P isoforms of PKC as well as other signalling proteins, including Src and phosphodiesterases (Yarwood et al., 1999). RACK-1 was found to enhance GABAa receptor phosphorylation by PKC, although it associates with GABAa receptor P subunits independently of PKC (Brandon et al., 2002). The functional modulation of GABAa receptors by muscarinic Ml receptors that activate PKC has also been shown to depend on binding of RACK-1 to P subunits (Brandon et al., 2002). In addition, PKC-dependent modulation of GABAergic transmission in the prefrontal cortex upon activation of 5 -HT4 receptors, has been demonstrated to depend on RACK-1 binding (Feng et al., 2001).

The pi and P3 subunits of the GABAa receptor are also phosphorylated by PKA (Brandon et al., 2002). A-kinase anchoring proteins (AKAPs) are involved in the targeting of PKA to specific substrates, and among these, AKAP 79/150 was shown to mediate binding of PKA to GABAa receptor pi and p3 subunits leading to PKA-dependent phosphorylation

(Brandon et al., 2003). The modulation of GABAa receptors by PKA has also been

- 3 5 - suggested to depend on association with another AKAP, the scaffold protein Yotiao (Lin et al., 1998).

The GABAa receptor |32 subunit is phosphorylated by the serine/threonine kinase Akt (or PKB). This results in an increase in receptor-mediated synaptic transmission, due to an increase in GABAa receptor insertion into the plasma membrane. Phosphorylation by Akt occurs at a phosphorylation site that is conserved in all P subimits, suggesting that Akt- mediated phosphorylation may also regulate pi and pS subunits (Wang et al., 2003). Recent evidence suggests that insulin can regulate expression of GABAa receptors at the cell surface via a phosphorylation-dependent mechanism which involves association between phosphoinositide 3-kinase (PI3-K) and the GABAa receptor P 2 subunit (Vetiska et al., 2007).

GAB A A receptor a subunits have been identified as substrates for phosphorylation by ERK kinases at the Thr-375 residue. In HEK293 cells transfected with aip2y2 GABAa receptors, ERK inhibition via inhibition of its upstream kinase, MEK, resulted in an enhancement of GABA-gated current amplitudes, which was abolished by mutation of Thr- 375 into a non-phosphorylatable residue (Bell-Homer et al., 2006).

CaMK Il-dependent phosphorylation of the GABAa receptor al subunit has been suggested to enhance the binding of allosteric modulators to these receptors (Chum et al., 2002). The addition of preactivated a-CaMK II has also been shown to increase the amplitudes of whole-cell GAB A currents in cerebellar granule neurones, as well as currents mediated by recombinant aip3 and aip3y2 GABAa receptors expressed in NG108-15 cells (Houston and Smart, 2006). It appears that in addition to directly phosphorylating the

GABAa receptor p3 subunit at Ser-383, CaMK II may be activating a tyrosine kinase that phosphorylates the y2S subimit at Tyr-365 and 367 as part of the same molecular mechanism (Houston et al., 2007).

Phospholipase C-related inactive protein type 1 (PRIP-1), a novel inositol 1,4,5- trisphosphate binding protein competes with the GABAa receptor y 2 subunit for its binding site on GABARAP (Kanematsu et al., 2002). A role for PRIP-1 in regulating GABAa

- 36 - receptor function was further suggested by altered GABAa receptor pharmacology and behaviour of PRIP-1 knock-out mice. Studies have shown both reduced effects of diazepam and a loss of Zn^^-induced inhibition of GABAa mediated effects, in addition to impaired motor co-ordination (Kanematsu et al., 2002). Additionally, PRIP-1 binds to the intracellular domains of p and to a lesser extent, y2 subunits (Terunuma et al., 2004). Comparison of PRIP-1 knock-out mice and wild-type mice showed that in wild-type mice, PPla counteracts PKA-mediated phosphorylation of P2 and P3 subunits, enhancing the expression at the plasma membrane (Terunuma et al., 2004; Yanagihori et al., 2006). Co- immunoprecipitation and pull-down assays indicate that PRIP is implicated in the clathrin/AP2-mediated internalisation of GABAa receptors (Kanematsu et al., 2007). It appears that the association of GABAa receptors with p subunits regulates the phosphorylation state of these subunits by recruitment of PPla, thereby modulating the cell surface expression. It has also been suggested that PRIP-1 and PRIP-2 are involved in

BDNF-dependent regulation of GABAa receptors by regulating the association of P subunits with PPl and PP2A (Kanematsu et al., 2006).

It has been established that some GPCRs may cause direct effects on GABAa receptor function, such as the dopamine D 5 receptor which directly binds to GABAa receptors. A protein-protein complex formation between the C-terminal domain of the D 5 receptor and the second intracellular loop of the GABAa receptor y 2 subunit allows rapid, mutual modulation of both GABAa a lp 2 y2 and dopamine Dg-mediated actions (Liu et al., 2000). This process occurs independently of established G-protein coupled receptor activation of intracellular signalling pathways. Activation of p-opioid receptors in periaqueductal gray neurones, a part of the descending pain control system, potentiates GABAa receptor current via PKA-dependent inhibition. It has been suggested that in the resting state, GABAa receptors are tonically inhibited by PKA. Opioid receptor activation leads to an inhibition of PKA, thus causing a potentiation of GAB A currents (Lee et al., 2003).

In summary, GABAa receptors are substrates for several protein kinases. A variety of studies have demonstrated that phospho-dependent regulation of GABAa receptors is of major importance in regulating their activity and subcellular distribution.

- 3 7 - 1.8 Presynaptic GAB Aa Receptors

The efficiency of neurotransmitter release can be influenced by a number of mechanisms, including the actions of receptors located on presynaptic nerve terminals defined as either autoreceptors or heteroreceptors. Autoreceptors are activated by the endogenous transmitter released from the same presynaptic terminal, thereby providing a positive or negative feedback type of regulation. In contrast, heteroreceptors are activated by neurotransmitters released from neighbouring presynaptic terminals due to their diffusion outside of the synapse (Kullmann, 2000). It is likely that the potency of neurotransmission is affected by activity of presynaptic receptors at the vast majority of synapses in the brain where they can mediate either facilitation or inhibition of presynaptic ion channels and/or synaptic vesicle exocytosis.

GAB A A receptors are found abundantly at inhibitory synapses, where they are located on dendrites and cell bodies precisely apposed to GABAergic nerve terminals. However, they are not limited only to these local postsynaptic zones, but are also found extrasynaptically, where they are generally tonically active (Brickley et al., 1996). In addition, GABAa receptors are expressed along axons where they function as components of axo-axonic synapses (Szabadics et al., 2006). The function and significance of these receptors is not yet fully understood, but may depend on the specific subunit composition of these receptors.

GABAa receptors are also present on presynaptic nerve terminals, as autoreceptors or heteroreceptors, where they have been demonstrated to either reduce or increase neurotransmitter release, depending upon the type of nerve terminals or the type of neuronal circuits involved. Presynaptic inhibition was first suggested following studies carried out in spinal motoneurones. These studies demonstrated that depression of EPSPs recorded across the postsynaptic membrane occurred without any change in the time course of these events, postsynaptic membrane potential or in the excitability of motoneurones (Frank and Fuortes, 1957). The same type of regulation was subsequently demonstrated in the crayfish neuromuscular junction, where this inhibition was shown to result from presynaptic GABAa receptor activation and Cl' mediated shunting of Na"^ currents (Dudel and Kuffler, 1961). However, GABAa receptors which are present on presynaptic

- 3 8 - terminals of the dorsal root ganglia inhibit release via a different mechanism. In this instance, it was shown that activation of GABAa receptors causes depolarisation of primary afferents, due to the Cl equilibrium potential being more positive in comparison to the resting membrane potential, leading to Cl" efflux (Eccles et al., 1961; Eccles et al., 1962; Eccles et al., 1963; Rudomin and Schmidt, 1999).

GABAa autoreceptors have been shown to reduce the K^-stimulated release of [^HJGABA from cerebellar neurones in dissociated cell culture (Pearce et al., 1982). Indeed, in a more recent study using cerebellar brain slices, Pouzat and Marty (1999) postulated that stellate and basket cell axons contain a large density of GABAa autoreceptors, although their effects on the release of GABA have not been directly demonstrated. GABA-mediated postsynaptic inhibitory currents recorded from CA3 pyramidal cells in rat hippocampal slices were decreased by GABAa receptor agonists, indicating that GABAa autoreceptors may participate in a negative feedback mechanism in these synapses (Axmacher and Draguhn, 2004). Two-photon imaging using the styryl dye FMI - 43 showed that the rate of vesicular release of GABA was decreased in these neurones under these conditions (Axmacher et al., 2004). In the suprachiasmatic nucleus, the presence of presynaptic

GABAa receptors was demonstrated by electron microscopy. These receptors, which contain a3 subunits, were subsequently shown to inhibit GABAergic transmission

(Belenky et al., 2003). GABAa receptors in dopaminergic neurones within the ventral tegmental area have recently been shown to facilitate GABAergic transmission (Xiao et al., 2007) as a result of membrane depolarisation caused by high intracellular Cl’ concentration

(Ye, 2000). In these studies, THIP, a ô subunit-containing GABAa receptor specific agonist, caused an increase in the frequency of spontaneous inhibitory postsynaptic currents

(sIPSCs), while , an antagonist of a4/a6 subunit-containing GABAa receptors, caused a decrease in sIPSC frequency, indicating the presence of a4/a6 and/or Ô subunits in these receptors (Xiao et al., 2007). GABA a autoreceptors also appear to be present in the cerebral cortex (Snodgrass, 1978; Mitchell and Martin, 1978; Brennan et al., 1981) and the substantia nigra (Arbilla et al., 1979).

A number of studies describe the actions of GABAa heteroreceptors in the modulation of presynaptic function. In mossy fibre terminals a biphasic dependence on membrane

- 3 9 - potential is exhibited by presynaptic action potential-dependent Ca^^ transients, which are altered by GABA a receptors. Antibodies against the a 2 subunit were shown to stain mossy fibre nerve terminals, although the exact subunit composition of functional GABAa receptors expressed on these terminals remains to be determined. These GABA a receptors have been postulated to play an important role in modulation of the flow of information to the hippocampus. When extracellular stimuli were delivered to the stratum lucidum while recording from granule cells in voltage clamp mode, the addition of muscimol reduced antidromic action potentials. had the opposite effect, suggesting that the receptors are tonically active (Ruiz et al., 2003). Presynaptic GABAa receptors have also been demonstrated to facilitate spontaneous glutamate release in rat CA3 pyramidal neurones (Jang et al., 2005). In these experiments, inhibition of voltage-gated Na"^ and Ca^"^ channels resulted in reduction of GABAa receptor-mediated facilitation, supporting the hypothesis that GABAa receptors depolarise these nerve terminals (Jang et al., 2005).

Activation of presynaptic GABAa receptors present on Schaffer collateral afferents by exogenously applied muscimol was shown to increase EPSC amplitude and reduce paired pulse facilitation. Additionally, extracellular recordings demonstrated that the excitability of these afferents was changed in response to muscimol. Additionally, muscimol was found to facilitate glutamate release from terminals attached to mechanically dissociated CAl neurones, demonstrating a direct depolarisation of glutamatergic Schaffer collateral terminals, (Jang et al., 2006). GABAa receptor-mediated depolarisation and subsequent facilitation of glutamate release in the ventromedial hypothalamus was shown to depend on the activity of the cation-chloride co-transporter NKCCl (Jang et al., 2001). In addition, muscimol-induced facilitation of spontaneous excitatory postsynaptic current (sEPSC) frequency was blocked by inhibition of voltage-gated Na"^ channels by TTX, as well as inhibition of VGCCS, indicating that GABAa receptor activity may lead to activation of voltage-gated Na"^ channels, followed by activation of VGCCs (Jang et al., 2001).

Depolarisation due to GABAa receptor activation was subsequently shown to be due to inward transport of Cl' by NKCCl, as muscimol-induced sEPSC facilitation was attenuated by inhibiting NKCCl with bumetanide (Jang et al., 2001). In the auditory brainstem, patch recordings from the Calyx of Held terminals in the medial nucleus of the trapezoid body demonstrated the presence of presynaptic GABAa receptors. Single-channel recordings displayed the presence of a number of conductance states, suggesting the possibility of a variety of receptor subtypes at these synapses (Turecek and Trussell, 2002). Activation of

- 4 0 - these receptors results in a depolarisation of the presynaptic plasma membrane, which was shown to facilitate glutamate release, by increasing Ca^"^ influx into the nerve terminal through VGCCs (Turecek and Trussell, 2001; Turecek and Trussell, 2002). Another area where presynaptic GABAa receptors have been identified is the locus coeruleus, where spontaneous glutamate release onto noradrenergic neurones has been shown to be increased in response to GABAa receptor activation (Koga et al., 2005). The effects of GABA a receptor activation in this study were shown to depend on the activity of NKCCl, demonstrating further that presynaptic GABAa receptors cause a plasma membrane depolarisation (Koga et al., 2005). An inhibitory effect of presynaptic GABAa receptors has been noted in basal ganglia, where they negatively regulate the release of glutamate.

However, it is unclear whether the observed effects are due to a direct action of GABA a heteroreceptors present on glutamatergic nerve terminals in the subthalamic nucleus, or due to indirect effects following inhibition of dopaminergic neurones in the substantia nigra (Hatzipetros and Yamamoto, 2006).

In the rat posterior pituitary, presynaptic GABAa receptors are present on presynaptic peptidergic nerve terminals. Activation of these receptors was found to cause a reduction in the release of oxytocin, arginine vasopressin and other hormones (Saridaki et al., 1989; Zhang and Jackson, 1993). Initial experiments showed that influx of Cl' through GABAa receptors causes a weak depolarisation, which blocks action potential propogation and limits release (Zhang and Jackson, 1993). Further studies showed that Eq\ was around -48 mV, indicating intraterminal Cl concentration is approximately 20 mM. Membrane shunting was demonstrated to play only a minor role in the inhibition of secretion, with the major determinant of action potential inhibition shown to be inactivation of Na"^ channels (Zhang and Jackson, 1995). Experiments using rat hippocampal synaptosomes have shown that activation of GABAa receptors can enhance noradrenaline release (Fung and Fillenz, 1983; Bonanno and Raiteri, 1987). This facilitation has been suggested to involve a localised depolarisation and opening of N-type VGCCs (Fassio et al., 1999).

Studies utilising GABAa receptor subunit specific antibodies have detected the presence of

GABAa receptor a and p subunits on presynaptic membranes in the substantia nigra and globus pallidus (Richards et al., 1987). They have also been identified at glutamatergic dendro-dendritic synapses of mitral cells in the olfactory bulb (Panzanelli et al., 2004).

-41 - In summary, accumulating evidence points to the presence of GABAa auto- and hetero­ receptors on presynaptic nerve terminals in a variety of brain regions. Activation of these receptors can modulate intraterminal Ca^^ concentration and neurotransmitter release, often as a result of depolarisation of the nerve terminal plasma membrane.

1.9 Cation co-transporters determine the functional outcome of GABAa receptor activation

Two electroneutral cation-Cl' cotransporters, NKCCl and KCC2, play important roles in modulating GABA a receptor activity, having been shown to affect GABAergic (and glycinergic) transmission via regulation of intracellular Cl" concentration [Cl"]i. The hyperpolarising effects of GABAa receptors are thought to be a result of Cl" extrusion by

KCC2 . Hence, the functional switch from excitatory to inhibitory function of GABA a receptors during development of the CNS was shown to correlate with up-regulation of KCC2 (Rivera et al., 1999; DeFazio et al., 2000). Additionally, Cl" accumulation by

NKCCl is thought to be responsible for the depolarising effects of presynaptic GABA a receptors in some brain regions (Jang et al., 2001; Koga et al., 2005).

NKCCl transports INa^ or K^ and ICI" ion into the cell, increasing [Cl"]i, whereas KCC2 transports IK^ and ICI" ion out of the cell and decreases [Cl"]i. NKCCl and NKCC2 have a molecular mass of -120 - 130 kDa. These proteins share a common topology of a large hydrophilic N-terminal (-20 - 30 kDa), a central hydrophobic membrane spanning region (-50 kDa) and an intracellular C-terminal domain (-50 kDa). The central region is thought to consist of 1 2 a-helical transmembrane segments, which are highly conserved between NKCCl and NKCC2 isoforms (Xu et al., 1994). These co-transporters are involved in the regulation of cell volume and maintenance of [Cl"]i. Their activity is regulated by changes in cell volume and [Cl"]i via phosphorylation/dephosphorylation-dependent pathways (Gamba, 2005). Both isoforms have N-linked glycosylation sites (Reshkin et al., 1993) and putative phosphorylation sites present in the N- and C-termini (Darman and Forbush, 2002). Three N-terminal threonine residues in NKCCl are phosphorylated in response to cell shrinkage and a decrease in [Cl"]i., leading to an increase in NKCCl activity (Darman and Forbush, 2002; Flemmer et al., 2002). NKCCl was shown to be expressed in the rat neocortex (Plotkin et al., 1997; Shimizu-Okabe et al., 2002).

- 4 2 - There are four KCC transporter isoforms, which have a similar structure and membrane topology to NKCCs and molecular mass of approximately 120 - 125 kDa (Gillen et al., 1996). They are termed KCCl, which is ubiquitous (Gillen et al., 1996), KCC2, which is neurone-specific and expressed in the cell body and processes of neurones (Lu et al., 1999; Williams et al., 1999), KCC3 and KCC4 which are also expressed in the CNS (Payne et al., 2003). KCC2 is inhibited by phosphorylation and activated by dephosphorylation (Darman and Forbush, 2002; Flemmer et al., 2002). KCC2 expression is low at birth but it increases during postnatal development of the CNS (Clayton et al., 1998; Lu et al., 1999).

During development GABA displays excitatory effects due to high [CF]i. The relationship between transmembrane Cl' concentration gradient and the reversal potential is extremely steep at physiological concentrations. This means that a change in [Cl']i as small as 12 mM is enough to cause the reversal potential to change from below to above the resting membrane potential, and in some instances even above the action potential threshold (Staley and Smith, 2001).

GABA is postulated to act as a self-limiting trophic factor during neural development causing changes in expression of the KCC2 co-transporter which correlate with the switch of GABAergic responses from being excitatory to inhibitory. This switch was shown to be delayed by chronic blockade of GABAa receptors and, conversely, accelerated by increased GABA a receptor activation (Ganguly et al., 2 0 0 1 ). The increase in depolarising GABA-mediated responses by NKCCl-mediated Cl" uptake has also been suggested to promote the formation of functional inhibitory synapses (Nakanishi et al., 2007). Additionally, the switch in GABAergic signalling from excitation to inhibition in developing neurones may also be mediated by endogenous nicotinic ACh activity. The mechanism responsible for this change is believed to involve a decrease in NKCCl and increase in KCC2 levels. These effects may be mediated directly by activation of a7-nACh receptors in neurones expressing these transporters or indirecty, acting through a7-nACh receptors in intemeurones (Liu et al., 2006). Down-regulation of NKCCl activity in response to secretion of maternal oxytocin during birth has been linked to a transient switch from GABA a receptor-mediated excitation to inhibition as part of the neuroprotective mechanism to insults and stress (Tyzio et al., 2006).

43 A number of studies have demonstrated that, due to NKCCl activity maintaining high

[Cl']i, the activation of presynaptic GABAa receptors can modulate neurotransmitter release via nerve terminal membrane depolarisation (Jang et al., 2001; Koga et al., 2005;

Xiao et al., 2007). Presynaptic GABAa receptor-mediated depolarisation may be due to the altered activity of CCCs in other brain regions, although not this has not been directly shown (Zhang and Jackson, 1995; Jackson and Zhang, 1995; Ruiz et al., 2003; Jang et al., 2005; Jang et al., 2006). Additionally reduced expression of KCC2 has been shown at axo­ axonic synapses, where GABAergic inputs evoke excitatory rather than inhibitory responses (Szabadics et al., 2006).

Cation-chloride co-transporters may play a prominent role in epileptogenesis. Altered levels of NKCCl and KCC2 expression have been shown to render some brain regions susceptible to seizures, due to the resultant excitatory actions of GABA (Dzhala et al., 2005; Palma et al., 2006; Sen et al., 2007; Munoz et al., 2007). In addition it has been demonstrated that NKCC1 mRNA and protein are transiently elevated in the choroid plexus following traumatic brain injury and directly contribute to brain oedema and neuronal damage (Lu et al., 2006).

1.10 Neurotransmitter release

The release of neurotransmitters occurs by the exocytosis of equally sized packets or quanta of transmitter, which are stored in individual small synaptic vesicles (Del Castillo and Katz, 1954). The arrival of an action potential at the nerve terminal opens Na"^ channels, resulting in depolarisation of the plasma membrane, opening of VGCCs and influx of Ca^"^. VGCCs are not evenly distributed in the presynaptic terminal, but are instead concentrated at active zones, forming microdomains of elevated intraterminal Ca^^ (Schoch and Gundelfinger, 2006). The rapid increase in [Ca^'^ji greatly increases the probability of membrane fusion, although terminals differ markedly in their Ca^^ sensitivity. Exocytosis can be triggered by [Ca^^ji as low as 5 - 10 p-M in some terminals or as high as 100 - 200 pM in others (Augustine, 2001). Not every action potential is converted into a secretory signal. In most terminals, only 10 % - 20 % of action potentials trigger release (Goda and Sudhof, 1997).

- 44 - A group of proteins referred to as SNARES (soluble N-ethylmaleimide sensitive factor [NSF]-attachment protein receptors) are associated with the regulation of synaptic vesicle exocytosis and neurotransmitter release. Syntaxin and SNAP-25 are known as target SNARES (t-SNARES) and synaptobrevin is referred to as a vesicular-associated SNARE (v-SNARE). Syntaxin 1 and 2 are 13 kDa transmembrane proteins, while SNAP-25 is found primarily on the cytosolic face of the plasma membrane (Murthy and De Camilli, 2003). Docking of synaptic vesicles to the plasma membrane occurs when nSecl, which is also known as Munc-18, dissociates from syntaxin, a process which also involves Munc-13 and Rab3-interacting molecule (RIM) (Pevsner et al., 1994; Augustin et al., 1999; Brose et al., 2000; Misura et al., 2000). Upon this, syntaxin binds SNAP-25 and synaptobrevin, bringing the SSV membrane and nerve terminal membrane in close proximity to each other (Koushika et al., 2001). SSVs then undergo membrane fusion, although the mechanism by which this process occurs is unclear. Vesicle fusion is dramatically increased by the increased levels of Ca^"^. This is believed to be due to the actions of synaptotagmins, which act as the main Ca^'^ sensor proteins presynaptically. Synaptotagmins are vesicular proteins, which bind Ca^"^, syntaxin, SNAP-25 and phospholipids (Li et al., 1995; Kee and Scheller, 1996; Schiavo et al., 1997; Chapman, 2002; Femandez-Chacon et al., 2002). When Ca^"^ binds to synaptotagmin, it causes buckling of the plasma membrane, thereby lowering the activation energy needed to promote membrane fusion. This results in fusion of docked SSVs, in conjunction with the ‘zippering’ of the parallel helices in the SNARE complex (Martens et al., 2007).

Considerable evidence suggests that synaptic vesicles undergo full fusion, completely incorporating into the plasma membrane as they release their stored neurotransmitter (Heuser, 1989). This fusion is followed by retrieval through clathrin-mediated endocytosis. After this classical full collapse fusion, vesicle retrieval and repriming requires several tens of seconds (Liu and Tsien, 1995; Ryan et al., 1993). However, other evidence supports the existence of a partial fusion mechanism as well, termed ‘kiss-and-run’, whereby vesicles release neurotransmitters by a transient fusion pore and then pinch off without fully collapsing into the plasma membrane. ‘Kiss-and-run’ forecasts that synaptic vesicles maintain their molecular identity, remain at the same site at the active zone, are rapidly reloaded with neurotransmitter and are once again ready for exocytosis (Fesce et al., 1994). ‘Kiss-and-run’ fusion remains controversial. While some studies suggest it to be the major

- 4 5 - mechanism of vesicle recycling at some synapses (Harata et al., 2006), others dispute this hypothesis (Granseth et al., 2006).

After fusion and neurotransmitter release, vesicles are endocytosed and recycled into distinct pools. The cycling of synaptic vesicles through exocytosis and endocytosis is fundamental to synaptic transmission. In clathrin-mediated endocytosis, the sorting of membrane proteins into clathrin-coated pits is believed to be driven by AP2, an adaptor complex that interacts directly with clathrin. AP2 has also been shown to bind directly to synaptotagmin (Zhang et al., 1994). Another adaptor protein, stonin, has been shown to interact with synaptotagmin (Fergestad and Broadie, 2001), and genetic deletion of stonin has been demonstrated to disrupt vesicle recycling (Fergestad et al., 1999; Stimson et al., 2001). Separation of the endocytic and plasma membranes is believed to be performed by the GTPase dynamin, a protein that is dephosphorylated during synaptic activity (Cousin and Robinson, 2001). Disruption of dynamin function has been shown to disrupt endocytosis at the late stage just prior to membrane separation (Koenig and Ikeda, 1989). The recruitment of syndapin, another protein important in synaptic vesicle endocytosis, was recently shown to be a result of dynamin dephosphorylation (Anggono et al., 2006). In addition to playing a prominent role in clathrin-mediated endocytosis, dynamin has been shown to be necessary for kiss-and-run events (Newton et al., 2006). Finally, the proteins involved in the endocytic process must be disassembled to allow the vesicle to be reused. Although little is currently understood, studies in synaptojanin knockout mice demonstrated accumulation of clathrin-coated vesicles and an increase in the time scale of vesicle re-use, suggesting the importance of phospholipid dephosphorylation in this process (Cremona et al., 1999).

The presynaptic terminal contains groups of synaptic vesicles which operate in a cycle, allowing their repeated use during prolonged activity. There are believed to be three distinct pools of vesicles, the readily releasable pool, the recycling pool and the reserve pool, each possessing different functional properties. At rest, a small proportion of vesicles

( ~ 1 %) which are immediately available for fusion, forming the readily-releasable pool, are docked at the active zone of the presynaptic nerve terminal. The recycling pool appears to contain between 5 - 20 % of synaptic vesicles, which recycle continuously during physiological stimulation (Harata et al., 2001; Richards et al., 2003). The reserve pool

- 4 6 - contains the majority of synaptic vesicles (-80 - 90 %) which participate in neurotransmitter release only during intense stimulation (Kuromi and Kidokoro, 2000; Delgado et al., 2000).

Neurotransmitter release can be regulated by presynaptic receptors via a number of mechanisms occurring at various stages of the release process. Activation of presynaptic receptors can inhibit release by activating/inhibiting Na^ or channels. This is observed in the crayfish neuromuscular junction, where activation of GABAa receptors causes Cl - mediated shunting of action potentials, reducing their duration and amplitude (Dudel and Kuffler, 1961). Depolarisation caused by presynaptic receptors can inactivate Na"^ channels if the depolarisation is subthreshold (Jackson and Zhang, 1995), whereas depolarisation can activate VGCCs and facilitate release by activating presynaptic Ca^^-dependent signalling pathways. Exocytosis can also be modulated at the level of SSVs by phosphorylation / dephosphorylation of synapsins by CaMK II / calcineurin respectively, thereby regulating the number of vesicles available at release sites (Llinas et al., 1991; Jovanovic et al., 2001). Alternatively, the activity of VGCCs can also be inhibited by presynaptic receptors, thereby decreasing Ca^"^ influx. One such example is the inhibition of N- and P/Q-type channels by

GABAb receptors which attenuates glutamate release (Huston et al., 1995). Finally, presynaptic receptors could impinge on neurotransmitter release via regulation of the release machinery itself. Muscarinic ACh receptors have been demonstrated to regulate release by directly interacting with SNAP-25 and syntaxin (Linial et al., 1997; Ilouz et al., 1999).

It is widely accepted that the modulation of synaptic strength occurs in part by modifications in neurotransmitter release mechanisms. Short-term plasticity has been attributed to rapid changes in [Ca^^Ji (Zucker and Regehr, 2002). Several proteins that are part of the presynaptic release machinery are known to be regulated by changes in [Ca^^Ji. These include synaptotagmins, Munc-13, RIM, and rabphilin (Augustin et al., 1999; Verhage et al., 2000). Numerous studies have indicated that phosphorylation of presynaptic proteins plays a prominent role in the regulation of synaptic strength (Micheau and Riedel, 1999). Protein kinases implicated in some aspects of presynaptic plasticity include CaMK II (Lisman et al., 2002), MAPK (Thomas and Huganir, 2004), PKA

- 4 7 - (Nguyen and Woo, 2003), PKC (Van der Zee and Douma, 1997) and CDK5 (Fischer et al., 2003).

1.11 Synapsins

Within the presynaptic terminals, a large number of proteins are specifically required to co­ ordinate the multiple steps of neurotransmitter release. However, synapsins occupy a special place among these proteins based on their exclusive presynaptic localisation and their well established regulation by multiple signal transduction cascades. Synapsins are a family of highly conserved neurone-specific phosphoproteins which are associated with synaptic vesicles (Greengard et al., 1993). They are only found presynaptically (De Camilli et al., 1983) and have been implicated in a number of neuronal functions, including recruiting synaptic vesicles to the reserve pool, regulating membrane fusion, stabilising synaptic vesicles, controlling synaptic vesicle clustering and mobilisation during nerve terminal activation, as well as in certain aspects of vesicle endocytosis. Synapsins are encoded by three genes, synapsin I, II and III, with a and b isoforms of synapsin I and II being generated by alternative splicing (Kao et al., 1999).

Investigations into the roles of synapsin have shown interactions with synaptic vesicles, actin, neurofilaments, microtubules and a number of signalling molecules. Under resting conditions, synapsins tether vesicles to each other and to the actin cytoskeleton, thereby forming a large pool of vesicles located at some distance from the release sites, also referred to as the “reserve pool”. Many biochemical properties of synapsins change upon phosphorylation at specific sites, including a reduction in the ability of synapsin to bind actin, and to associate with synaptic vesicles (De Camilli et al., 1990). Based on these findings, the current model of synapsin-dependent regulation of synaptic vesicles trafficking points to phosphorylation of synapsin during synaptic activity, leading to dissociation from vesicles, allowing them to migrate from the “reserve pool” of vesicles to the “readily releasable pool”, which then undergo exocytosis at the plasma membrane and release neurotransmitters (Greengard et al., 1993). When stimulation is terminated, synapsins are dephosphorylated and once more associate with the reserve pool of vesicles.

- 4 8 - Synapsin I was originally identified as a brain-specific protein which could act as a substrate for multiple protein kinases (Johnson et al., 1971), and several phosphorylation sites have now been identified (Fig. 2). Phosphorylation site 1 (P-site 1, Ser-9), which is located in domain A, is phosphorylated by cyclic AMP-dependent protein kinase (PKA), and calcium/calmodulin-dependent kinase (CaM kinase) I and IV. This phosphorylation decreases the binding affinity of synapsin to actin (Babler and Greengard, 1987), without affecting binding to synaptic vesicles (Schiebler et al., 1986). CaM kinase II phosphorylates Ser-566 and Ser-603 (Czemik et al., 1987), referred to as P-sites 2 and 3, which respond to the influx of Ca^'^ during nerve terminal activity (Huttner and Greengard, 1979; Sihra et al., 1989). This results in a major conformational change which reduces the binding affinity of synapsin for synaptic vesicles (Schiebler et al., 1986) and abolishes its interactions with actin (Babler and Greengard, 1987). Phosphorylation of synapsins by CaMK II occurs rapidly in response to action potential arrival and influx of Ca^'^ through VGCCs. This process is facilitated by a direct interaction between a CaMK II and synapsins shown to occur at the surface of synaptic vesicles (Benfenati et al., 1992). Thus, phosphorylation of CaMK Il-specific sites in synapsins provides us with a highly sensitive functional read-out of changes in [Ca^^ji concentration at rest as well as during synaptic activity.

P-sites 4 and 5 (Ser-62 and Ser-67), which are located in domain B are phosphorylated by extracellular signal-regulated kinases (ERKs) 1 and 2. P-site 6 (Ser-549), located in domain D is phosphorylated by ERK kinases, cyclin-dependent kinase (cdk) 1 and 5 (Jovanovic et al., 1996; Matsubara et al., 1996). Phosphorylation at these sites causes a slight reduction in the interaction of synapsin with actin, but no change in the ability of synapsin to bind to synaptic vesicles (Jovanovic et al., 1996). P-site 7 is specifically phosphorylated by cdk 5 (Matsubara et al., 1996; Jovanovic et al., 1996). Synapsin I is dephosphorylated at P-sites 1, 2 and 3 by protein phosphatase 2A (PP2A), (Jovanovic et al.,

2001). P-sites 4, 5 and 6 are dephosphorylated in response to Ca^'*' entry during nerve terminal activity, by the protein phosphatase 2B (calcineurin) (Jovanovic et al., 2001 ;Chi et al., 2003).

- 4 9 - ERK1&2 Cdk5 I P-site 4 Psite 5 P-site 6 Site 7 (Ser-62) (Ser-67) (Ser-549) (Ser-551)

rat-syn la

rat-syn-lb

P-site 1 Site 2 Site 3 (Ser-9) (Ser-566) (Ser-603)

PKA CaMKI/IV CaMKII

Figure 2. Domain model of rat synapsin la and Ib showing location of phosphorylation sites. Synapsins isolated from different species show a high degree of sequence similarity within domains A, C and E. Domains B and D, although very variable between different synaptic isoforms, contain highly conserved phosphorylation sites.

- 5 0 - Ultrastructural studies using Lamprey giant axon synapses have demonstrated the association of synapsins with endocytic vesicles, suggesting a regulatory role in vesicle recycling (Bloom et al., 2003).

Decreases in the level of synapsin expression are associated with a number of disease states such as schizophrenia, bipolar disorder (Vawter et al., 2002) and Alzheimer’s disease (Qin et al., 2004). Huntington’s disease (Lievens et al., 2002) and schizophrenia have also been associated with abnormal phosphorylation of synapsin I (Porton et al., 2004). Synapsins are also thought to be involved in neuronal development, having been shown to participate in the formation and maintenance of synaptic contacts, in addition to regulation of neurite elongation and branching (Ferreira and Rapoport, 2002).

1.12 Voltage-gated Ca^^ Channels

Voltage-gated calcium channels (VGCCs) play a critical role in controlling the release of neurotransmitters by regulating the influx of Ca^"^ into the nerve terminal and thereby Ca^"^- dependent synaptic vesicle fusion. Together with and Na"^ channels, they form a superfamily of proteins of voltage-gated ion channels (Catterall, 1995). VGCCs are composed of four or five subunits: a, p, Ô and y. The a l subunit is the largest (190 - 250 kDa), containing the conduction pore, the voltage sensor, channel regulation sites and the

gating mechanism. It has 4 domains (I - IV), each with 6 membrane spanning sections (SI -

S6 ). The S4 section acts as a voltage sensor, while ion conductance and selectivity is

determined by the pore loop between S5 and S 6 (Mikami et al., 1989). The a2 subunit is entirely extracellular and is linked with the plasma membrane by a disulphide bond with the transmembrane ô subunit (Tanabe et al., 1987; Witcher et al., 1993). The P subunit of VGCCs is entirely intracellular (Castellano et al., 1993a; Castellano et al., 1993b). Although differing pharmacological and electrical properties of VGCCs are derived mainly from the diversity of a l subtypes, p subunits can have profound effects on calcium channel function, including alteration of kinetic and voltage-dependent properties (Singer et al., 1991; De Waard et al., 1994; Brice et al., 1997). The yl subunit is present in skeletal muscle channels (Takahashi et al., 1987) while the localisation of other y-like subunits (y2 -

8 ) and their association with VGCCs is unknown (Letts et al., 1998; Moss et al., 2002).

-51 - channel subtypes have diverse physiological and pharmacological properties and are expressed in different neuronal subcellular compartments (Tsien et al., 1995). T-type Ca^"^ channels are also known as low voltage-activated (LVA) Ca^^ chaimels as they are activated by weak depolarisation, activating and inactivating at 20 - 30 mV more negative potentials than high voltage-activated (HVA) channels. The threshold for T-type channel activation has been reported to be between -75 and -60 mV. T-type channels also have a small single-channel conductance (6 -8 pS), resistance to current rundown and a low sensitivity to classical blockers of HVA channels (Huguenard, 1996; Perez-Reyes, 2003). They have been identified in cardiac (Bean, 1985; Nilius et al., 1985) and neuronal tissues (Carbone and Lux, 1984), modulating important cellular functions such as cell excitability, contraction, secretion, growth, differentiation and proliferation. In the CNS they are located mainly on the soma and dendrites of neurones (Huguenard, 1996; Craig et al., 1999; Gasparini et al., 2001). T-type Ca^"^ channels have been implicated in regulation of cardiac pacemaking, cardiac hypertrophy, slow-wave sleep, epilepsy and nociception. They can be targeted by neurotransmitters, hormones and neuroactive drugs (Perez-Reyes, 2003). Three different a l subunit genes encoding for T-type channels have been identified, alG, alH, a l l (Cav3.1 - 3.3) (Craig et al., 1999; Ertel et al., 2000).

Ca^"^ chaimels designated as HVA channels include L-, N-, P, Q- and R-type Ca^^ channels. L-type channels were so named because of their large single channel conductance (Nowycky et al., 1985), and are generally defined by their relatively slow activation and inactivation kinetics (Tanabe et al., 1988). L-type channels are blocked by dihydropyridines (DHPs) (Yatani and Brown, 1985). There are four VGCC a subunits, a lS , alC , alD , a lF (Cayl.l - 1.4), that generate L-type currents (Ertel et al., 2000; Lipscombe et al., 2002). However, evidence exists that not all L-type channels fit the traditional criteria. Cay 1.3 channels have been shown to be less sensitive to DHPs, have lower activation thresholds (approximately -55 mV), activate relatively quickly and are not long-lasting (Xu and Lipscombe, 2001; Koschak et al., 2001).

N-type (neuronal) channels are also referred to as Cay2.2 channels. They are highly sensitive to an irreversible block by co-conotoxin GVIA (co-CgTx), a peptide isolated from the marine cone snail Conus geographus (Plummer et al., 1989). They have intermediate

-52 inactivation kinetics and can be difficult to distinguish from L-type channels at the single channel level without the use of DHPs. Their conductance (-20 pS) is only slightly lower than L-type conductance (-25 pS) (Fox et al., 1987; Plummer et al., 1989). N-type Ca^"^ currents arise from a lB subunits (Williams et al., 1992; Ertel et al., 2000).

P- and Q-type Ca^^ channels (alA/Cav2.1) are difficult to distinguish pharmacologically from each other due to the lack of specific blockers. P-type channels were originally characterised in cerebellar Purkinje cells (Regan, 1991). They are specifically blocked by co-agatoxin IV A (co-Aga IVA), a toxin isolated from the venom of the funnel web spider Agelenopsis aperta, at concentrations from 100 nM (Mintz et al., 1992). They can also be inhibited by co-Conotoxin MVIIC (co-CTx MVIIC) at higher concentrations (Zhang et al., 1993; McDonough et al., 1996), but are insensitive to co-CTx GVIA. They display inactivation kinetics which are slower than those of the N-type Ca^"^ channels, showing no decay during 0.1 sec depolarisations (Randall and Tsien, 1995). Q-type Ca^'^ channels were first described in cerebellar granule cells and can be distinguished from P-type channels based on channel inactivation kinetics, showing approximately 35 % inactivation during 0.1 sec depolarisation. Q-type channels are also blocked by high concentrations of co-CTx MVIIC (Zhang et al., 1993; McDonough et al., 1996). Although it has been suggested that P/Q-type currents arise from alternative splicing of Cay2.1 subunits (Catterall, 1998; Bourinet et al., 1999), it appears that their differences may be due to post-translational modifications or modulation by proteins associated with the chaimels (Tsunemi et al., 2002). N-, P- and Q-type VGCCs are utilised for neurotransmitter release in various types of nerve terminals and are localised at the transmitter release sites (Dunlap et al., 1995; Stanley, 1997).

Many neurones display a component of HVA current that is resistant to DHPs, CgTx, co- Aga IVA and co-CTx MVIIC, known as the R (resistant) current. R-type channels, also known as a lE (Cay2.3) channels (Ertel et al., 2000), often activate and inactivate at more negative voltages than other HVA channels (Zhang et al., 1993). These channels are inactivated by the synthetic peptide toxin SNX-482, which is derived from the venom of the tarantula Hysterocrates gigas (Newcomb et al., 1998). R-type channels appear to participate in synaptic vesicle exocytosis, mediating 26 % of the total Ca^"^ influx in the

- 5 3 - calyx of Held terminals following presynaptic activation (Wu et al., 1998). They also contribute to transmitter release at the neuromuscular junction (Pagani et al., 2004). R-type channels also mediate cholinergic transmission in cultured superior cervical ganglion neurones (Mochida et al., 2003). They appear to be localised to the exocytotic machinery in some instances, controlling 55% of the release of catecholamines in adrenal chromaffin cells (Albillos et al., 2000), and can contribute to fast glutamatergic synaptic transmission at mossy fibre synapses (Gasparini et al., 2001). R-type chaimels have also been shown to participate in long term potentiation (LTP) (Dietrich et al., 2003).

1.13 Modulation of neurotransmitter release by presynaptic neurotransmitter receptors

Neurotransmitter release is modulated by a number of presynaptic neurotransmitter receptors, functioning either as auto- or hetero-receptors. Metabotropic receptors which are located presynaptically and modulate the release of neurotransmitters include metabotropic glutamate receptors (mGluRs), histamine, adenosine and muscarinic acetylcholine (mACh) receptors. lonotropic receptors that have been shown to regulate neurotransmitter release via presynaptic mechanisms include glycine, 5 -HT3, AMPA, kainate, NMD A, nicotinic acetylcholine (nACh), P2X, and TRPV1 receptors

1.13.1 Presynaptic G-Protein Coupled Receptors

1.13.1.1 Metabotropic Glutamate Receptors

Metabotropic glutamate receptors (mGluRs) are divided into 3 subgroups based on their sequence homology and signal transduction systems (Nakanishi, 1992). Group I contains mGluRl and mGluR5, which are coupled to Gq proteins and cause stimulation of phospholipase C (PLC) via an increase in hydrolysis of phosphatidylinositol bisphosphate

(PIP2) to inositol-triphosphate (IP 3) and diacylglycerol (DAG) (Pin and Duvoisin, 1995). Activation of presynaptic Group I mGluRs has been shown to cause an increase in glutamate release (Herrero et al., 1998). Group II contains mGluR2 and mGluR3, which are negatively coupled to adenylate cyclase through pertussis toxin-sensitive Gi proteins, leading to a reduction in intracellular cAMP levels (Pin and Duvoisin, 1995). Group II mGluRs have been shown to modulate glutamate release in the substantia nigra (Wang et

54 al., 2005), globus pallidus (Poisik et al., 2005), dentate gyrus (Dietrich et al., 2002) and from CAl hippocampal intemeurones (Price et al., 2005). Group III contains m01uR4, m01uR6, mOluR? and mOluRS, which couple to Gi/o proteins leading to reduction of cyclic adenosine monophosphate (cAMP) levels (Okamoto et al., 1994). They act as autoreceptors to regulate glutamate release, possibly by inhibiting VGCCs (Guo and Ikeda, 2005), or as heteroreceptors to reduce GAB A release in various brain regions (Lafon-Cazal et al., 1999).

1.13.1.2 GABAb receptors

GABAb receptors are coupled to Gai/o subunits which cause inhibition of adenylyl cyclase (Hill, 1985). Additionally, direct interaction with the Gpy subunit can either activate inwardly rectifying channels or inhibit VGCCs, both pre- and postsynaptically, in excitatory and inhibitory synapses in the central nervous system (Ong and Kerr, 2000;

Vacher and Bettler, 2003). Activation of presynaptic GABAb receptors by baclofen decreases glutamate release from rat cerebrocortical synaptosomes (Bonanno and Raiteri, 1992; Pende et al., 1993; Perkinton and Sihra, 1998). This inhibition of glutamate release has been shown to be due to decreased conductance of N- and P/Q-type VGCCs which are coupled to exocytosis (Huston et al., 1995; Dittman and Regehr, 1996). GABAb autoreceptors have been demonstrated to attenuate G ABA release via inhibition of P/Q- type VGCCs (Chen and van den Pol, 1998). A reduction of GAB A release by baclofen, as a result of an increase in conductance, has also been reported to occur in some GABAergic presynaptic nerve terminals. These effects are mediated via direct actions of activated G-proteins (Misgeld et al., 1989; Thompson and Gahwiler, 1992). A similar type of inhibition has also been shown in glutamatergic nerve terminals (Kubota et al., 2003).

Presynaptic GABAb receptors and subsequent Gi/o activation may also inhibit production of cAMP (Sakaba and Neher, 2003). They are also regulated by PKC activity, as part of a mechanism which appears to fine-tune neurotransmitter release (Perkinton and Sihra, 1998).

1.13.1.3 Histamine receptors

In hippocampal synaptosomes, activation of presynaptic histamine Hi and H% receptors, which are linked to Gq and Gs proteins respectively, causes an increase in evoked glutamate

- 5 5 - release due to an increase in [Ca^'^Ji (Rodriguez et al., 1997). Contrastingly, histamine H 3 receptors are coupled to Gi/o proteins (Takeshita et al., 1998) and reduce entry of Ca^^ through VGCCs (Molina-Hemandez et al., 2001). Electrophysiological and neurochemical evidence shows that glutamate release is decreased by these receptors in the hippocampus and striatum (Brown and Reymann, 1996; Brown and Haas, 1999; Doreulee et al., 2001;

Molina-Hemandez et al., 2001). H 3 receptors inhibit glutamate release from thalamic nerve terminals (Garduno-Torres et al., 2007).

1.12.1.4 Adenosine receptors

Stimulation of presynaptic adenosine Ai receptors can attenuate adenosine release (Yawo and Chuhma, 1993a), glutamate release (Prince and Stevens, 1992; Scanziani et al., 1992; Arrigoni et al., 2001) and G ABA release (Arrigoni et al., 2001). They are coupled to Gi/o proteins and modulate transmitter release by inhibition of N- and P/Q-type VGCCs (Yawo and Chuhma, 1993; Ambrosio et al., 1997). The activation of presynaptic Ai receptors inhibits release of GAB A in hippocampal CAl neurones via a cAMP- and PKA-dependent pathway (Jeong et al., 2003). Adenosine Ai receptor activation, can also inhibit cAMP/PKA-induced potentiation of glutamate release (Wang and Sihra, 2003).

Conversely, activation of presynaptic A 2A receptors enhances hippocampal glutamate release by attenuating the tonic effect of inhibitory presynaptic Ai receptors (Lopes et al.,

2002). It has recently been demonstrated that heteromerisation of Ai receptors and A 2A receptors allows adenosine to fine-tune the modulation of glutamate release. A 2A activation lowers the affinity of the Ai receptor for agonists, imparting a mechanism by which glutamate release can be either inhibited or facilitated by differing concentrations of adenosine (Cimela et al., 2006).

1.13.1.5 Muscarinic acetylcholine receptors

Muscarinic acetylcholine receptors are either coupled to Gq-proteins (Mi, M 3, M5) and

linked to DAG/IP 3, or to Gi/o, and regulation of cAMP (M 2, M4). Mi receptors attenuate GAB A release in the amygdala, nucleus accumbens and striatum (Sugita et al., 1991) as

well as in the visual cortex (Kimura and Baughman, 1997). M 2 receptors act as autoreceptors and inhibit the release of ACh in the hippocampus and cerebral cortex

(Kitaichi et al., 1999; Zhang et al., 2002a). M 3 receptors facilitate glutamate release in the

- 5 6 - amygdala, nucleus accumbens and striatum (Sugita et al., 1991). M 3 receptors also increase the release of GAB A in the striatum (Zhang et al., 2002a). Presynaptic inhibition of glutamatergic transmission is mediated by M 3 receptors in the rat mesencephalon (Grillner et al., 1999). M4 receptors inhibit ACh release in the hippocampus and cerebral cortex

(Zhang et al., 2002a), and G ABA release in the striatum (Zhang et al., 2002a). M 5 receptors facilitate dopamine release in the striatum (Zhang et al., 2002b).

1.13.2 Presynaptic ligand-gated ion channels

1.13.2.1 Glycine receptors

Glycine receptors are ligand-gated anion channels which are related closely to GABAa receptors and are composed of a (1 - 4) and P subunits (Kuhse et al., 1995). Presynaptic glycine receptors inhibit the release of vasopressin in the neurohypophysis (Hussy et al., 2001). Additionally, presynaptic glycine receptors are reported to be present on GABAergic nerve terminals in the ventral tegmental area where they inhibit G ABA release (Ye et al., 2004). They facilitate glutamate release in the auditory brainstem (Turecek and Trussell, 2001), and in the cerebellum (Kawa, 2003). Glycine autoreceptors increase glycine release in the spinal cord (Jeong et al., 2003) and cerebellar neurones (Kawa, 2003). The enhancement of neurotransmitter release by glycine receptors correlates with the depolarisation of nerve terminals, likely due to high [Cl'h (Ehrlich et al., 1999; Rivera et al., 1999; Kilb et al., 2002).

1.13.2.2 Nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine (nACh) receptors are cation channels composed of a (o2 - oclO) and P (P2 - P4) subunits, which can be assembled into heteropentamers or homopentamers (oc7 - a9) (Gotti and Clementi, 2004). Presynaptic nACh receptors can modulate neurotransmitter release in a number of brain regions (Wonnacott, 1997; Dajas- Bailador and Wonnacott, 2004). Immuno-gold labelling demonstrated the presence of a l subunits on presynaptic nerve terminals in the prefrontal cortex (Lubin et al., 1999). Facilitation of glutamate release by presynaptic nACh receptors was first shown in the chick, where they increase the frequency of spontaneous EPSCs (McGehee et al., 1995). Presynaptic nACh receptors can promote neurotransmitter release either by causing membrane depolarisation and opening of VGCCs, or as a result of Ca^"^ influx through the

- 5 7 - receptor itself. The release of [^H] noradrenaline from hippocampal synaptosomes following activation of a3|34 nACh receptors does not require VDCCs (Kulak et al., 2001). However, p2-subunit-containing nACh receptor-evoked release of dopamine from striatal synaptosomes is mediated by VGCCs (Soliakov and Wonnacott, 1996; Kulak et al., 2001). An alternative mechanism by which presynaptic nACh receptors can modulate neurotransmitter release has been observed in the striatum, where dopamine release was shown to be modulated by PKC (Soliakov and Wonnacott, 2001). GAB A release has been shown to be facilitated predominantly by cx3p2- or a6p2- and possibly a7-containing nACh receptors in the mouse superficial superior colliculus as a result of Ca^"^ influx through the receptor complex (Endo et al., 2005). In the CA3 region of the hippocampus, Ca^'^ influx through presynaptic nACh receptors causes release of Ca^"^ from intracellular stores and initiates glutamate release (Sharma and Vijayaraghavan, 2003), while activation of presynaptic nACh receptors containing a l subunits in CAl pyramidal neurones also enhances glutamate release (Liu et al., 2003).

1.13.2.3 5 HT3 receptors

Serotonin 5HT] receptors are Ca^^-permeable ion channel receptors belonging to the same family as the GABAa receptor. Presynaptic 5 HT3 receptor activation causes an enhancement in [Ca^^li in synaptosomes following Ca^^ influx through VGCCs in the striatum (Ronde and Nichols, 1998), hippocampus, amygdale and cerebellum (Nayak et al., 1999). Studies utilising brain slices and synaptosomes have demonstrated facilitation of neurotransmitter release by 5 HT3 receptors in the hippocampus, hypothalamus, frontal cortex and raphe nucleus (Martin et al., 1992; Blier and Bouchard, 1993; Haddjeri and

Blier, 1995). 5 HT3 receptors appear to act as autoreceptors in the hippocampus and raphe nuclei. Dopamine release is increased by 5 HT3 receptors in the striatum (Blandina et al., 1989; Zazpe et al., 1994), while noradrenaline release is increased in the hippocampus and neocortex (Allgaier et al., 1995). The frequency of spontaneous GABA-mediated IPSPs recorded from hippocampal CAl pyramidal neurones is increased by the addition of 5HT, an effect which can be reduced by the addition of the 5 HT3 receptor antagonist tropisetron (Ropert and Guy, 1991). Similarly, an increase in frequency and amplitude of GABA- mediated IPSCs caused by activation of 5 HT3 receptors has been demonstrated in CAl

- 5 8 - pyramidal neurones (Turner et al., 2004), and in mechanically dissociated hippocampal (Katsurabayashi et al., 2003) and amygdala (Koyama et al., 2000) neurones.

1.13.2.4 NMDA receptors

NMDA (N-methyl-D-aspartate) receptors are ionotropic glutamate receptors permeable to Ca^"^, Na"^, and K^. NMDA receptor activation requires binding of glycine to an allosteric site on the receptor, distinct from that of the agonist binding site. Activation also requires membrane depolarisation to remove Mg^'^ block of the ion channel (Mayer et al., 1984; Nowak et al., 1984). The activation of presynaptic NMDA receptors has been shown to increase the frequency of mIPSCs and decrease the amplitude of evoked IPSCs in cerebellar Purkinje, basket and stellate neurones (Glitsch and Marty, 1999). In layer II of the entorhinal cortex, NR2B-containing NMDA receptors mediate tonic facilitation of glutamate release (Berretta and Jones, 1996; Yang et al., 2006). NMDA heteroreceptors modulate release of dopamine (Wang, 1991) and noradrenaline (Wang et al., 1992). Presynaptic NMDA receptors enhance glutamate release in layer V neurones of the visual cortex (Sjostrom et al., 2003). In addition, in layer II/III pyramidal neurones NMDA receptor glycine binding sites regulate glutamate release (Li and Han, 2007). Activation of presynaptic NMDA receptors in the rat spinal cord dorsal horn results in an inhibition of glutamate release (Bardoni et al., 2004). It has been proposed that presynaptic NMDA receptors facilitate neurotransmitter release by increasing [Ca^^ji (Woodhall et al., 2001)

1.13.2.5 AMPA receptors

AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are composed of GluRl GluR2, GluR3 and GluR4 subunits which assemble in various stoichiometries, forming channels permeable to Na"^ and K^. They have been identified presynaptically by immunocytochemical methods (Martin et al., 1998) and have been shown to facilitate glutamate release (Sherman et al., 1992; Barnes et al., 1994; Chittajallu et al., 1996). Spontaneous release of G ABA is increased in the developing cerebellum following the activation of presynaptic AMPA receptors (Bureau and Mulle, 1998). In the mature cerebellum, activation of climbing fibres decreases the amplitude of evoked IPSCs recorded from Purkinje cells by causing a spillover of synaptically released glutamate and activation

- 5 9 - of presynaptic AMPA receptors (Satake et al., 2000; Satake et al., 2004). Direct recordings from the calyx of Held terminals have demonstrated the presence of presynaptic AMPA receptors that mediate inward current, causing a decrease in eEPSC amplitude (Takago et al., 2005).

1.13.2.6 Kainate receptors

Kainate receptors are ionotropic glutamate receptors. They are composed of five different subunits, GluR5, GluR 6 , GluR7, KAl and KA2 (Hollmann and Heinemann, 1994). They are permeable to Na"^ and K^. In the hippocampus, presynaptic kainate receptors can act to either enhance or depress neurotransmitter release. A reduction in glutamate release from Schaffer collaterals onto CAl neurones in hippocampal slices is caused by kainate receptor activation (Chittajallu et al., 1996). Similarly, in GABAergic hippocampal intemeurones, a reduction in synaptic transmission by kainate receptors has also been observed (Clarke et al., 1997; Rodriguez-Moreno et al., 1997). Kainate receptor-mediated modulation of neurotransmitter release is due to ionotropic actions causing nerve terminal depolarisation (Kerchner et al., 2001; Schmitz et al., 2001). However, facilitation of glutamate release in mossy fibre terminals by presynaptic kainate receptors is reported to be due to stimulation of adenylyl cyclase, reflected in the cAMP-mediated activation of PKA (Rodriguez- Moreno and Sihra, 2004). In the cortex, it has been suggested that kainate receptors located on presynaptic terminals decrease GAB A release (Ali et al., 2001). However, kainate receptors located on glutamatergic terminals in the cortex were shown to facilitate glutamate release (Perkinton and Sihra, 1999). Evidence has also been presented for the role of presynaptic kainate receptors in the cerebellum where these receptors either facilitate or depress transmission at parallel fiber synapses. Low-frequency stimulation of parallel fibers facilitates synapses onto stellate cells and Purkinje cells, whereas high- frequency stimulation results in depression of stellate cell synapses although still causing facilitation of Purkinje cell synapses (Delaney and Jahr, 2002). Kainate receptors in the hypothalamus increase the frequency but not the amplitude of GABA-mediated mIPSCs, pointing to a presynaptic site of action (Liu et al., 1999).

- 6 0 - 1.13.2.7 P2X receptors

P2X receptors are ATP-gated cation channels that are permeable to Na"^, and Ca^^ ions (North and Barnard, 1997), with permeability to Ca^^ differing between receptor subtypes and subunit composition (Evans, 1996). Immunostaining has demonstrated that P2X receptors are present on presynaptic nerve terminals in the olfactory bulb (Le et al., 1998), brain stem nuclei and in the dorsal horn of the spinal chord (Vulchanova et al., 1996). P2X receptors enhance the release of glutamate at dorsal root ganglion-dorsal horn synapses in culture (Gu and MacDermott, 1997) and in spinal cord slices (Nakatsuka and Gu, 2001). In addition, both spontaneous and evoked GABAergic transmission is facilitated by presynaptic P2X receptors in cultured dorsal horn neurones (Hugel and Schlichter, 2000).

1.13.2.8 Vanilloid receptors

The vanilloid receptor 1 (TRPVIR), is a noxious stimuli-gated and ligand-gated ion channel, permeable to Ca^^ and Na’^, leading to an increase in neuronal excitability (Van Der Stelt and Di Marzo, 2004). Increased release of GAB A in the hippocampus was suggested to be caused by the activation of presynaptic TRPVl recepetors (Huang et al., 2002), although the lack of evidence for functional presynaptic TRPVi receptors in this brain region has also been reported (Kofalvi et al., 2006). In the hypothalamus, activation of presynaptic TRPVl receptors has been shown to enhance both glutamatergic and GABAergic transmission. Given the permeability of these receptors, the facilitation could be explained by an increase in [Ca^'^Ji (Karlsson et al., 2005).

1.14 Isolated Nerve Terminals (Synaptosomes)

Synaptosomes are a well-established biochemical preparation of highly purified nerve terminals which have been ‘pinched off’ from their axons during brain homogenisation. Differential centrifugation of homogenised tissue in sucrose results in a crude ‘P2’ pellet. Contamination of this pellet by free mitochondria, myelin and broken postsynaptic membranes can be removed by purification using discontinuous Percol gradients (Dunkley et al., 1986; Dunkley et al., 1988). Once synaptosomes have been prepared, they remain

-61 - viable for 6 - 8 hours. As with intact nerve terminals, they measure 0.5 - 1 jiM in diameter on average. They contain an intact plasma membrane, mitochondria, a large population of SSVs and functional neurotransmitter transporters and receptors (Maycox and Jahn, 1990). Synaptosomes are able to produce ATP and are therefore metabollically active. They maintain a resting plasma membrane potential of -60 mV and -70 mV in media with low concentrations of K^. Synaptosomes can also maintain low cytosolic Ca^"^ concentrations (Gray and Whittaker, 1962; Nicholls et al., 1987; Nicholls, 2003).

A number of preparations can be used to measure neurotransmitter release. Brain slices are used for electrophysiological and neurochemical investigations, having the advantage of containing intact neuronal circuitry. However, this can also be a disadvantage as the modulation of neurotransmitter release by neurotransmitter receptors cannot be studied in complete isolation from their postsynaptic effects. Primary neuronal cell cultures have the advantage of highly homogenous populations of neurones, although to distinguish between pre- and post-synaptic mechanisms is often impossible. Synaptosomes are the simplest system with which to measure the release of neurotransmitters and directly correlate this process with the activity of presynaptic-specific signalling mechanisms. Neurotransmitter release can be electrically stimulated, although this release is largely independent of Ca^"^ (Bradford, 1970a; Bradford, 1970b; Bradford et al., 1973). Release can also be elicited by direct entry of Ca^^ with the addition of Ca^^ ionophores such as ionomycin, thereby bypassing the delimited mechanisms involving voltage-gated ion channels (McMahon and Nicholls, 1991; Sihra et al., 1992). Finally, depolarisation of synaptosomes by biochemical agents such as 4-aminopyridine (4AP), a channel blocker, or by the presence of high concentrations of KCl, can be utilised to study the Ca^^-dependent release of neurotranmitters from synaptosomes (Nicholls and Sihra, 1986; Tibbs et al., 1989), and its modulation by various types of presynaptic receptors in isolation from functional postsynaptic components.

The aims of this study are to establish the presence of presynaptic GABAa receptors in the rat neocortex, determining their effects on neurotransmitter release and elucidating their mechanism of action.

- 6 2 - 1.15 Aims

• To characterise the expression of functional GABAa receptors in isolated cerebrocortical nerve terminals.

• To investigate the regulation of presynaptic signalling pathways by GABAa receptors.

• To examine the regulation of neurotransmitter release from isolated cerebrocortical

nerve terminals by presynaptic GABAa receptors.

• To examine the modulation of membrane potential and intraterminal Ca^"^

concentration by presynaptic GABAa receptors.

• To determine the mechanism by which presynaptic GABAa receptors produce their effects by investigating their functional coupling to cation co-transporters, voltage- gated Ca^"^ channels and synaptic vesicle exocytosis.

-63 CHAPTER TWO

64 2. Materials and Methods

2.1 Preparation of synaptosomes

Synaptosomes were prepared from the cortices, hippocampi or cerebra of 2 month old (150 - 200 g) male Sprague-Dawley rats as described previously (Nicholls and Sihra, 1986; Nicholls et al., 1987; Nicholls and Sihra, 1986; Sihra and Nicholls, 1987). The animal was killed and the brain rapidly removed. The cortices were carefully dissected and homogenised in 320 mM sucrose in a Potter-Elvejhem tissue grinder. This consisted of a smooth glass mortar and Teflon pestle (0.1 - 0.15 mm clearance) which was rotated at 900 rpm with 8 up/down strokes. The homogenate was then centrifuged at 3,020 x g (5,000 rpm, JA-20) for 2 min at 4 °C. The Pi pellet, which contains cell bodies, nuclei, blood vessels, myelin and connective tissue, was discarded and the supernatant. Si, was centrifuged at 14,600 x g (11,000 rpm, JA-20) for 12 min at 4 °C. The supernatant, S 2, was discarded and the P 2 pellet, which is a crude synaptosomal suspension, resuspended in 2 ml of 320 mM sucrose with a Bounce homogeniser. A total volume of 8 ml was made up with the sucrose medium and the suspension mixed by gentle inversion. Due to contamination of the P 2 pellet with free mitochondria, myelin and other broken membranes, the suspension was further purified using a Percoll gradient method (Dunkley et al., 1986; Dunkley et al., 1988c). Percoll gradients were prepared by layering 2.5 ml of 23 %, 10 % and 3 % Percoll into 4 polycarbonate tubes, then layering 2 ml of the synaptosomal resuspension on top of each gradient. The tubes were then centrifuged at 35,100 x g

(16,500 rpm, JA-20) for 6 min at 4 °C. Three layers were formed in the gradient, with the 3 % layer containing myelin and light broken membranes, and the 23 % layer containing mitochondria. Synaptosomes were purified as a single band in the 10 % Percoll layer. The synaptosomal layer was harvested, transferred into HBM buffer (140 mM NaCl, 5 mM

KCl, 5 mM NaHCOs, 1.2 mM NaH2P0 4 , 1 mM MgCl2, 10 mM glucose, 1 mg/ml BSA and 10 mM HEPES, pH 7.4) and centrifuged at 27,216 x g (15,000 rpm, JA-20) for 10 min. The resulting final synaptosomal pellet was resuspended in 2-3 ml HBM buffer with the use of a Bounce homogeniser. Bradford assay (Bradford, 1976) was used to determine the synaptosomal protein concentration. The required amount of protein for each experiment

- 6 5 - was then added to 8 ml HBM buffer and subjected to a final spin at 3,020 x g (5,000 rpm,

JA-20) for 10 min, 4 °C. Pellets were then kept on ice and used within 6 hours.

2.2 Biochemical Assays

2.2.1. Bradford Protein Assay

The Bradford protein assay (Bradford, 1976) is based on the colour change of Coomassie Brilliant Blue G-250 dye after binding to proteins in solution. Under acid conditions, the dye is most stable as a doubly-protonated red form. The dye binds mainly to basic (especially arginine) and aromatic amino acid residues and upon protein binding it is most stable as an unprotonated, blue form. The absorbance of this form of the reagent is measured at 590 nm. The binding of Bradford reagent is sensitive to influence from non protein sources, such as detergents and salts. The response also varies with the composition of the protein. The linearity of the assay is established using increasing concentrations of a standard, usually bovine serum albumin (BSA),

The Bradford assay was carried out using Bio-Rad protein assay solution (diluted 1:5, Bio- Rad) to determine synaptosomal protein concentration prior to synapsin phosphorylation, glutamate release, membrane potential and intraterminal Ca^^ experiments. BSA was used to generate a standard curve (5, 10, 15 and 20 |ig). The concentration of synaptosomal samples (5 |il and 10 pi) were tested in duplicates and measured using a Beckman Coulter DU 800 Spectrophotometer.

2.2.2 Bicinchoninic acid (BCA) Protein Assay

The principle of the bicinchoninic acid (BCA) assay relies on the formation of a Cu^"^- protein complex under alkaline conditions, followed by reduction of the Cu^"^ to Cu^ by amides. The amount of reduction is proportional to the amount of protein present. BCA forms a purple-blue complex with Cu^ in alkaline environments and the reduction of alkaline Cu^"^ by proteins can be measured at an absorbance of 562 nm. The BCA assay is less variable than the Bradford assay because the protein-reagent complex is more stable. It is also less susceptible to detergents and salts, and is applicable over a broad range of protein concentrations.

- 66 - The BCA assay was used to determine the amount of synaptosomal protein loaded onto SDS-polyacrylamide gels following phosphorylation experiments. Assays included a series of duplicate standard solutions of 1, 2, 3, 4, 5, 10, 15 and 20 pg BSA to generate a standard curve, and duplicates of 5 pi of synaptosomal samples. Samples were heated at 37 °C for 30 min following the addition of 2 ml of working BCA reagent (Pierce, Rockford IL). Absorbance was measured at 562 nm using a Beckman Coulter DU 800 Spectrophotometer.

2.2.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a method used to separate proteins (Raymond and Weintraub, 1959; Davis, 1964; Omstein, 1964). SDS is an anionic detergent that binds to proteins and denatures them. It is used in combination with heat and 2 -mercaptoethanol, a reducing agent, to dissociate proteins before gel loading. It binds to proteins at a constant ratio of 1.4 g SDS per 1 g protein, so that the amount of SDS bound is always proportional to the molecular mass of the protein. SDS confers a negative charge to proteins. Due to the large amount of negatively charged SDS, all denatured proteins have a constant mass:charge ratio, ensuring proteins move through polyacrylamide gels according to their molecular mass. A discontinuous gel system was used (Omstein, 1964; Davis, 1964), where a large pore gel, (stacking gel) was layered on top of a separating gel. The high degree of resolution was achieved using a discontinuous buffer system: stacking gel buffer (125 mM Tris, pH 6 .8 ), separation gel buffer (325 mM Tris, pH 8 .8 ) and electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1

% (w/v) SDS, pH 8 .6 ). The stacking gel contained 3.37 % (v/v) acrylamide, 0.1 % (v/v) bis-acrylamide, 0.1 % (w/v) SDS and 125 mM Tris-HCl (pH 6 .8 ) and was polymerised using 1 |il/ml TEMED and 5 p.l/ml APS. The separating gels contained 10 % (v/v)

acrylamide, 0.2 % (v/v) bis-acrylamide, 0.1 % (w/v) SDS and 325 mM Tris-HCl (pH 8 .8 ) and was polymerised using 0.5 pl/ml TEMED and 2.5 |il/ml APS. The stacking gel contains chloride ions (leading ions) which migrate more quickly through the gel than the protein sample, while glycine ions in the electrophoresis buffer (trailing ions) migrate more slowly. Proteins are then trapped in a sharp band between these ions. When the proteins enter the separating gel, they are separated based on their molecular mass as they are sieved through the separating gel in a zone of uniform voltage and pH.

- 6 7 - Following polymerisation, gels were loaded with protein samples (amounts were determined using the BCA protein assay). For blots using anti-phosphosynapsin I P-Site 1, anti-phosphosynapsin I P-Site 3, anti-synapsin and anti-total synapsin I antibodies, 10 pg protein was loaded, whereas for blots using anti-phospho-CaMK II, anti-GABAAR al/2, anti-GABAAR p2/3, anti-GABAAR y2, and anti-GAD antibodies, 100 pg protein was loaded. Gels were run at a constant voltage (80 mV through the stacking gel and 150 mV through the separating gel) at room temperature. Rainbow molecular weight markers (Amersham) ranging between 14.3 kDa and 250 kDa were used to visibly label the gel during electrophoresis so that the positions of migrating proteins could be identified.

2.2.4 Quantitative Immunobloting

After proteins had been separated by SDS-PAGE, proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane (Protran, Schleischer and Schuell) (Burnette, 1981) in electrotransfer buffer containing 23 mM Tris, 192 mM glycine and 20 % (v/v) methanol overnight at a constant current of 200 mA, at room temperature. Membranes were labeled with Ponceau S (0.2 % (w/v) Ponceau Red, 3 % (v/v) trichloroacetic acid and 3 % (v/v) sulfosalycilic acid) to visualise the transferred proteins and then washed to remove Ponceau S.

Membranes were incubated for 30 min at room temperature in blocking buffer containing Tris-buffered saline (TBS) (50 mM Tris, pH 7.4, 200 mM NaCl), 0.05 % (v/v) Tween-20 and 1.5 % (w/v) nonfat dry milk to reduce non-specific binding of antibodies to the membrane. Membranes were incubated with a primary antibody in blocking buffer for 90 min to detect the protein of interest. Membranes were rinsed twice with blocking buffer, followed by 2 long washes ( 1 0 min each) to remove any excess, unbound antibody. Membranes were then incubated for 1 hr with ^^^I-Protein A or ^^^I-labelled anti-rabbit secondary antibody (Amersham Biosciences), diluted in blocking buffer, followed by 2 rinses and 2 washes (10 min each) with the blocking buffer. Membranes were then washed with TBS containing 0.05 % Tween-20 two times. Membranes were air-dried and exposed to a Phosphorlmager screen (Kodak). Antibody binding was visualised using

6 8 Phosphorlmager scanning (Amersham Biosciences) and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

2.2.5 Data Analysis

Following phosphoimager scanning, bands were highlighted using uniform-sized boxes in order to reduce errors due to differential contribution of backround radioactivity levels. A box of the same size was also used to measure the levels of background radioactivity, which was then subtracted from each sample value. The densitometry measurement for each band was then normalised to the 37 °C control.

Normalised Value (%)= Sample Value - Background ^ 31 °C Control - Background

Normalised values were used to preclude any blot to blot variability and day to day changes in phosphorimager sensitivity. Data were subsequently analysed in Microcal Origin and Microsoft Excel. Statistical analysis was carried out using One-way ANOVA followed by post-hoc LSD test.

- 6 9 - 2.2.6 Reagents

Name Abbreviation Source 3,3 '-dipropylthiadicarbocyanine DiSC3(5) Molecular Probes iodide 4-aminopyridine 4AP Sigma-Aldrich 5a-Pregnan-3P-ol-20-one 3p- Allopregnanolone Sigma-Aldrich acetate Acrylamide (30%) National Diagnostic Ammonium peroxodisulphate APS VWR BaClz VWR Baclofen Sigma-Aldrich Bicuculline methiodide Bicuculline Sigma Bicuculline methiodide Bicuculline Sigma Bis-acrylamide National Diagnostic Bovine serum albumin BSA Sigma-Aldrich Bradford Reagent VWR Bumetanide Sigma-Aldrich CaClz VWR Diazepam Sigma-Aldrich Ethylene glycol-bis (p-amino- BGTA Sigma-Aldrich ethyl ether) N, N, N’, tetra acetic acid Fluothane ICI Pharmaceuticals, UK Fura-2 acetoxymethyl ester (cell Fura-2 AM Molecular Probes permeable) GABase from Pseudomanas GABase Sigma-Aldrich fluorescens Gelatin VWR Glucose VWR Glycerol Sigma-Aldrich Glycine VWR H2O (HPLC grade) VWR Hepes VWR Ionomycin, Streptomyces Ionomycin Calbiochem Conglobatus Isoguvacine hydrochloride Isoguvacine Tocris KCl Sigma-Aldrich KH2PO4 VWR L-Glutamate Dehydrogenase GDH Sigma-Aldrich (type II from bovine liver) L-glutamic acid Glutamate Sigma-Aldrich Methanol VWR MgCl2 VWR

-70 MgS04 VWR Muscimol hydrochloride Muscimol Calbiochem N-(6-aminohexyl)-5-chloro- W7 Tocris I-naphthalenesulfonamide hydrochloride N, N, N’, N’- TEMEDVWR tetramethylethylethylenediamine NaCl VWR NaH2P04 VWR NaHCOg VWR NiCli VWR Paraformaldehyde Sigma-Aldrich Pentobarbitone Sagatal, Rhone Merieux Percoll VWR Picric acid Sigma-Aldrich Picrotoxin Sigma-Aldrich Ponceau S Solution Sigma-Aldrich SNX-482 Alemone Laboratory Sodium dodecyl sulphate SDS VWR Sucrose VWR Tris(hydroxymethyl)methylamine Tris VWR Nifedipine Sigma-Aldrich P-nicotinamide adenine NADP+ Sigma-Aldrich dinucleotide phosphate (O-Agatoxin IV A co-AgalVA Alemone Laboratory co-Conotoxin GVIA co-CTxGVIA Alemone Laboratory

2.2.7 Antibodies

Antibodies used for immunoblotting were anti-phosphorylated P-site 3 synapsin (RU19, 0.5 }ig/ml; Czemik et al., 1991), anti-phosphorylated P-site 1 synapsin (G257, 0.5 iig/ml), anti- synapsin (G304, 0.5 |ig/ml), all gifts from the laboratory of Professor Paul Greengard, Rockefeller University, New York), anti-total synapsin I antibody (1 p-g/ml; Synaptic Systems), anti-GABAAR ocl/2 (0.5 pg/ml), anti-GABAAR P2/3 (20 |ig/ml), anti-GABAAR p3 (0.21 |ig/ml), anti-GABAAR 7 2 (l|ig/ml; Chemicon), anti-glutamic decarboxylase (GAD, 1 pg/ml; Chemicon), anti-vGlut 1 (in serum: used at 1:5000; Chemicon), anti- phospho-Thr^^^-CaMK II (l|ig/ml; Phosphosolutions), ^^^I-labelled Protein A (0.05 jiCi/ml;

-71 Amersham Bioscience), ^^^I-labelled secondary anti-rabbit antibody (0.05 jiCi/ml; Amersham Bioscience), goat anti-rabbit IgG conjugated to Alexa 555 (2.67 |ig/ml; Molecular Probes), goat anti-mouse IgG conjugated to Alexa 555 (2.67 |ig/ml; Molecular Probes) and goat anti-guinea-pig IgG conjugated to Cy5 (2 pg/ml; Chemicon).

2.3 Immunohistochemistry

2.3.1 Anaesthesia and dissection

All procedures carried out in this study were according to British Home Office regulations with regard to the Animal Scientific Procedures Act 1986. In order to aid animal handling, male rats were anaesthetised by inhalation with fluothane. Once the righting reflex was no longer evident, animals were anaesthetised by an intra-peritoneal injection of sodium pentobarbitone (60 mg/kg). The toe pinch reflex was used to monitor the level of anaesthesia; when this reflex was no longer present, the rats were perfused transcardially with ice cold oxygenated modified Artificial Cerebrospinal Fluid (mACSF) (248 mM

sucrose, 25.5 mM NaHCOs, 3.3 mM KCl, 1.2 mM KH 2PO4 , 1 mM MgS0 4 , 2.5 mM CaCb,

and 15 mM D-glucose, equilibriated with 95% O 2 and 5% CO 2). Sodium pentobarbitone (60 mg/L) was added to the mACSF to maintain the anaesthesia. The perfusion was carried out in order to remove blood from the tissue and improve its preservation. For the perfusion, a large incision was made in the abdomen below the sternum. To gain access to the thoracic cavity, Spencer Wells forceps were clamped to the base of the sternum and the diaphragm excised. The rib cage was cut along both sides to reveal the heart. A butterfly needle (Butterfly - 21) connected to a beaker of mACSF was inserted into the left ventricle and the right atrium cut open with scissors. Once the liquid flowing from the right atrium became clear, the animal was decapitated with sharp scissors. An incision was made in the scalp using a scalpel blade to reveal the skull. Each of the skull plates were then removed using flat nose pliers to expose the brain. The dura was removed with a scalpel and the brain gently scooped out of the skull and immersed in fresh ice cold oxygenated mACSF. The whole brain was placed on top of a sheet of filter paper moistened with cold mACSF and positioned on a Petri dish filled with ice. A block of cortex was cut, stuck onto the

- 7 2 - chuck of a vibroslice (Campden Instruments, Loughborough, UK), and 450 - 500 jim slices were cut.

2.3.2 Immunohistochemistry

Histology and immunofluorescence were performed as described previously (Hughes et al., 2000). In order to preserve the tissue by cross linking proteins and maintain the relative positions of tissue structures without the leakage of important constituents, slices were fixed overnight in depolymerised 4 % paraformaldehyde and 0.2 % saturated picric acid solution in 0.1 M phosphate buffer (PB) pH 7.2, at 4 °C. As fixatives containing high concentrations of glutaraldehyde are known to increase levels of background labelling during fluorescence microscopy (Totterdell et al., 1992), slices of tissue were immersed in a glutaraldehyde-free fixative solution.

After washing in PB, slices were laid out flat in a petri dish and embedded in 3-5 mm of molten gelatin (38 - 40 °C), in order to provide a firm and stable media to support the tissue during sectioning on the Vibratome. The gelatin solution (12 % gelatin (w/v) made up in distilled water) was then hardened at 4 °C for 30 min. Blocks containing the gelatin- embedded tissue were fixed for 30 min in the same fixative solution used to fix the slices, then rinsed three times in 0.1 M PB before being cut in series to a thickness of 50 pm using a Vibratome.

Prior to the permeabilisation of cell membranes by freeze-thawing above liquid nitrogen the sections were incubated in PB-based cryoprotectant solutions: 2 x 10 min in 10 %

sucrose (w/v), 2 x 2 0 min in 2 0 % (w/v) sucrose with 6 % (v/v) glycerol, followed by 2 x 30 min in 30 % (w/v) sucrose and 12 % (v/v) glycerol. The sections were then carefully placed in envelopes of aluminium foil ensuring that they were flat, that they did not overlap and that excess cryoprotectant was removed. By taking such precautions the risk of sections shattering during the freeze-thawing cycles could be minimised. Sections were frozen by holding them just above liquid nitrogen for 45 sec and thawed until all the resulting condensation on the aluminium had disappeared. This rapid freeze-thawing cycle was repeated three times for each slice.

- 7 3 - After washing the sections in PB (3 x 5 min) to remove cryoprotectant medium, the sections were incubated in 1 % (w/v) sodium borohydride (NaBILt) solution in 0,1 M PB for 30 min. The liberation of ions from this solution terminates the cross-linking actions of the aldehyde fixative and blocks endogenous peroxidase activity in the tissue (KOSAKA ET AL., 1987}, which results in a reduction of general background fluorescence. The sections were again washed in PB (5 x 5 min) to remove traces of NaBEU prior to incubation with 1 % (w/v) BSA in 0.1 M phosphate buffered saline (PBS) for 30 min. BSA (Sigma) was used as a blocking solution to reduce non-specific binding of antibodies.

Sections were incubated with primary antibody solutions between 14 and 16 h at 4 °C. The following day the sections were washed (5x5 min) in PBS and incubated in a mixture of secondary antibodies tagged with different fluorescent markers. Each slice was incubated in a cocktail of fluorescently-tagged markers, diluted in PB containing 1% (w/v) BSA, comprising goat anti-rabbit IgG conjugated to Alexa 488 (diluted 1:750; Chemicon), goat anti-mouse IgG conjugated to Alexa 555 (diluted 1:750; Chemicon) and goat anti-guinea- pig IgG conjugated to Cy5 (diluted 1:750; Chemicon). Immunoreactivity was visualised using a Zeiss LSM 510 Meta laser scanning confocal microscope.

2.4 Functional assays

2.4.1 Measurement of glutamate release

Glutamate release from synaptosomes can be measured continuously on-line using an enzyme-linked fluorometric technique (Nicholls and Sihra, 1986). This technique was initially developed using guinea-pig synaptosomes prepared from the cerebral cortex, but has since been used to study release from rat and mouse brains (Sihra et al., 1992; Li et al., 1995; Rodriguez-Moreno and Sihra, 2004). Released glutamate is metabolised to a- ketoglutarate and ammonia by the addition of glutamate dehydrogenase (GDH), with nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. GDH is added in large amounts in order to efficiently process the low levels of vesicular glutamate. The oxidative

- 7 4 - deamination causes reduction of NADP to NADPH, which is fluorescent, emitting a maximum fluorescent signal at 460 nm when excited at 340 nm. The increase in NADPH fluorescence therefore can be correlated with the amount of released glutamate, with the reaction predicting a direct 1:1 molar ratio between NADPH produced and glutamate released. NADP^ is used instead of the physiological cofactor NAD"*" as the reduced NADPH is not recognised by mammalian oxidases and therefore is not reoxidised. However, it is equally effective for the reduction process (Sanchez-Prieto et al., 1987). A high concentration of NADP^ ensures that the equilibrium of the reaction tends towards the production of NADPH. By adding 2.5 nmol exogenous glutamate after each experiment and monitoring the subsequent fluorescence change, the response can be calibrated and the evoked glutamate release expressed as nmol/mg synaptosomal protein.

X Ex 340 nM X Em 460 nM

GLUTAMATE + H^O a-KETOGLUTARATE + NH,

NADP NADPH + H+

Figure 3. Glutamate is metabolised to a-ketoglutarate and anunonia. Reduction of NADP to NADPH emits a maximum fluorescent signal at 460 nm when excited at 340 nm.

Synaptosomes (0.1 mg) were resuspended in 1.5 ml HEM containing 1 mg/ml BSA, transferred to a cuvette and placed in a Perkin-Elmer LS-5 spectrofluorimeter (Perkin- Elmer, Emeryville, CA) at 37 °C with stirring. Glutamate dehydrogenase (GDH, 50 Units/ml) and NADP^ (2 mM) were added immediately, followed by 1 mM CaCL after 3 min. To stimulate the release of glutamate, 4AP, KCl or ionomycin were added after 10 min. The addition of any other drugs is detailed in the results section and in figure legends. The addition of an exogenous glutamate standard (2.5 nmol) after 15 min enabled the calculation of glutamate release. Data was accumulated at 2 sec intervals and analysed

- 7 5 - using Lotus 1-2-3, Microcal Origin and Microsoft Excel, with traces being aligned vertically and horizontally at the point of stimulation in order to allow direct comparisons of the effects of various additions on glutamate release. Statistical analysis was carried out using One-way ANOVA followed by post-hoc LSD test.

^ , Sample fluorescence 2.5 nmol Glutamate Release = ------^------x ------Glutamate standard fluorescence Synaptosomal protein (mg/ml)

2.4.2 Measurement of membrane potential

In order to directly measure changes in synaptosomal membrane potential, carbocyanine dyes, such as DiSCsCS) can be utilised. DiSC3(5) is a lipophilic cationic dye that accumulates in hyperpolarised membranes and is translocated into the lipid bilayer by an electrophoretic mechanism. Upon depolarisation, release of the dye from the membrane bilayer occurs, and is indicated as an increase in fluorescence due to sensitivity of the dye to intracellular and extracellular environments (Hargittai et al., 1991). DiSC3(5) can inhibit respiration (Smith et al., 1981) and may therefore be relatively cytotoxic at high concentrations (Anderson et al., 1993).

Synaptosomes (0.1 mg) were resuspended in 1.5 ml HBM containing 1 mg/ml BSA, transferred to a cuvette and placed in a Perkin-Elmer LS-5 spectrofluorimeter at 37 °C with stirring. After 1 min, 4 |iM DiSC3(5) was added, followed by 1 mM CaCl 2 after 3 min, and allowed to equilibrate. Muscimol (1 - 500 jiM) was added after 7 min of incubation. Depolarisation was triggered by the addition of 1 or 3 mM 4AP or 10 mM KCl after 10 min. Fluorescence was measured at an excitation wavelength of 651 nm and emission wavelength of 675 nm, and data accumulated at 2 sec intervals. Cumulative data was analysed using Lotus 1-2-3 and results expressed as fluorescence units. Further analysis was carried out using Microcal Origin and Microsoft Excel. Statistical analysis was carried out using One-way ANOVA followed by post-hoc LSD test.

- 7 6 - 2.4.3 Measurement of intraterminal calcium concentration

Fura-2 is a fluorescent Ca^"^ chelator used in order to determine intraterminal free Ca^^ concentrations. Binding of Ca^"^ depends on the free Ca^'^ concentration and the dissociation constant (Kd) for the chelator. With fura-2, half maximal binding occurs at 224 nm at 37 °C. The excitation spectrum of fura-2 changes following binding of Ca^"^, with an increase in fluorescence at 340 nm and a decrease at 380 nm. This allows the proportion of free fura-2 and Ca^^-fura-2 (and therefore the Ca^^ concentration) to be calculated from the ratio of emission intensity following excitation at 340 nm and 380 nm. An important characteristic of this ratiometric determination is that the signal is independent of dye loading or variations in the size of the cell. Fura-2 is hydrophilic and therefore membrane impermeable due to five carboxyl groups. When these carboxyl groups are esterified to form acetoxymethyl (AM) esters, the resultant fura-2-AM is hydrophobic enough to cross the plasma membrane. Non-specific esterases cleave off the AM groups once in the cell cytoplasm, releasing fura-2. After a 30 min incubation period, most of the original fura-2 AM will be accumulated in the cytoplasm as free fura-2.

Synaptosomes (0.1 mg) were resuspended in 1 ml HBM containing 1 mg/ml BSA, 0.1 mM CaClz and 5 |iM Fura-2 AM (Molecular Probes) for 30 min at 37 °C with stirring. The synaptosomes were centrifuged in a microcentrifuge for 30 sec at 10,000 rpm. The resulting pellet was resuspended in 1.5 ml HBM containing 1 mg/ml BSA, transferred to a cuvette and placed in a Perkin-Elmer LS-5 spectrofluorimeter at 37 °C with stirring. CaCl 2 (1 mM) was added after 3 min, followed by depolarisation with 1 mM 4AP after 10 min. The addition of any other drugs is detailed in the results section and in figure legends. At the end of each experiment, calibration was performed as described previously (Sihra et al., 1993), using 0.1 % (w/v) SDS to lyse synaptosomes. This was followed by the addition of 0.5 M Na-EGTA/1 M Tris, at a pH known to neutralise the increase in [H"^] caused by EGTA binding to 1 mM Ca^^. Upon lysis, fura-2 binds saturating amounts of CaCli (1 mM), giving a maximum fluorescence. Na-EGTA subsequently chelates Ca^"^, allowing measurement of minimum fluorescence levels. Fluorescence measmements are accumulated at excitation wavelengths of 340 nm and 380 nm (emission wavelength 505 nm) at 3.5 second intervals.

- 7 7 - Cytosolic free concentrations were then calculated using the following calculation (Grynkiewicz et al., 1985).

V^max “ ^b2

Kd = 224 iiM at 37 °C R = Experimental fluorescence ratio value (340 nM/380 nM) Rmin = minimum fluorescence ratio value Rmax = maximum fluorescence ratio value

Sf2 = minimum fluorescence value at 380nm

Sb 2 = maximum fluorescence value at 380nm

Cumulative data was analysed using Lotus 1-2-3. Further analysis was carried out using Microcal Origin and Microsoft Excel. Statistical analysis was carried out using One-way ANOVA followed by post-hoc LSD test.

-78 CHAPTER THREE

79 3. Regulation of presynaptic Ca^^-dependent signalling pathways and synapsin I phosphorylation by G A B A a receptors

3.1 Introduction

Neuronal Ca^"^ signalling is tightly controlled to ensure proper functioning of numerous Ca^^-dependent processes (Berridge et al., 1998; Berridge et al., 2000). Changes in intraterminal Ca^"^ levels are an important factor in modulating presynaptic function, regulating neurotransmitter release and acting as a second messenger in neuronal signalling mechanisms. Cytosolic Ca^^ signals come from two main sources, from the extracellular surroundings through plasma membrane delimited Ca^^ channels and from internal stores in the endoplasmic reticulum where Ca^'^ is released through inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. The activation of IP3R and RyR channels is promoted by cytosolic Ca^^, resulting in Ca^^-induced Ca^"^ release, a regenerative process which enables these pathways to interact (Friel and Tsien, 1992; Fagni et al., 2000). Growing evidence links disruptions in Ca^^ signalling to Alzheimer’s disease (Stutzmann, 2005), Parkinson’s disease (Sheehan et al., 1997) Huntington’s disease (Huang et al., 1995), exocitotoxicity (Choi, 1987; Carriedo et al., 1996; Van Den Bosch et al., 2000) and cerebral ischaemia (Bano and Nicotera, 2007).

Ca^^ has been shown to regulate the activity of a number of molecules in presynaptic nerve terminals, including calmodulin (CaM; LaPorte et al., 1980), Ca^^ /calmodulin-dependent protein kinases (CaMKs) I, II and IV (Hook and Means, 2001), Ca^Vphospholipids- dependent protein kinase C (PKC; Parfitt and Madison, 1993), Ca^^/calmodulin-dependent protein phosphatase 2B/calcineurin (Klee and Haiech, 1980) and Ca^^-dependent protease calpain (Friedrich, 2004). Ca^"^ has been shown to directly modulate the K isoform of Ras, a small GTPase (Villalonga et al., 2001). Ras activity is also regulated in a Ca^^-dependent manner by guanine nucleotide exchange factors (GEFs) containing Ca^^-binding EF-hand domains (Walker et al., 2003). Ca^^ influx activates synaptic vesicle-associated tyrosine kinase Src (Bamekow et al., 1990), which phosphorylates other synaptic vesicle proteins such as synaptophysin (Bamekow et al., 1990). Other Ca^^-sensitive proteins expressed in presynaptic nerve terminals include rabphilin (Ubach et al., 1999), a protein which interacts with the GTP-binding protein Rab3A (Shirataki et al., 1993), synaptotagmin I, a Ca^"^-

- 8 0 - binding protein which mediates fusion and recycling of synaptic vesicles at the active zone (Geppert et al., 1994; Fukuda et al., 1995; Llinas et al., 2004), Munc-13, a cytoplasmic protein associated with synaptic vesicle priming (Betz et al., 2001; Dulubova et al., 2005) and phospholipase C (PLC; Betz et al., 2001). All three types of PLCs, p, y and ô possess EF-hand domains, which can bind Ca^^, although the PLC Ô is the most sensitive to Ca^'^ (Rhee and Bae, 1997). Presynaptic voltage-dependent Ca^^ channels are also subject to feedback regulation by Ca^"^ via Ca^^-dependent facilitation and inactivation (Borst and Sakmann, 1998; Cuttle et al., 1998) following direct binding of Ca^^ and calmodulin (Lee et al., 1999).

Calmodulin is a regulatory protein that confers Ca^'^ sensitivity on, and modulates the activity of a number of molecules. Binding of Ca^'^ generally enhances the affinity of CaM for its target proteins by inducing a conformational change and thereby exposing a hydrophobic domain which is the main site of interaction between CaM and its target proteins (LaPorte et al., 1980).

CaMK II is a ubiquitous enzyme which is activated in response to changes in intracellular calcium concentrations. It regulates numerous processes such as gene expression, calcium homeostasis (Braun and Schulman, 1995; Heist and Schulman, 1998), apoptosis (Wright et al., 1997), receptor and ion-channel regulation (McGlade-McCulloh et al., 1993) and release of neurotransmitters (Llinas et al., 1991). CaMK II has an autoinhibitory domain and a catalytic domain. Binding of Ca^VCaM activates the kinase by causing a conformational change which removes the constraint of the autoinhibitory domain on the catalytic domain and allows autophosphorylation of a single residue (Thr-286 in the a isoform and Thr-287 in the p isoform). This leads to an increase in the kinase affinity for CaM (Yang and Schulman, 1999). By binding CaM, the kinase becomes calcium- independent, and remains in this state until it is dephosphorylated by protein phosphatases or phosphorylated on additional (autoinhibitory) residues (Meyer et al., 1992). CaMK II is able to respond rapidly to Ca^"^ elevation induced by activation of voltage- or transmitter- dependent channels, or by release from intracellular stores. It is highly enriched at synaptic sites and is present in presynaptic terminals and in postsynaptic densities (Ouimet et al., 1984; Kelly et al., 1984). These findings have lead to a number of functional studies

-81 - focusing on the role of CaMK II in synaptic transmission. CaMK II has been shown to enhance the release of neurotransmitters when introduced into rat brain synaptosomes or squid giant terminal (Sihra et al., 1989; Nichols et al., 1990; Llinas et al., 1991). Conversely, CaMK II activation correlates with the stimulation of neurotransmitter release in synaptosomes (Gorelick et al., 1988; Tsutsui et al., 1994).

Protein phosphatase 2B (PP2B), also known as calcineurin, is a Ca^Vcalmodulin-dependent Ser/Thr phosphatase. It is the major calcium-regulated protein phosphatase in neurones. PP2B is activated by Ca^"^ following an interaction with calmodulin, which binds to the catalytic A subunit, and by calcium itself, which binds to the regulatory B subunit. Presynaptically, PP2B inhibits the release of glutamate from rat cortical synaptosomes (Sihra et al., 1995). PP2B can dephosphorylate N- and L-type Ca^^ channels (Lukyanetz et al., 1998) and has been shown to modulate the regulation of Ca^'^ entry by a negative feedback mechanism (Burley and Sihra, 2000). In addition, PP2B dephosphorylates voltage-gated sodium channels (Chen et al., 1995).

One of the best characterised substrates for Ca^^-dependent kinases and phosphatases are the synapsins (section 1.11, Fig. 2), a family of neurone-specific and synaptic vesicle- associated proteins with an exclusive presynaptic localisation (Hilfiker et al., 1999).

We hypothesised that the activity of presynaptically localised GABAa receptors may regulate intraterminal Ca^"^ concentration ([Ca^'^ji) and thereby the activity of presynaptic CaMK II in association with synapsin I, causing a change in phosphorylation state of P- sites 2 and 3. We therefore employed a P-site 3-specific antibody in quantitative immunoblotting experiments as a biochemical read-out of changes in intraterminal Ca^"^ in response to GABAa receptor-specific agonists and antagonists.

To detect the activity of presynaptic GABAa receptors in isolation from the large postsynaptic pool, we employed a well-characterised biochemical procedure to purify presynaptic nerve terminals from the rat neocortex (Dunkley et al., 1986; Dunkley et al., 1988). The isolated nerve terminal preparation (synaptosomes) represents a mixture of different types of nerve terminals, the majority of which are glutamatergic and GABAergic (Beaulieu, 1993). On incubation at 37 °C synaptosomes show metabolic competence and

- 8 2 - display most of the features of intact nerve terminals, including spontaneous and evoked neurotransmitter release. Our preliminary measurements using high pressure liquid chromatography (HPLC) have indicated that the concentration of extrasynaptosomal G ABA is in a range of 0.5 - 1 jiM in synaptosomal suspension at the concentration of 1 mg/ml {Jovanovic, J., and. Sihra, T.S., unpublished Based data). on this finding, we hypothesised that functional GABAa receptors, if present on isolated nerve terminals, are likely to be in partially occupied and activated state, given that the K

We aimed to demonstrate the presence of functional GABAa receptors on presynaptic rat neocortical nerve terminals by measuring changes the in Ca^'^-dependent phosphorylation of synapsin I by CaM kinases in response to GABAa receptor-specific agonists and antagonists.

-83 3.2 Methods

3.2.1 Preparation of synaptosomes

Synaptosomes were prepared as described in Chapter 2 (section 2.1).

3.2.2 Drug-incubation protocols

Synaptosomes were kept at 4 °C and resuspended to a final concentration of 0.8 mg/ml in HBM. The drug of interest was added and the first fraction of 100 pi was collected in order to monitor the phosphorylation of synapsin I in inactive synaptosomes (Sample 1, data not shown). The synaptosomal suspension was transferred to 37 °C. After 5 min, 1 mM CaCh was added and a further 100 pi fraction collected after 9 min (Sample 2, basal samples). At 11 min, 1 mM 4AP was added and the final fractions collected at 12 min (Sample 3, 4AP samples). Immediately after collection, synaptosomes were lysed using the addition of 20 pi of 10 % SDS to yield the final concentration of 1.5 % (w/v) SDS in these samples. Experiments were carried out in the absence and presence of increasing concentrations of GABase (0.02 - 0.4 units/ml), bicuculline methiodide (10 - 100 pM) or picrotoxin (1 - 100 pM) (Protocol 1). In separate experiments, 1 mM Ca^"^ was added after 5 min and the first fraction of 100 pi was collected 7 min from the start of the incubation (Sample 1). Muscimol (1 - 500 pM) or isoguvacine (1 - 500 pM) were added after 9 min and the second fraction of 100 pi (Sample 2) was collected after 11 min. This was followed by the adition of 1 mM 4AP after 13 min. The final fraction of 100 pi (Sample 3) was collected at 15 min (Sample 3, Protocol 2).

- 8 4 - P R O T O C O L 1 4 4 9’ 11' 1 2 ’ I I I I H------1-----1 Î Î t A 4»C II 37°C

BICUCULUNE CaCU PICROTOXIN G A B ase

PR O T O C O L 2 SAIVPLE 2 SAIUPLE 3 4 4 11' 13' 15' t Î 4°C 37®C CaCU 4AP MUSCIMOL ISOGUVACINE

Schematic 1. Drug incubation protocols used for phosphorylation experiments in Chapter 3.

3.2.3 SDS-PAGE and immunoblotting

The protein concentration of collected fractions was estimated using the BCA asay. Equal amounts of synaptosomal proteins were separated by SDS/PAGE and transferred onto nitrocellulose membrane. Quantitative immunoblotting was carried out using anti-P-site 3 or anti-P-site 1 antibody at 0.5 pg/ml concentration, anti-P-Thr286-CaMK II antibody (1 |ig/ml) and anti-total syn I antibody (1 jig/ml). Immunoreactivity was detected by ^^^I- labelled anti-rabbit secondary antibody and phosphoimager spectometry. Data was analysed as described in Chapter 2 (section 2.2.4).

3.2.4 Immunohistochemistry

Immunohistochemistry was carried out as described in Chapter 2 (section 2.3).

8 5 - 3.3 Results

3.3.1 Modulation of Ca^^-dependent phosphorylation of synapsin I by GABase

We began our studies by determining whether the removal of tonic GABAa receptor activity, due to the presence of relatively high concentration of ambient GAB A in the synaptosomal suspension, affects the levels of intraterminal Ca^"^, thereby affecting Ca^"^- dependent phosphorylation of synapsin I. To achieve this, we incubated synaptosomes in the presence of increasing concentrations of GABase, an enzyme complex containing GABA-aminotransferase and succinic semi aldehyde dehydrogenase, which catalyses the breakdown of GAB A to succinic semi aldehyde and further to succinate (Hopkins et al., 1992).

Synaptosomes (0.8 mg/ml) were incubated with increasing concentrations of GABase (0.02 - 0.4 units/ml) and samples collected under basal conditions prior to and 1 min following depolarisation with 4AP as described in section 3.2.1 (Protocol 1). Samples were subjected to SDS-PAGE and immunoblotting. Immunoblotting with anti-P-site 3 antibody revealed a dose-dependent decrease in CaMK Il-dependent P-site 3 phosphorylation of synapsin I under basal conditions. As previously shown (Jovanovic et al., 2001), phosphorylation of P-site 3 was increased following depolarisation of synaptosomes with 4AP. However, in the presence of 4AP and increasing concentrations of GABase P-site 3 phophorylation was decreased in a dose-dependent manner (Fig. 4, P-site 3 Syn I).

Immunoblotting with anti-P-site 1 antibody, reflecting the activity of CaMK I/IV and PKA also showed a decrease in phosphorylation with increasing concentrations of GABase (0.02 - 0.4 U/ml) under basal conditions. Following depolarisation with 4AP, synapsin I phosphorylation at P-site 1 was increased as shown previously (Jovanovic et al., 2001). However, the level of synapsin I P-site 1 phosphorylation in the presence of 4AP and increasing concentrations of GABase was reduced in a dose-dependent maimer (Fig. 4, P- site 1 Syn I).

We next used an anti-synapsin I antibody to determine if the presence of GABase caused any change in the total amount of synapsin I present in synaptosomal samples. GABase

- 8 6 - ______Basal______4-AP______

97.5- a WWW* P-site 3 syn I

66 —

Ww %# M# # # M ^ b P-site 1 syn I

6 6 -

97.5- 4b Syn I

6 6 -

6 6 - ^ P-CaMK 4 5 - a

0 0.02 0.1 0.2 0.4 0 0.02 0.1 0.3 0.4 [GABase] (Units/ml)

Figure 4. Presynaptic activity of CaM kinases and Ca^-dependent phosphorylation of synapsin la and Ib are reduced in the absence of extrasynaptosomal G ABA. Immunoblotting of synaptosomal samples incubated with increasing concentrations of GABase (0.02, 0.1, 0.2, 0.4 U/ml) under basal conditions and following depolarisation with 1 mM 4AP, was carried out with anti-P-site 3 (n = 5), anti-P-site 1 (n = 1), anti-synapsin I (n = 4) and anti-P-Thr286-CaMK II (n = 2) specific antibodies. The binding of primary antibodies was detected by incubation with ’^^I-labelled anti-rabbit secondary antibody and analysed using phosphorimager spectometry. Positions of molecular weight markers are shown (kDa).

- 8 7 - [GABase] (U/ml) [GABase] (U/ml) 0.02 0.1 0.2 0.02 0.1 0.2

B asai 4 AP [GABase] (U/ml) [GABase] (U/ml) 0.02 0.1 0.2 0.4 0.02 0.1 0.2 0.4 120 120

100 100 90 90

80 c 80 70 70 60 60 50 50 40 f B asai 40 4 AP e G A B ase

P-Site 3 Phosphorylation ® o CaMKII

Succinic Acid + G lutam ic Acid + GABA NADP + H*

Figure 5. Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is decreased in the presence of GABase. Synaptosomes were incubated in the absence or presence of increasing doses of GABase (0.02, 0.1, 0.2, 0.4 U/ml) prior to depolarisation with 1 mM 4/VP. Immunoblotting was carried out with anti-P-site 3 antibody followed by ‘^I-labelled secondary antibody and quantified using phosphorimager spectometry. a. Dose-dependent decrease in synapsin I P-site 3 phosphorylation under basal conditions. *P<0.05 compared to 100 % 37 °C controls (paired data sets in grey. One-way ANOVA with post-hoc LSD test; n = 5). h. Dose-dependent decrease in synapsin I P-site 3 phosphorylation following depolarisation with 4AP. P<0.05 compared to 100 % 4AP control (paired data sets in grey. One-way ANOVA with post-hoc LSD test; n = 5). c. Total synapsin I is unaffected by increasing concentrations of GABase under basal conditions (n =5). d. Total synapsin I is unaffected by increasing concentrations of GABase following depolarisation with 4/VP (n =5). e. Schematic depiction of presynaptic GABAa receptor-mediated regulation of synapsin I phosphorylation at CaMK Il-dependent P-site 3 in the presence of GABase. had no significant effect on the total levels of synapsin I under basal conditions: 0.02 U/ml (91.9 ± 8.0 % of control, mean ± S.E.M., n = 3), 0.1 U/ml (108.7 ± 7.1 % of control, mean ± S.E.M., n = 4), 0.2 U/ml (101.3 ± 7.4 % of control, mean ± S.E.M., n = 4) and 0.4 U/ml (96.9 ± 6.0 % of control, mean ± S.E.M., n = 4). Furthermore, no significant difference was detected in the presence of 4AP: 0.02 U/ml (92.3 ± 7.8 % of control, mean ± S.E.M., n = 3), 0.1 U/ml (93.0 ± 8.3 % of control, mean ± S.E.M., n = 4), 0.2 U/ml (89.5 ± 1.0 % of control, mean ± S.E.M., n = 4) and 0.4 U/ml (89.1 ± 4.4 % of control, mean ± S.E.M., n = 4)). In addition, no change in the level of synapsin I was detected upon the addition of 4AP in the absence of GABase, (98.2 ± 9.8 % of control, mean ± S.E.M., n = 4) (Fig. 4, Syn I; Fig. 5).

Finally, we investigated the effect of GABase on Ca^^-dependent autophosphorylation of CaMK II. Immunoblotting with an anti-phospho-CaMK Il-antibody, specific for both phospho-Thr286 and phospho-Thr287 present in the a and p isoform of CaMK II, respectively revealed an apparent dose-dependent inhibition of both isoforms with increasing concentrations of GABase under basal conditions. The autophosphorylation of CaMK II a and P was increased following the addition of 4AP as shown previously (Gorelick et al., 1988). However, the autophosphorylation of CaMK II was decreased in the presence of increasing concentrations of GABase (Fig. 4, P-CaMK II).

Quantitative data analysis revealed that GABase caused a reduction in the Ca^^-dependent phosphorylation of synapsin I by CaMKII at P-site 3, with 0.02 U/ml (92.4 ± 1.0 % of control, mean ± S.E.M., n = 3) and 0.4 U/ml (59.8 ± 7.7 % of control, mean ± S.E.M., n = 6) of GABase causing a significant decrease under basal conditions (Fig. 5a). Under depolarising conditions in the presence of 4AP, synapsin I phosphorylation at P-site 3 was increased to 531.8 ± 107.2 % control (data not shown). However, under these conditions and in the presence of GABase, a significant decrease in synapsin I P-site 3 phosphorylation was detected with 0.2 U/ml (81.4 ± 5.1 % of 4AP alone, mean ± S.E.M., n = 5) and 0.4 U/ml of GABase (42.8 ± 5.9 % of 4AP alone, mean ± S.E.M., n = 5) (Fig. 5b).

8 9 - In summary as schematically depicted in the Fig. 5c, degradation of extrasynaptosomal GABA by GABase results in a dose-dependent inhibition of presynaptic CaMK II activity and a decrease in P-site 3 phosphorylation of synapsin I, reflecting a reduction in [Ca^^]i.

3.3.2 Modulation of Ca^^-dependent phosphorylation of synapsin I by bicuculline

To determine if the observed regulation of the presynaptic CaMK Il/synapsin I signalling pathway by extrasynaptosomal GABA is mediated by GABAa receptors, we incubated synaptosomes with increasing concentrations of bicuculline, a GABAa receptor specific competitive antagonist.

Synaptosomes were incubated with increasing concentrations of bicuculline (10 - 100 jiM) and samples collected under basal conditions prior to and 1 min following depolarisation with 4AP as described in section 3.2.1 (Protocol 1). Samples were subjected to SDS-PAGE and immunoblotting. Immunoblotting with anti-P-site 3 antibody revealed a dose- dependent decrease in CaMK Il-dependent P-site 3 phosphorylation of synapsin I under basal conditions. As previously shown (Fig. 4; Jovanovic et al., 2001), phosphorylation of P-site 3 was increased due to depolarisation of synaptosomes with 4AP. However, in the presence of 4AP and increasing concentrations of bicuculline, P-site 3 phosphorylation was decreased in a dose-dependent manner (Fig. 6, P-site 3 Syn I).

Immunoblotting with anti-P-site 1 antibody, reflecting the activity of CaMK I/IV and PKA also showed a decrease in phosphorylation with increasing concentrations of bicuculline (10 - 100 |iM) under basal conditions. Following depolarisation with 4AP, synapsin I phosphorylation at P-site 1 was increased as shown previously (Fig. 4; Jovanovic et al., 2001). The level of synapsin I P-site 1 phosphorylation in the presence of 4AP and increasing concentrations of bicuculline was reduced in a dose-dependent manner (Fig. 6, P-site 1 Syn I).

Immunoblotting with anti-synapsin I antibody was employed to determine if the presence of bicuculline caused any change in the total amount of synapsin I present in synaptosomal samples. Bicuculline (10 - 100 p,M) appeared to have no effect on the total levels of synapsin I either under basal conditions or in the presence of 4AP. In addition, no change

- 9 0 - ______Basal______4-AP______

##########A b P-site3 syn I

66 -

# » # # # » # » ^ b P-site1synl

6 6 -

97.5- _ mw. mmm ^ a Atb Syn I 6 6 -

66 - P-CaMK II * 4 '^ «Mir *-***. 3 B dm##» OC 45 -

0 10 20 50 100 0 10 20 50 100

[Bicuculline] (^iM)

Figure 6. Presynaptic activity of CaM kinases and Ca"-dependent phosphorylation of synapsin I are reduced in the presence of GABAa receptor antagonist hicuculline. Immunoblotting of synaptosomal samples incubated with increasing concentrations of bicuculline (10, 20, 50, 100 pM) under basal conditions and following depolarisation with 1 mM 4AP, was carried out with anti-P-site 3 (n = 5), anti-P-site 1 (n = 4), anti synapsin I (n = 2) and anti-PThr286- CaMK II (n = 1 ) specific antibodies. The binding of primary antibodies was detected by incubation with ’^^I-labelled anti-rabbit secondary antibody and analysed using phosphorimager spectometry. Positions of molecular weight markers are shown (kDa).

-91 - [Bicuculline] (|iM) [Bicuculline] (|liM)

10 20 50 100 10 20 50 100 ro 100' m 100 o t o -C ^ 80 80

B o l o 60 60

wc CL m 40 40 >

Bicuculline PHOSPHORYLATIONi'

C a M K II

GABA

I I RECEPTOR

Figure 7. Ca^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is decreased in the presence of hicuculline. Synaptosomes were incubated in the absence or presence of increasing doses of bicuculline (10, 20, 50, 100 pM) prior to depolarisation with 1 mM 4AP. Immunoblotting was carried out with anti-P-site 3 antibody followed by '^^I-labelled secondary antibody and quantified using phosphorimager spectometry. a. Dose-dependent decrease in synapsin I P-site 3 phosphorylation under basal conditions. P<0.05 compared to 100 % 37 °C control (paired data sets in grey. One-way ANOVA with post-hoc LSD test; n = 5). h. Dose- dependent decrease in synapsin I P-site 3 phosphorylation following depolarisation with 4AP. P<0.05 compared to 100 % 4AP control (paired data sets in grey. One-way ANOVA with post-hoc

LSD test; n = 5). c. Schematic depiction of presynaptic GABA a receptor-mediated regulation of synapsin I phosphorylation at CaMK Il-dependent P-site 3 in response to bicuculline.

- 9 2 - in the level of synapsin I was detected upon the addition of 4AP in the absence of bicuculline (Fig. 6, Syn I).

Finally, we investigated the effect of bicuculline on Ca^^-dependent autophosphorylation of CaMK II. Immunoblotting with anti-phospho-CaMK II antibody, (anti-phospho- Thr286/Thr287 antibody) revealed an apparent dose-dependent inhibition of both a and p isoforms of CaMK II with increasing concentrations of bicuculline under basal conditions. The autophosphorylation of both isoforms of CaMK II was increased following the addition of 4AP as shown previously (Fig. 4; Gorelick et al., 1988). However, the autophosphorylation of CaMK II was decreased in the presence of increasing concentrations of bicuculline (Fig. 6, P-CaMK II).

Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 was decreased by bicuculline in a dose-dependent manner, with statistically significant decreases under basal conditions determined at all concentrations tested (10 pM: 73.1 ±8.9 % of control, mean ± S.E.M; n = 5; 20 pM: 72.4 ± 8.6 % control, mean ± S.E.M; n = 5; 50 pM: 55.8 ± 10.3 % of control, mean ± S.E.M; n = 5; and 100 pM: 65.0 ± 7.9 % control, mean ± S.E.M; n = 5; Fig. 7a). Synapsin I phosphorylation at P-site 3 was potently increased in the presence of 4AP to 315.6 ± 98 % of control (mean ± S.E.M; n = 4, data not shown). However, under these conditions and in the presence of 50 pM bicuculline, a significant decrease in synapsin I P-site 3 phosphorylation was detected (77.3 ± 7.7 % of 4AP alone, mean ± S.E.M; n = 5; Fig. 7b).

In summary, as schematically depicted in Fig. 7c, inhibition of GABAa receptors by a competitive antagonist bicuculline results in a dose-dependent decrease in presynaptic CaMK II activity, as well as a decrease in P-site 3 phosphorylation of synapsin I, reflecting a reduction in [Ca^^Ji under these conditions.

3.3.3 Modulation of Ca^'^-dependent phosphorylation of synapsin I by picrotoxin

To further examine the regulation of the presynaptic CaMK Il/synapsin I signalling pathway by tonically active GABAa receptors, we incubated synaptosomes in the presence of increasing doses of picrotoxin, a GABAa receptor Cl' channel blocker.

- 9 3 - Synaptosomes were incubated with increasing concentrations of picrotoxin (1 - 100 jiM) and samples collected under basal conditions prior to and 1 min following depolarisation with 4AP, as described in section 3.2.1 (Protocol 1). Immunoblotting with anti-P-site 3 antibody revealed a dose-dependent decrease in CaMK Il-dependent P-site 3 phosphorylation of synapsin I under basal conditions. As previously shown (Fig. 4; Jovanovic et al., 2001), phosphorylation of P-site 3 was increased following depolarisation of synaptosomes with 4AP. However, in the presence of 4AP and increasing concentrations of picrotoxin, P-site 3 phophorylation was decreased in a dose-dependent manner (Fig. 8, P-site 3 Syn I).

Immunoblotting with anti-P-site 1 antibody, reflecting the activity of CaMK I/IV and PKA also seemed to show a decrease in phosphorylation with increasing concentrations of picrotoxin (1 - 100 pM) under basal conditions. Following depolarisation with 4AP, synapsin I phosphorylation at P-site 1 was increased as previously reported (Fig. 4; Jovanovic et al., 2001). However, the level of synapsin I P-site 1 phosphorylation in the presence of 4AP and increasing concentrations of picrotoxin was reduced in a dose- dependent manner (Fig. 8, P-site 1 Syn I).

Immunoblotting with an anti-synapsin I antibody was employed to determine if the presence of picrotoxin caused any change in the total amount of synapsin I present in synaptosomal samples. Picrotoxin (1 - 100 pM) had no apparent effect on the total levels of synapsin I either under basal conditions or in the presence of 4AP. In addition, no change in the level of synapsin I was detected upon the addition of 4AP in the absence of picrotoxin (Fig. 8, Syn I).

Finally, we investigated the effect of picrotoxin on the Ca^^-dependent autophosphorylation of CaMK II. Immunoblotting with anti-phospho-CaMK II antibody, (anti-phospho- Thr286/Thr287 antibody) appeared to reveal a dose-dependent inhibition of both the a and (3 isoforms of CaMK II with increasing concentrations of picrotoxin under basal conditions. The autophosphorylation of both isoforms of CaMK II was increased following the addition of 4AP as shown previously (Fig. 4; Gorelick et al., 1988). Nevertheless, the

- 9 4 - ______Basal______4-AP______

mmmmmmrn^ 11 P-Sites syn I

6 6 -

97.5 - a m mm #^####0 #00t b P-Sitel syn I

6 6 -

7.5- j = Synl

6 6 -

66 - ^ R »- ^ 3 „ P-CaMK II

0 1 5 10 20 50 100 0 1 5 10 20 50 100

[Picrotoxin] (|iM)

Figure 8. Presynaptic activity of CaM kinases and Ca^-dependent phosphorylation of synapsin I are reduced in the presence of GABAa receptor antagonist picrotoxin. Immunoblotting of synaptosomal samples incubated with increasing concentrations of picrotoxin (1,5, 10, 20, 50, 100 pM) under basal conditions and following depolarisation with ImM 4AP, was carried out with anti-P-site 3 (n = 5), anti-P-site 1 (n = 4), anti-synapsin I (n = ) and anti-PThr286- CaMK II (n = 1 ) specific antibodies. The binding of primary antibodies was detected by incubation with ’^^I-labelled anti-rabbit secondary antibody and analysed using phosphorimager spectometry. Positions of molecular weight markers are shown (kDa).

- 9 5 - [Picrotoxin] (jiM) [Picrotoxin] (|iM) 5 10 20 50 100 5 10 20 50 100

WQ.

CL Ô ^ P 90 CO o 80

CL ^

U) Basal 4AP

Picrotoxin PHOSPHORYLATION C v lf'

C a M K II

GABA

ih

Figure 9. Ca^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is decreased in the presence of picrotoxin. Synaptosomes were incubated in the absence or presence of increasing doses of picrotoxin (1,5, 10, 20, 50, 100 pM) prior to depolarisation with 1 mM 4AP. Immunoblotting was carried out with anti-P-site 3 antibody followed by ‘^^I-labelled secondary antibody and quantified using phosphorimager spectometry. a. Dose-dependent decrease in synapsin I P-site 3 phosphorylation under basal conditions. P<0.05 compared to 100 % 37 °C control (paired data sets in grey. One-way ANOVA with post-hoc LSD test; n = 5). b. Dose- dependent decrease in synapsin I P-site 3 phosphorylation following depolarisation with 4AP. P<0.05 compared to 100 % 4AP control (paired data sets in grey. One-way ANOVA with post-hoc

LSD test; n = 5). c. Schematic depiction of presynaptic GABAa receptor-mediated regulation of synapsin I phosphorylation at CaMK Il-dependent P-site 3 in response to picrotoxin.

- 9 6 - autophosphorylation of CaMK II was decreased in the presence of increasing concentrations of picrotoxin (Fig. 8, P-CaMK II).

Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 was inhibited by picrotoxin in a dose-dependent manner, with statistically significant decreases under basal conditions determined at concentrations of 50 pM (54.9 ± 5.3 % control, mean ± S.E.M., n = 5) and 100 pM picrotoxin (61.6 ± 5.8 % control, mean ± S.E.M., n = 5) (Fig. 9a). Synapsin I phosphorylation at P-site 3 was potently increased in the presence of 4AP to 573.3 ± 113.9 % of control (mean ± S.E.M.; n = 5, data not shown). However, under these conditions and in the presence of picrotoxin, a significant decrease in synapsin I P- site 3 phosphorylation was detected in the presence of 5 pM (86.1 ±3.7 % of 4AP alone, mean ± S.E.M.; n = 5), 50 pM (70.3 ± 6.4 % of 4AP alone, mean ± S.E.M.; n = 5) and 100 pM picrotoxin (62.9 ± 8.5 % of 4AP alone, mean ± S.E.M.; n = 5) (Fig. 9b).

Inhibition of the tonic activity of GABAa receptors by the Cl" channel blocker picrotoxin results in a dose-dependent decrease in presynaptic CaMK II activity, as well as a decrease in P-site 3 phosphorylation of synapsin I, due to a reduction in intraterminal Ca^"^ levels (Fig. 9c).

3.3.4 Modulation of Ca^^-dependent phosphorylation of synapsin I by isoguvacine

As the inhibition of tonically active GABAa receptors in synaptosomes causes a decrease in Ca^^-dependent phosphorylation of synapsin I, we tested if the further activation of

GABAa receptor in the presence of isoguvacine, a GABAa receptor specific agonist, results in any change in synapsin phosphorylation.

Synaptosomes were incubated with increasing concentrations of isoguvacine and samples collected under basal conditions prior to and 2 min following depolarisation with 4AP, as described in section 3.2.1 (Protocol 2). Immunoblotting with anti-P-site 3 antibody revealed a dose-dependent increase in CaMK Il-dependent P-site 3 phosphorylation of synapsin I under basal conditions. As previously shown (Fig. 4; Jovanovic et al., 2001), phosphorylation of P-site 3 was increased following depolarisation of synaptosomes with

-97 ______Basal______4-AP______

i b P-site3 syn I 6 6 -

97.5 - « « . . i M p l H I I M a i M i b P-site1 syn I 66 —

s y m

66 -

66 - ■ mm P-CaMK a 45 -

0 1 10 50 100 500 0 1 10 50 100 500 [Isoguvacine] (^iM)

Figure 10. Presynaptic activity of CaM kinases and Ca^-dependent phosphorylation of synapsin I are increased in the presence of GABAa receptor agonist isoguvacine. Immunoblotting of synaptosomal samples incubated with increasing concentrations of isoguvacine (1, 10, 50, 100, 500 pM) under basal conditions and following depolarisation with 1 mM 4AP, was carried out with anti-P-site 3 (n = 3), anti-P-site 1 (n = 1), anti-synapsin I (n = 2) and anti-PThr286- CaMK II (n = 2) specific antibodies. The binding of primary antibodies was detected by incubation with ‘^^I-labelled anti-rabbit secondary antibody and analysed using phosphorimager spectometry. Positions of molecular weight markers are shown (kDa).

- 9 8 - Basal 4AP

^ 2 500 | q 400 S o 140

10 50 100 500 1 10 50 100 500 [Isoguvacine] ()iM) [Isoguvacine] (|aM)

Isoguvacini

C aM K II

GABA.

Figure 11. Ca^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is increased in the presence of isoguvacine. Synaptosomes were incubated for 4 min in the absence or presence of increasing doses of isoguvacine (1, 10, 20, 50, 100 and 500 pM) before depolarisation with 4AP. Immunoblotting was carried out with anti-P-site 3 antibody followed by ‘^^I-labelled secondary antibody and quantified using phosphorimager spectometry. a. Dose- dependent increase in synapsin I P-site 3 phosphorylation under basal conditions. P<0.05 compared to 100 % 37 °C controls (One-way ANOVA with post-hoc LSD test; n = 3). b. Dose- dependent increase in synapsin I P-site 3 phosphorylation following depolarisation with 4AP. P<0.05 compared to 100% 4AP control (One-way ANOVA with post-hoc LSD test; n = 3). c.

Schematic depiction of presynaptic GABA a receptor mediated regulation of synapsin I phosphorylation at CaMK Il-dependent P-site 3 in the presence of isoguvacine.

- 9 9- 4AP. In the presence of 4AP and increasing concentrations of isoguvacine, P-site 3 phophorylation was further increased in a dose-dependent manner (Fig. 10, P-site 3 Syn I).

Immunoblotting with anti-P-site 1 antibody, reflecting the activity of CaMK I/IV and PKA also seemed to show an increase in phosphorylation with increasing concentrations of isoguvacine (1 - 500 |iM) under basal conditions. Following depolarisation with 4AP, synapsin I phosphorylation at P-site 1 was increased as previously reported (Fig. 4; Jovanovic et al., 2001). In addition, the level of synapsin I P-site 1 phosphorylation in the presence of 4AP and increasing concentrations of isoguvacine was further increased in a dose-dependent manner (Fig. 10, P-site 1 Syn I).

Immunoblotting with an anti-synapsin I antibody was employed to determine if the presence of isoguvacine caused any change in the total amount of synapsin I present in synaptosomal samples. Isoguvacine (1 - 500 p,M) had no apparent effect on the total levels of synapsin I either under basal conditions or in the presence of 4AP. In addition, no change in the level of synapsin I was detected upon the addition of 4AP in the absence of isoguvacine as determined in previous experiments (Fig. 10, Syn I).

Immunoblotting with an anti-phospho-CaMK II antibody revealed an apparent dose- dependent enhancement of CaMK II autophosphorylation with increasing concentrations of isoguvacine under basal conditions. While the autophosphorylation of CaMK II a and (3 isoforms was significantly increased following the addition of 4AP, it was further potentiated in the presence of isoguvacine (Fig. 10, P-CaMK II).

Quantitative analysis indicated that, under basal conditions, a significant increase in CaMK II - dependent phosphorylation of synapsin I resulted from incubation with 50 p,M (578.8 ± 107.2 % control, mean + S.E.M., n = 3) and 500 jiM (999.9 ± 308.3 % control, mean + S.E.M., n = 3) isoguvacine (Fig. 11a). P-site 3 phosphorylation was potently increased in the presence of 4AP to 500.9 ± 110.4 % of basal control level (mean ± S.E.M.; n = 3, data not shown), and further enhanced by 1 |iM (160.0 ± 13.4, mean ± S.E.M., n = 3), 100 jiM (175.0 ± 18.4, mean ± S.E.M., n = 3) and 500 jiM isoguvacine (153.5 ± 15.6, mean + S.E.M., n = 3) (Fig. 1 lb).

- 100 - In summary as depicted in Fig. 11c, activation of GAB A a receptors by the specific agonist isoguvacine results in a dose-dependent enhancement of presynaptic CaMK II activity and an increase in P-site 3 phosphorylation of synapsin I, reflecting an increase in [Ca^^Ji.

3.3.5 Modulation of Ca^^-dependent phosphorylation of synapsin I by muscimol

The activity of presynaptic CaM kinases and Ca^^-dependent phosphorylation of synapsin I was further investigated in the presence of muscimol, a GAB A A receptor-specific agonist. Synaptosomes were incubated with increasing concentrations of muscimol (1 - 500 pM) and samples collected under basal conditions prior to and 2 min following depolarisation with 4AP, as described in section 3.2.1 (Protocol 2). Immunoblotting with anti-P-site 3 antibody revealed a dose-dependent increase in CaMK Il-dependent P-site 3 phosphorylation of synapsin I under basal conditions. As shown in previous figures (Fig. 4, 6, 8, 10), phosphorylation of P-site 3 was increased following depolarisation of synaptosomes with 4AP. In the presence of 4AP and increasing concentrations of muscimol, P-site 3 phophorylation was further increased in a dose-dependent manner (Fig. 12, P-site 3 Syn I).

Immunoblotting with anti-P-site 1 antibody, reflecting the activity of CaMK I/IV and PKA also showed an increase in phosphorylation with increasing concentrations of muscimol (1 - 500 |iM) under basal conditions. Following depolarisation with 4AP, synapsin I phosphorylation at P-site 1 was increased as previously reported (Fig. 4, 6, 8, 10). In addition, the level of synapsin I P-site 1 phosphorylation in the presence of 4AP and increasing concentrations of muscimol was further increased in a dose-dependent manner (Fig. 12, P-site 1 Syn I).

Immunoblotting with anti-synapsin I antibody seemed to indicate that the presence of muscimol caused no change in the total amount of synapsin I present in synaptosomal samples either under basal conditions or in the presence of 4AP. In addition, no change in the level of synapsin I was detected upon the addition of 4AP in the absence of muscimol as determined in previous experiments (Fig. 12, Syn I).

101 - Control______4-AP______9 7 .5 - i b P-site3synl

6 6 -

— i h P-sitelsvnl

6 6 -

9 7 .5 - # # # # Syn 6 6 -

66 - P-CaMK II 45 -

0 1 10 50 100 500 0 1 10 50 100 500

[Muscimol] (^iM)

Figure 12. Presynaptic activity of CaM kinases and Ca“^-dependent phosphorylation of synapsin I are increased in the presence of GABAa receptor agonist muscimol. Immunoblotting of synaptosomal samples incubated with increasing concentrations of muscimol (1, 10, 50, 100, 500 pM) under basal conditions and following depolarisation with ImM 4AP, was carried out with anti-P-site 3 (n = 5), anti-P-site 1 (n = 4), anti-synapsin I (n = 1) and anti-PThr286- CaMK II (n = 1 ) specific antibodies. The binding of primary antibodies was detected by incubation with ’^^I-labelled anti-rabbit secondary antibody and analysed using phosphorimager spectometry. Positions of molecular weight markers are shown (kDa).

- 1 0 2 - a. b. Basal 4AP

^ 250 o o

1 10 50 100 500 1 10 50 100 500 [Muscimol] (|iM) [Muscimol] (|iM)

C.

M uscim ol

C a M K II

g a b a '

I I RECEPTOR

Figure 13. Ca^^-dependent phosphorylation of synapsin I at CaMK Il-dependent P-site 3 is increased in the presence of muscimol. Synaptosomes were incubated for 4 min in the absence or presence of increasing doses of muscimol (1, 10, 20, 50, 100 and 500 pM) before depolarisation with 4AP. Immunoblotting was carried out with anti-P-site 3 antibody followed by ‘^^I-labelled secondary antibody and quantified using phosphorimager scanning, a. Dose-dependent increase in synapsin I P-site 3 phosphorylation under basal conditions. P<0.05 compared to 100 % 37 °C controls (One-way ANOVA with post-hoc LSD test; n = 5). b. Dose-dependent increase in synapsin I P-site 3 phosphorylation following depolarisation with 4AP. P<0.05 compared to 100 % 4AP control (One-way ANOVA with post-hoc LSD test; n = 5). c. Schematic depiction of presynaptic GABAa receptor-mediated regulation of synapsin I phosphorylation at CaMK Il- dependent P-site 3 in the presence of muscimol.

- 10 3 - Immunoblotting with anti-phospho-CaMK II antibody revealed an apparent dose-dependent enhancement of CaMK II autophosphorylation with increasing concentrations of muscimol under basal conditions. While the autophosphorylation of both a and p isoforms of CaMK II was significantly increased following the addition of 4AP, it was further potentiated in the presence of muscimol (Fig. 12, P-CaMK II).

Quantification of these experiments revealed that, under basal conditions, a significant increase in CaMK Il-dependent phosphorylation of synapsin I resulted from incubation with 500 pM muscimol (302.2 ± 68.4 % control, mean ± S.E.M., n = 6) under basal conditions (Fig. 13a). P-site 3 phosphorylation was potently increased in the presence of 4AP to 999.2 ±91 % of control basal level (mean ± S.E.M.; n = 6, data not shown), and further enhanced by 10 pM (118 ± 5.7 % 4AP alone, mean ± S.E.M., n = 6), and 100 pM muscimol (133.7 ± 12.1 % 4AP alone, mean ± S.E.M., n = 6) (Fig. 13b).

In summary as depicted in Fig. 13c, activation of GABAa receptors by specific agonist muscimol, caused a dose-dependent enhancement of presynaptic CaMK II activity and an increase in P-site 3 phosphorylation of synapsin I, reflecting an increase in [Ca^"^]i.

3.3.6 Expression of GABAa receptor subunits in purified cerebrocortical synaptosomes

The expression of several GABAa receptor subunits was characterised using immunoblot analysis of different fractions collected during the preparation of synaptosomes, with the final fraction being highly enriched in presynaptic proteins (Dunkley et al., 1988a).

GABAa receptor subunit-specific antibodies revealed the presence of a l, a2, |33 and y2 subunits in the homogenate (H), crude synaptosomal fraction (P2) and purified synaptosomes (S, Fig. 14). The al, a2, p2 and p3 subunits were expressed at high levels in each fraction. The y2 subunit appeared specifically enriched in the synaptosomal fraction in comparison with the homogenate and P2 fraction, displaying the highest level of immunoreactivity. Similar enrichment of GABA synthesising enzyme glutamic acid decarboxylase (GAD 65/67), and three major synapsin isoforms (synapsin la, Ila and Ilia) were detected in the fraction of purified synaptosomes (Fig. 14). GAD, which is known to be expressed in presynaptic nerve terminals, and synapsins, which are expressed solely in

104 - GABA^R

a1/2 p2/3 p3

H P2 S H P2 S H P2 S H P2 S 97.5 -

66 —

4 5 -

3 0 - 'i;Ë

Syn l/ll/lll GAD65/67

H P2 S H P2 S 97.5 —

66 —

45 —

•t 30 —

Figure 14. GABAa receptor subunits, synapsin and GAD are abundantly expressed in rat cerebrocortical synaptosomes. GABA a receptor subunits (al, a2, P2, ps and y2) are abundantly expressed in rat cerebrocortical synaptosomes, as are the synapsins and GABA-producing enzyme GAD. Immunoblotting of fractions collected at three different stages of synaptosome preparation was carried out with anti-GABAA a 1/2 (0.5 |ig/ml), anti-GABAA P2/p3 (20 pg/ml), anti-GABAA

P3 (0.21 |Lig/ml), anti-GABAA 72 (1 p.g/ml), anti-synapsin (0.5 pg/ml) and anti-GAD (1 |ig/ml) specific antibodies. The specific enrichment of synapsin, GAD and the y2 subunit in the fraction of highly purified synaptosomes was detected (H- homogenate, P2- crude synaptosomal fraction, S- purified synaptosomes). The binding of primary antibodies was detected by incubation with labelled anti-rabbit secondary antibody and analysed using phosphoimager spectometry.

- 10 5 - presynaptic nerve terminals, display the highest levels of expression in the fraction of purified synaptosomes. These results indicate that a number of GABAa receptor subunits are indeed expressed presynaptically at significant levels.

3.3.7 Synapsin I phosphorylated by CaMK II at P-site 3 is localised to glutamatergic nerve terminals

Synapsins are unique structural and functional presynaptic markers expressed ubiquitously in all presynaptic nerve terminals in the CNS (De Camilli et al., 1983). In order to determine whether Ca^^-dependent phosphorylation of synapsin I occurs universally throughout the rat neocortex or only in certain populations of nerve terminals, imunohistochemical analysis was carried out using fixed neocortical slices.

Triple-labelling experiments were carried out with anti-P-site 3 antibody (in green) in combination with an anti-GAD antibody (in red), specifically labelling GABAergic nerve terminals, and an anti-vesicular-glutamate transporter (v-Glut, in blue) antibody, specifically labelling glutamatergic nerve terminals. Terminal-specific staining was detected with all three antibodies. High magnification (x 126) analysis using confocal microscopy revealed an exclusive co-localisation of P-site 3 synapsin I with the glutamatergic marker v-Glut 1 in all the layers (I-VI) of the rat neocortex (Fig. 15-17). P- site 3 synapsin I was mainly absent from GAD-positive terminals as indicated by limited co-localisation between the green (P-syn I) and red (GAD) staining (Fig. 16, Fig. 17, Fig. 18). Furthermore, no co-localisation was detected between GAD and v-Glut, confirming the specificity of antibodies used in our experiments (Fig. 15 -17). Together, these experiments demonstrate that phosphorylation of synapsin I by CaMK II occurs mainly in glutamatergic nerve terminals.

-106 V-GLUT P-SYN GAD

jr- . ■ ■> • ..."

V-GLUT/ P-SYN P-SYN / GAD

V-GLUT/P-SYN/GAD

Figure 15. Ca^^-dependent phosphorylation of synapsin I at P-site 3 occurs in glutamatergic nerve terminals in neocortical layers I - III. Immunohistochemistry of brain slices from rat neocortex layers I - III was carried out using anti-vGlut 1 (in blue), anti-P-site 3 (in green) and anti- GAD (in red) antibodies. Phosphosynapsin I is present in glutamatergic nerve terminals as indicated by co-localisation between the blue and green fluorophores. Immunoreactivity was visualised using a Zeiss LSM 510 Meta laser scanning confocal microscope (Scale bar = 10 pM ).

- 1 0 7 - V-GLUT P-SYN GAD

V-GLUT/ P-SYN P-SYN / GAD

V-GLUT/P-SYN/GAD

Figure 16. Ca^-dependent phosphorylation of synapsin I at P-site 3 occurs in glutamatergic nerve terminals in neocortical layer IV. Immunohistochemistry of brain slices from rat neocortex layer IV was carried out using anti-vGlut 1 (in blue), anti-P-site 3 (in green) and anti-GAD (in red) antibodies. Phosphosynapsin I is present in glutamatergic nerve terminals as indicated by co­ localisation between the blue and green fluorophores. Immunoreactivity was visualised using a Zeiss LSM 510 Meta laser scanning confocal microscope (Scale bar = 10 pM ).

- 108 - V-GLUT P-SYN GAD

» V ' ’

s .'vV: V-GLUT / P-SYN P-SYN / GAD

V-GLUT/P-SYN/GAD

Figure 17. Ca^-dependent phosphorylation of synapsin I at P-site 3 occurs in glutamatergic nerve terminals in neocortical layers V and VI. Immunohistochemistry of brain slices from rat neocortex layers V and VI was carried out using anti-vGlut 1 (in blue), anti-P-site 3 (in green) and anti-GAD (in red) antibodies. Phosphosynapsin I is present in glutamatergic nerve terminals as indicated by exclusive co-localisation between the blue and green fluorophores. Immunoreactivity was visualised using a Zeiss LSM 510 Meta laser scanning confocal microscope (Scale bar = 10 p M ).

- 1 0 9 - 3.4 Discussion

The presence of functional GABAa receptors at presynaptic sites has been demonstrated in some areas of the CNS, where they play an important role in regulation of intraterminal Ca^^ homeostasis (Turecek and Trussell, 2001; Ruiz et ah, 2003). The work in this chapter explored the hypothesis that functional GABAa receptors are also present in presynaptic terminals in the rat neocortex where they may regulate the activity of Ca^^-dependent signalling pathways as a consequence of changes in [Ca^^h. Specifically, we characterised changes in Ca^^-dependent phosphorylation of synapsin I by CaM kinases in response to

GABAa receptor-specific agonists, muscimol and isoguvacine, and antagonists, bicuculline and picrotoxin. The results presented in this chapter infer that activation of GABAa receptors in isolated nerve terminals may cause an increase in [Ca^'^h and stimulation of synapsin I phosphorylation by CaM kinases. The immunohistochemical localisation of CaMK Il-dependent phosphorylation of synapsin I to glutamatergic nerve terminals indicates that biochemically characterised changes in synapsin I phosphorylation, in response to inhibition or activation of GABAa receptors, occur in glutamatergic nerve terminals. Together, these findings suggest that presynaptic GABAa receptors present at excitatory glutamatergic nerve terminals may play a prominent role in regulation of Ca^"^- dependent processes involved in glutamate release.

The observed changes phosphorylation of synapsin I at P-site 3 in presynaptic nerve terminals suggests that GABAa receptor activation may cause a depolarisation of the plasma membrane. This contradicts the classically described mechanism of action of

GABAa receptors, whereby the consequence of their activation is plasma membrane hyperpolarisation. However, GABAa receptors have been shown to cause membrane depolarisation in various brain regions. It is well documented that in developing and injured neurones, GABAa receptors activation leads to a depolarisation of plasma membrane (Owens et al., 1996; Chen et al., 1996). In addition, GABAa receptors which are situated at presynaptic sites in some areas of the CNS also cause a depolarisation of the presynaptic membrane, modulating [Ca^'^h and neurotransmitter release (Ruiz et al., 2003; Jang et al., 2005).

-110 Tonic activity of presynaptic GABAa receptors has been demonstrated in various brain regions (Farrant and Nusser, 2005), and is consistent with our observations that breakdown of endogenous GABA by GABase in synaptosomal preparation leads to a decrease in the activity of presynaptic CaM kinases and phosphorylation of synapsin I, without affecting the total levels of this protein. The observed changes in synapsin I phosphorylation at P- site 3 are a direct consequence of changes in the activity of CaM kinase II, as opposed to being due to changes in the activity of protein phosphatase 2A known to dephosphorylate these sites in vitro and in synaptosomes (Jovanovic et al., 2001). This is supported by immunoblotting with an anti-phospho-CaMK II antibody which demonstrated that changes in autophosphorylation of Thr 286/7 in the o/p isoforms of CaMK II closely parallel changes in synapsin I phosphorylation by this kinase.

Under resting conditions the addition of drugs which act as agonists (isoguvacine and muscimol), or antagonists (bicuculline and picrotoxin) at GABAa receptors resulted in opposing changes in CaMK-mediated phosphorylation of synapsin I, viz. agonists produced a dose-dependent increase while antagonists caused a dose-dependent decrease in the state of phosphorylation. Although qualitatively similar to changes induced by depolarisation of synaptosomes with 4AP, these changes were quantitatively less prominent, suggesting that only a small population of nerve terminals contains functional GABAa receptors.

Alternatively, GABAa receptor activation may lead to a small change in plasma membrane potential sufficient to activate only some but not all of VGCCs present in synaptosomes. Incubation of synaptosomes with 1 mM 4AP is well known to trigger both glutamate and GABA release which could lead to a further increase in CaMK-dependent phosphorylation of synapsin I. Under these conditions, the majority of GABAa receptor subtypes with a relatively high affinity for agonists are predicted to be activated. Therefore, the addition of muscimol or isoguvacine in the presence of 4AP could activate only those receptor subtypes with a relatively low affinity for agonists. It is well established that functional properties of GABAa receptors can vary significantly depending on their subunit composition (Hevers and Luddens, 1998). It is of interest to note that in some cases significant effects on CaMK Il-dependent phosphorylation of synapsin I were observed with lower (1 pM) and higher (100 - 500 pM) concentrations of GABAergic agonists and antagonists, although not with intermediate concentrations. The apparent bimodal responses suggest that at least two populations of GABAa receptors may be tonically active - I l l - in synaptosomes, possibly due to differing subunit compositions. Tonic activation of GAB A A receptors has been reported in various other brain regions and is thought to play a crucial role in network excitability and signal processing (Farrant and Nusser, 2005).

In order to determine the cellular distribution of P-site 3 phosphosynapsin I, we performed triple labelling immunohistochemical experiments using rat neocortical slices. Our results showed that P-site 3 phospho-synapsin I is scarcely present in GABAergic nerve terminals but instead, is largely localised to glutamatergic nerve terminals.

Therefore, changes in CaMK Il-dependent phosphorylation of synapsin I in response to GAB A A receptor specific agonists and antagonists described in this chapter seem to occur in the population of glutamatergic nerve terminals in the synaptosomal preparation.

Together, these results strongly indicate that GABAa receptors are present in glutamatergic nerve terminals in the neocortex where they may function as heteroreceptors to regulate the level of synaptic excitation.

- 112 3.5 Conclusions

Presynaptic GABAa receptors are tonically active, as indicated by changes in the CaMK Il-dependent phosphorylation of synapsin I under basal conditions in response to receptor antagonists.

Activation of presynaptic GABAa receptors appears to cause nerve terminal depolarisation rather than hyperpolarisation, resulting in an increase in intraterminal Ca^"^ concentration and subsequent increase in CaMK Il-dependent phosphorylation of synapsin I.

CaMK Il-phosphorylated synapsin I is present predominantly in glutamatergic nerve terminals. This indicates that the regulation of Ca^^-dependent

phosphorylation of synapsin I by GABAa receptor ligands described in this chapter occurs is relevant to changes occurring in glutamatergic nerve terminals and establishes these receptors as functional presynaptic heteroreceptors.

- 113 - CHAPTER FOUR

-114 4. Modulation of glutamate release by presynaptic GABAa receptors

4.1 Introduction

The release of a neurotransmitter into the synaptic cleft is the final outcome of a highly coordinated cascade of events starting with the arrival of an action potential into the presynaptic nerve terminal. At rest, presynaptic nerve terminals maintain a membrane potential of approximately -70 mV, which results from an unequal distribution of Na^, and Cr across the plasma membrane (Hodgkin and Huxley, 1952). Arrival of an action potential results in activation of voltage-dependent Na"^ channels and influx of Na"^ which depolarises the plasma membrane. When the plasma membrane potential has reached approximately -40 mV, opening of voltage-gated Ca^"^ channels (VGCCs) occurs, leading to a sudden increase in the concentration of Ca^"^ within the nerve terminal. Influx of Ca^"^ through VGCCs located in the vicinity of small synaptic vesicles, where the neurotransmitter is stored, causes a fusion of these vesicles with the plasma membrane, a process also known as synaptic vesicle exocytosis. The neurotransmitter is released through the pore which is formed due to either an incomplete or complete fusion of synaptic vesicles with the plasma membrane.

Isolated nerve terminals in suspension (synaptosomes) retain the basic functional features of intact nerve terminals including the ability to release neurotransmitter in a highly controlled Ca^^-dependent manner. Neurotransmitter release from synaptosomes can be evoked biochemically by a variety of methods, including the inhibition of channels with a specific blocker, 4-aminopiridine (4AP; Tibbs et al., 1989), disruption of the electrochemical gradient of across the plasma membrane by increasing its extracellular concentration (Nicholls and Sihra, 1986), prolonged activation of Na^ channels by veratridine (Robinson and Dunkley, 1983), or via a direct influx of Ca^"^, mediated by Ca^"^ ionophores such as ionomycin (McMahon and Nicholls, 1991). While the first three drugs cause depolarisation of the synaptosomal plasma membrane, leading to activation of VGCCs and influx of Ca^"^ into the nerve terminal (Tibbs et al., 1989; McMahon and Nicholls, 1991), they differ significantly with respect to the properties of depolarisation caused. While 4AP evokes a repetitive firing of TTX-sensitive Na"^ channels and an asynchronous depolarisation, high extracellular KCl triggers a ‘clamped’ depolarisation and

- 115 - a fast inactivation of Na"^ channels (Nicholls and Coffey, 1994). In contrast, veratridine causes depolarisation by preventing voltage-gated Na"*" channel inactivation (McMahon and Nicholls, 1991). Glutamate release triggered by the addition of Ca^"^ ionophores is a result of direct Ca^^ entry without depolarisation of the nerve terminal and VGCC activation. Ionomycin is an electroneutral Ca^‘^/2H'^ exchanger which causes Ca^"^ entry by randomly inserting into the plasma membrane (Blau and Weissmann, 1988).

The presence of many types of neurotransmitter receptors on presynaptic nerve terminals has been demonstrated in various regions of the CNS, where they often play an important role in regulating the properties of neurotransmitter release. If presynaptically localised receptors bind a neurotransmitter released within the same synapse, they are referred to as presynaptic auto-receptors. Activation of auto-receptors therefore allows a positive or negative feed-back regulation of the release by the same neurotransmitter. In contrast, if receptors localised presynaptically bind a neurotransmitter released from the neighbouring synapses, they are referred to as hetero-receptors, and they represent a means by which one type of neurotransmitter regulates the release of another. Given that neurotransmitter release results from a co-ordinated function of many components, the regulation of this process can be quite complex, occurring at multiple levels, including plasma membrane depolarisation, activation of VGCCs, synaptic vesicle trafficking and exocytosis.

The regulation of neurotransmitter release by different types of presynaptic G protein coupled receptors (GPCRs) is now well established. GPCRs located on presynaptic nerve terminals can modulate neurotransmitter release by regulating the function of voltage-gated ion channels involved in depolarisation and Ca^"^ entry, or synaptic vesicle trafficking and exocytosis. The mechanisms underlying these processes include a direct interaction with activated G proteins, or activation of second messengers and protein phosphorylation pathways (Engelman and MacDermott, 2004).

Ligand-gated ion channels, also known as ionotropic receptors, produce their effects on membrane excitability in a direct manner via opening of their ion channels (Miyazawa et al., 2003). Many types of these receptors, such as nicotinic acetylcholine (nACh), NMD A, AMPA, kainate, P2X and glycine receptors, have been shown to reside on presynaptic nerve terminals in specific areas of the CNS where they regulate neurotransmitter release as

- 116 - either auto- or hetero-receptors (MacDermott et al., 1999). GABAa receptors have also been reported to regulate neurotransmitter release in some areas of the CNS. Specific examples of how various presynaptic receptors modulate neurotransmitter release in different brain regions are described in Section 1.13.

As our results in Chapter 3 demonstrate that activation of presynaptic GABAa receptors regulate intraterminal Ca^^ levels in glutamatergic nerve terminals isolated from the rat neocortex, it was of interest to investigate whether the activity of these GABAa receptors has any functional significance for the regulation of glutamate release. GAB A A receptors contain several binding sites for a large variety of pharmacologically important agents which potently modify the activity of these receptors. Functional interaction between allosteric binding sites and agonist binding sites can influence the equilibrium binding of GABA in a complex manner leading to either a reduction or increase. On that basis, allosteric modulators are classified as negative, neutral and positive modulators of GABAa receptor function. Agents which can cause a positive allosteric modulation of GABAa receptors include benzodiazepines and neurosteroids (See Section 1.4), and the effects of these drugs on glutamate release have been investigated.

Presynaptic GABAb receptors are well-established regulators of glutamate release and it was of interest to investigate in this chapter a possible functional cross-talk between these receptors and the presynaptic GABA a receptors, given that the activation of both receptor types is likely to occur simultaneously in response to GABA. Moreover, a direct interaction between GABAa receptor t2S subunit and the GABAb receptor R2 subunit has been characterised and shown to increase the internalisation of GABAb receptors in response to agonist binding (Balasubramanian et al., 2004).

Work in this chapter aimed to examine the effects of activation and allosteric modulation of presynaptic GABAa receptor activation on synaptosomal glutamate release.

117 - 4.2 Methods

4.2.1 Synaptosomal preparation

Synaptosomes were prepared as described in Chapter 2 (section 2.1).

4.2.2 Glutamate Release Assay

Glutamate release was measured on-line using Perkin-Elmer LS-5 spectrofluorimeter as described in Chapter 2 (section 2.4.1). This enzyme-linked assay monitors the accumulation of NADPH, which is produced due to the oxidation of released glutamate by the exogenous glutamate dehydrogenase (GDH) (Nicholls and Sihra, 1986; Nicholls et al., 1987). The excitation and emission of NADPH were measured at 1 = 340 and 460 nm, respectively. The addition of a known amount of glutamate (2.5 nmol) at the end of each assay allows the quantification of the released glutamate from synaptosomes as described in detail in section 2.4.1. Experiments were analysed as described in Chapter 2 (section 2.4.1) using Lotus 1-2-3, MicroCal Origin and Microsoft Excel. Statistical analysis was carried out using One-way ANOVA followed by post-hoc LSD test.

4.2.3 Drug-incubation protocols

Synaptosomes were kept at 4 °C after purification. Synaptosomal pellets were resuspended to a final concentration of 0.07 mg/ml in HBM and transferred to 37 °C at the start of each assay. CaClz was added at 3 min and the secretagogue (4AP, KCl, ionomycin) was added at 10 min after the start of incubation (Protocol 1). Ca^^-independent release was measured in the absence of CaCb and in the presence of 200 pM EGTA, which were added at the start of incubation. Experiments were carried out in the absence and presence of increasing concentrations of muscimol (10 - 500 pM) or isoguvacine (1 - 500 pM), which were added 7 min after the start of incubation (Protocol 1). In separate experiments, picrotoxin (1 - 100 pM) was added from the start of experiments. Allosteric modulators of GABA a receptors, diazepam (300 nM) and allopregnanolone (100 nM - 30 pM), and the GABA b receptor

- 118 agonist baclofen were added immediately following the addition of muscimol at 7 min (Protocol 2).

PROTOCOL 1

H -h -f îtî 4"C II 37”C PICROTOXIN CaCI, MUSCIMOL/ 4AP/ ISOGUVACINE KCl/ IONOMYCIN

GLUTAMATE STANDARD PROTOCOL 2

O' A A Î Î % 4"C 37'C |j CaCI, MUSCIMOL 4AP DIAZEPAM/ ALLOPREGNANOLONE/ BACLOFEN

Schematic 2. Drug incubation protocols used for glutamate release experiments in Chapter 4.

- 119 4.3 Results

4.3.1 Regulation of glutamate release by presynaptic GABAa receptors

Neocortical synaptosomes were resuspended at the concentration of 0.07 mg/ml, and incubated in the presence of muscimol (10 - 500 |iM) for 3 min prior to the addition of 1 mM 4AP to trigger glutamate release. Under control conditions, 4AP-evoked glutamate release was 28.2 ± 1.6 nmol/mg/5 min. When synaptosomes were incubated with increasing concentrations of muscimol, glutamate release measured was as follows: 28.4 ± 5.0 nmol/mg/5 min with 10 pM, 27.3 ±1.6 nmol/mg/5 min with 20 pM, 26.5 ±1.9 nmol/mg/5 min with 50 pM and 25.6 ±1.5 nmol/mg/5 min with 100 pM muscimol. A maximum inhibition of glutamate release was seen with 200 pM muscimol (21.8 ± 1.6 nmol/mg/5 min, 77.2 % of control release). A significant decrease in 4AP evoked glutamate release was also caused by 500 pM muscimol (22.6 ±1.8 nmol/mg/5 min, 80.3 % of control) (Fig. 18).

The release of glutamate in the presence of Ca^"^ and 4AP occurs primarily due to the exocytosis of small synaptic vesicles. Synaptosomes also contain a pool of cytosolic glutamate, some of which is released following depolarisation with 4AP and KCl (Nicholls et al., 1987) due to a reversal of plasma membrane glutamate transporter activity. This Ca^'^-independent release of glutamate was measured by incubating synaptosomes in the absence of CaCl 2 and in the presence of 200 pM EGTA. Under these conditions, Ca^"^- independent glutamate release evoked by 4AP was 8.0 ± 1.9 nmol/mg/5 min in controls, and it was not altered by the presence of 200 pM muscimol (8.0 + 1.8 nmol/mg/5 min) (Fig. 19). This indicates that GABAa receptor activation does not affect the activity of glutamate transporters, but instead it causes a reduction in Ca^'^-dependent release of glutamate mediated by SSV exocytosis.

Synaptosomes were also incubated with increasing concentrations of isoguvacine (1 - 500 pM). Under control conditions, 4AP-evoked glutamate release was 26.9 ±1.6 nmol/mg/5 min. In the presence of isoguvacine, 4AP-evoked glutamate release was as follows: 21.0 ± 2.4 nmol/mg/5 min with 1 pM, 24.6 ± 2.0 nmol/mg/5 min with 10 pM, 26.0 ±1.3

- 1 2 0 - 35 Control Control O) 30 lOuM lOOiaM 200^M 25 20|iM 50|jM SOOpM 20

15 Muscimol 4AP Muscimol 4AP

10

5

0

1---- 1---- r 1-----1—I----r Time (50sec/division)

35

U) ^ S I 30

25 = s

20

0 10 100 Log,o [Muscimol] (p.M)

Figure 18. Muscimol causes a dose-dependent inhibition of 4AP-evoked glutamate release. Synaptosomes were incubated in the presence of ImM CaClz and glutamate release evoked by the addition of ImM 4AP. Muscimol was added 3 min prior to the addition of 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis. Indicates significant differences at p<0.05 using One-way ANOVA followed by post-hoc LSD test (10 pM, n

= 3; 20 jiM, n = 4; 50 pM, n = 5; 100 pM, n = 6; 200 pM, n = 6; 500 pM, n = 6). a. On-line glutamate release in the absence or presence of increasing doses of muscimol, b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of increasing doses of muscimol.

- 121 - 10 Control O) Muscimol I0 E

5 Muscimol 4AP ï 1 0) oc

I 0 E S 3 O

Time (50 sec/division)

^O) 10 Io E

ï 3 Q) 5 oc B (0 E iS 3 ü

Control Muscimol

Figure 19. Muscimol has no effect on Ca^-independent release of glutamate in the presence of 4AP. Synaptosomes were incubated in the absence of CaCb and in the presence of 200 pM

EGTA, and glutamate release evoked by the addition of ImM 4AP. Muscimol (200 pM) was added 3 min prior to the addition of 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis (n = 4, One-way ANOVA followed by post-hoc LSD test), a. On-line Ca^"^- independent glutamate release in the absence or presence of muscimol, b. Cumulative glutamate release values following 5 min of incubation with 4AP in the absence or presence of muscimol.

- 122 - 30 Control Control O) lOO^M I 25 0 200^M E 20 500|j M 0) s 15 V oc Isoguvacine 4AP isoguvacine 4AP ü 10 ns E 15 5 O

I I r Time (50 sec/division)

I 30

i . c 0) t ' t flj(A I 20 OC s I 15 ü

10 1 10 100 Log,o [isoguvacine] (|j,M)

Figure 20. Isoguvacine causes a dose-dependent inhibition of 4AP-evoked glutamate release. Synaptosomes were incubated in the presence of ImM CaCb and glutamate release evoked by the addition of 1 mM 4AP. Isoguvacine was added 3 min prior to the addition of 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis. * Indicates significant differences at p<0.05 using One-way ANOVA followed by post-hoc LSD test (1 pM, n

= 6; 10 pM, n = 4; 20 pM, n = 4; 50 pM, n = 6; 100 pM, n = 7; 200 pM, n = 7; 500 pM, n = 7). a. On-line glutamate release in the absence or presence of increasing doses of isoguvacine. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of increasing doses of isoguvacine.

- 123 - nmol/mg/5 min with 20 juM, and 24.8 ±1.6 nmol/mg/5 min with 50 |iM isoguvacine. A significant reduction of glutamate release was detected in the presence of 100 pM (21.0 ± 1.9 nmol/mg/5 min, 80.2 % of the control), 200 pM isoguvacine (20.5 ±1.0 nmol/mg/5 min, 76.3 % control, and 500 pM isoguvacine (23.2 ±1.5 nmol/mg/5 min, 86.2 % control) (Fig. 20).

To confirm that muscimol-induced inhibition of 4AP-evoked glutamate release is mediated by GABAa receptors, we incubated synaptosomes in the presence of 50 pM and 100 pM picrotoxin before the addition of muscimol. Glutamate release caused by addition of 4AP was 29.3 ± 0.4 nmol/mg/5 min in controls, and 24.1 ±1.3 nmol/mg/5 min in the presence of 200 pM muscimol, representing 82.3 % of the average control level. In the presence of 50 pM picrotoxin prior to the addition of 200 pM muscimol, the decrease in glutamate release observed was partial (27.1 ± 0.9 nmol/mg/5 min), representing only 92.7 % of the control release. In the presence of 100 pM picrotoxin, the reduction in release caused by muscimol was completely abrogated (29.4 ± 0.8 nmol/mg/5 min, 100.3 % of the control release) (Fig.

21).

The effect of picrotoxin on 4AP-evoked glutamate release in the absence of muscimol was also tested. In these experiments glutamate release measured under control conditions was 25.2 ±1.3 nmol/mg/5 min. In the presence of two different doses of picrotoxin, 50 pM and 100 pM, the 4AP-evoked release of glutamate was 26.0 ± 0.8 nmol/mg/5 min and 25.6 ± 1.1 nmol/mg/5 min, respectively, indicating that no significant effect was observed (Fig. 22). These results indicate that the endogenous levels of GABA in the synaptosomal suspension at 0.07 mg/ml may not be high enough to cause a tonic activation of presynaptic

GABA a receptors, as was observed in synaptosomal suspension at the concentration of 0.8 mg/ml in experiments presented in Chapter 3.

GAB A A receptors have been demonstrated to reside on mossy fibre terminals in the CA3 region of the hippocampus (Ruiz et al., 2003). Hippocampal synaptosomes were prepared as described in Chapter 2. To test if the presence of functional presynaptic GABAa receptors can be detected in hippocampal synaptosomes, the effect of muscimol on 4AP- evoked glutamate release was assessed. Glutamate release evoked by 1 mM 4AP was

124 - 35 Control Control cn Muscimol ? 30 Muscimol 1GG|iM Picrotoxin 255GpM Picrotoxin 25

Muscimol 4AP M uscim ol 4AP

1 b Time (50 sec/division) Time (50 sec/division)

Control Muscimol Muscimol + Control Muscimol Muscimol + 5GpM Picrotoxin 1GG|iM Picrotoxin

Figure 21. Muscimol-induced decrease of 4AP-evoked glutamate release is inhibited by picrotoxin. Synaptosomes were incubated in the presence of ImM CaCb and glutamate release evoked by the addition of ImM 4AP. Picrotoxin (50 pM and 100 pM) was added at the start of incubation. Muscimol (200 pM) was added 3 min prior to the addition of 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis. Indicates significant differences at p<0.05 using One-way ANOVA followed by post-hoc LSD test (n = 4). a. On-line glutamate release in the absence or presence of muscimol or muscimol/picrotoxin. b. Cumulative glutamate release values following 5 min of incubation with 4AP in the absence or presence of muscimol or muscimol/picrotoxin.

- 1 2 5 - 30 Control Control o) 25 Picrotoxin Picrotoxin

20

15 4AP 4AP

ca E 23 Ü

Time (50 sec/division) Time (50 sec/division)

? 30

3, 25

w 15

Control 50)jM Picrotoxin Control lOOfiM Picrotoxin

Figure 22. Picrotoxin has no effect on 4AP-evoked glutamate release. Synaptosomes were incubated in the presence of CaCb and in the absence or presence of 50 jitM or 100 |iM picrotoxin from the start. Glutamate release was evoked by the addition of 1 mM 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis (n = 3, One-way ANOVA followed by post-hoc LSD test), a. On-line glutamate release in the absence or presence of picrotoxin. b. Cumulative glutamate release values following 5 min of incubation with 4AP in the absence or presence of picrotoxin.

- 12 6 - O) 20 Control 1 Muscimol o Muscimol + Picrotoxin E 15 c ï 10 Muscimol 4AP S o ÛC 5 E 2 3 0 ü

Time (50 sec/division) 3I 20 I

0)1 10 d> Œ Î 5 E S 3 o ^ Control Muscimol Muscimol 4- Picrotoxln

Figure 23. Muscimol-induced decrease in 4AP-evoked glutamate release from hippocampal synaptosomes, inhibited by picrotoxin. Hippocampal synaptosomes were incubated in the presence of ImM CaCb and glutamate release evoked by the addition of ImM 4AP. Picrotoxin (100 pM) was added at the start of incubation. Muscimol (200 pM) was added 3 min prior to addition of 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis. * Indicates significant differences at p<0.05 using One-way ANOVA followed by post-hoc LSD test (n = 5). a. On-line glutamate release in the absence or presence of muscimol or rmuscimol/picrotoxin. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol or muscimol/picrotoxin.

- 127 - Control Muscimol

Muscimol 4AP m 10

Time (50 sec/division)

I» O) I 1 " a>w g 10

Î 5 E 2 3 O 0 Control Muscimol

Figure 24. Muscimol has no effect on 4AP-evoked glutamate release from cerebellar synaptosomes. Cerebellar synaptosomes were incubated in the presence of ImM CaCb and glutamate release evoked by the addition of ImM 4AP. Muscimol (200 |iM) was added 3 min prior to addition of 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis (One-way ANOVA followed by post-hoc LSD test, n = 5). a. On-line glutamate release in the absence or presence of muscimol, b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol.

- 128 - 17.2 ± 0.7 nmol/mg/5 min. In the presence of muscimol, glutamate release was reduced to 13.4 ± 0.8 nmol/mg/5 min representing 77.8 % of the control release (Fig. 24). To confirm that the observed inhibition of glutamate release in hippocampal synaptosomes was due to the activity of GAB A A receptors, we incubated synaptosomes in the presence of 100 pM picrotoxin prior to the addition of 200 pM muscimol. However, in the presence of muscimol and picrotoxin together, glutamate release was 17.0 ±1.3 nmol/mg/5 min, indicating that picrotoxin, by blocking GAB A A receptor gating, eliminates muscimol- dependent inhibition of glutamate release (Fig. 23).

Presynaptic GABAa receptor activity has also been described in the cerebellum (Pouzat and Marty, 1999). Cerebellar synaptosomes were prepared as described in Chapter 2. Glutamate release evoked by 1 mM 4AP was 17.5 ±1.6 nmol/mg/5 min under control conditions. In the presence of 200 pM muscimol, glutamate release was 18.6 ±1.3 nmol/mg/5 min, indicating that no significant effect of GABAa receptor activation on glutamate release was observed (Fig. 24).

An alternative way to trigger glutamate release from synaptosomes is by increasing the extracellular concentration of KCl from 5 mM to 10 mM for a partial depolarisation, or 30 mM for a complete depolarisation of the plasma membrane, in the presence of 1 mM CaClz. Glutamate release from cortical synaptosomes evoked by 10 mM KCl was 19.7 ± 1.3 nmol/mg/5 min under control conditions. In the presence of 200 pM muscimol, the release was reduced to 15.0 ± 1.0 nmol/mg/5 min which represents 76.1 % of the control release (Fig. 25). These data indicate that glutamate release triggered by a partial depolarisation of synaptosomes with 10 mM KCl is inhibited by the activation of presynaptic GABA a receptors. When synaptosomes were depolarised by 30 mM KCl, glutamate release was significantly higher reaching the level of 24.6 ±1.3 nmol/mg/5 min under control conditions, as described previously (Sihra et al., 1992; Cousin and Robinson, 2000). However, in the presence of 200 pM muscimol, glutamate release was not altered, reaching the level of 25.1 ±1.3 nmol/mg/5 min (Fig. 26). These results suggest that the inhibition of glutamate release by presynaptic GABAa receptors may depend on the level of depolarisation or nerve terminal activity.

129- 30 O) Control — Muscimol Io 25 E c 20 0) tn o 15 0) ce KClMuscimol î 10 E S 5 3 O 0

Time (50 sec/division) ^ 30 Ë 25 E o 20 c_E 0) (A 15 S 0) OC 10 î E B 3 ü Control Muscimol

Figure 25. Muscimol causes an inhibition of glutamate release triggered by 10 mM KCl. Cortical ynaptosomes were incubated in the presence of 1 mM CaCb and glutamate release evoked by the addition of 10 mM KCl. Muscimol (200 pM) was added 3 min prior to addition of KCl. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis. • Indicates significant differences at p<0.05 using One-way ANOVA followed by post-hoc LSD test (n = 5). a. On-line glutamate release in the absence or presence of muscimol, b. Cumulative glutamate release following 5 min of incubation with KCl in the absence or presence of muscimol.

- 130 - Control Muscimol

c 20

S 15 KClMuscimol

Time (50 sec/division)

c 30 £ % 25

Io E 20

Ï 15 o 0) Œ 10 Î E 5 iS3 Ü 0 Control Muscimol

Figure 26. Muscimol has no effect on glutamate release triggered by 30 mM KCl. Cortical synaptosomes were incubated in the presence of 1 mM CaCb and glutamate release evoked by the addition of 30 mM KCl. Muscimol (200 p-M) was added 3 min prior to addition of KCl. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis (One-way ANOVA followed by post-hoc LSD test, n = 5). a. On-line glutamate release in the absence or presence of muscimol, b. Cumulative glutamate release following 5 min of incubation with KCl in the absence or presence of muscimol.

- 131 - Control 3 1 0 Muscimol

8

6 lonomycin Muscimol 4

2

0

Time (50 sec/division)

^ 0 Control Muscimol

Figure 27. Muscimol has no effect on glutamate release triggered by ionomycin. Synaptosomes were incubated in the presence of 1 mM CaCIi and glutamate release evoked by the addition of 5 pM ionomycin. Muscimol (200 pM) was added 3 min prior to addition of ionomycin. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis (One-way ANOVA followed by post-hoc LSD test, n = 4). a. On-line glutamate release in the absence or presence of muscimol, b. Cumulative glutamate release following 5 min of incubation with ionomycin in the absence or presence of muscimol.

- 132 - Glutamate release can also be triggered by the addition of Ca^^ ionophore ionomycin, which allows direct entry of Ca^"^ into the synaptosome and synaptic vesicle exocytosis independently of the activation of VGCCs. Glutamate release evoked by 5 jiM ionomycin was 8.3 ± 0.5 nmol/mg/5 min under control conditions as described previously (McMahon and Nicholls, 1991; Sihra et al., 1992). In the presence of 200 |iM muscimol, release was not changed, reaching the level of 7.8 ± 0.7 nmol/mg/5 min following the addition of ionomycin (Fig. 27). This indicates that the mechanism underlying GABAa receptor- mediated inhibition of glutamate release involves the activity of ion channels which play a role in stimulus/exocytosis coupling rather than affecting synaptic vesicle trafficking or the exocytotic machinery.

4.3.2 Regulation of glutamate release by allosteric modulators of GABAa receptors

We next examined whether presynaptic GABAa receptors residing on glutamatergic nerve terminals are sensitive to diazepam, an of GABAa receptors known to increase their activity. In these experiments glutamate release evoked by 1 mM 4AP was 26.5 ±0.6 nmol/mg/5 min under control conditions. In the presence of 300 nM diazepam, glutamate release was slightly decreased to 24.7 ±0.8 nmol/mg/5 min (92.9 % of control), although this was not statistically significant. In the presence of 200 |iM muscimol, glutamate release was inhibited to 22.7 ± 0.7 nmol/mg/5 min (85.7 % of control release). However, a small decrease in glutamate release to 21.5 ± 3.0 nmol/mg/5 min detected in the presence of both drugs was not significantly different from a decrease in release caused by muscimol alone (94.7 %) (Fig. 28).

In order to investigate whether presynapic GABAa receptors are sensitive to neurosteroids, which are also well-described allosteric modulators of GABAa receptors, synaptosomes were preincubated with increasing doses (100 nM - 30 jiM) of allopregnanolone both in the absence and presence of 200 |iM muscimol. In control experiments glutamate release evoked by 1 mM 4AP was 25.1 ± 0.6 nmol/mg/5 min (Fig. 29). The release was not significantly altered by 100 nM allopregnanolone (24.9 ± 0.8 nmol/mg/5 min, 99.2 % control), 300 nM allopregnanolone (24.3 ±1.2 nmol/mg/5 min, 96.8 % control), 1 |iM allopregnanolone (23.4 ±1.9 nmol/mg/5 min, 93.2 % control) or 3 jiM allopregnanolone

- 133 - Control Diazepam Muscimol Diazepam / Muscimol

20 Diazepam / « ^ 15 0) O) Muscimol 4AP Muscimol 4AP

n I I I Time (50 sec/division)

If.20

siC5

0 Control Muscimol Diazepam Muscimol / Diazepam

Figure 28. Muscimol-induced inhibition of 4AP-evoked glutamate release in the presence of diazepam. Synaptosomes were incubated in the presence of ImM CaCl 2 and glutamate release evoked by the addition of ImM 4AP. Muscimol (200 pM) and diazepam (300 nM) were added 3 min prior to addition of 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis. Indicates significant differences at p<0.05 using One-way ANOVA followed by post-hoc LSD test (n = 4). a. On-line glutamate release in the absence or presence of muscimol or muscimol/diazepam, h. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol or muscimol/diazepam.

- 134 - - nM Allopregnanolone Control lOOnM Allopregnanolone Control 300 - nM Allopregnanolonejn o io n e i Muscimol lOOnM Allopregnanolone Muscimol 300 / Muscimol i:: / Muscimol Alio/ Alio/ i " im ol 4AP M u sc im o l 4AP M u sc im o l 4AP M u sc im o l 4AP =: 10 I 5 I I lo

I I I I I III IT I I I 1 I I I I I I I I I I l l I I I T im e (50 sec/division) T im e (50 sec/division) « 30 3|25 i r a #20 Ih II:: II: h u C on M use l lM use l Alio lM/A

— 30 Control luM Allopregnanolone Control - 3pM Allopregnanolone I. Muscimol ipM Allopregnanolone Muscimol - 3pM Allopregnanolone ^ / M uscimol / Muscimol

Alio/ Alio/ I M u sc im o l 4AP M u sc im o l 4AP M u sc im o 4AP M u sc im o l 4AP f // I ® |i 1/ I 0 T I I I I I I I I I I I I I I I I I I “ I I I I I I I I I I I I I I I I I I I T im e (50 sec/division) T im e (50 sec/division) 0 30 2% 20 15 ifi1 iio l i s llll ^ -5 llll C on M use C on M use

Control lOpM Allopregnanolone Control 30pM Allopregnanolone Muscimol l 0pM Allopregnanolone Muscimol 30pM Allopregnanolone / Muscimol / Muscimol S20 Alio/ Alio/ M u sc im o l 4AP M u sc im o l 4AP M u sc im o l 4AP

T--1 Tim e (50 sec/division) Tim e (50 sec/division)

l i ” K 120 f I 15

0 llll Con M use l iM use b Alio Figure 29. Muscimol-induced inhibition of 4AP-evoked glutamate release in the presence of allopregnanolone. Synaptosomes were incubated in the presence of ImM CaC^ and glutamate release evoked by the addition of ImM 4AP. Muscimol (200 pM) and allopregnanolone (a. 100 nM, b. 300 nM, c. 1 pM, d. 3 pM, e. 10 pM, f. 30 pM) were added 3 min prior to the addition of 4AP. Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP are presented as bar graphs and used for statistical analysis. Indicates significant differences at p<0.05 using One-way ANOVA followed by post-hoc LSD test (n = 4). On-line glutamate release in the absence or presence of muscimol, allopregnanolone or muscimol/allopregnanolone is presented above the bar graphs. Con = control. Muse = muscimol. Alio = allopregnanolone, M/A = muscimol -t- allopregnanolone.

- 1 3 5 - 30 Control Baclofen Muscimol Baclofen / Muscimol

Baclofen / Muscimol 4AP Muscimol 4AP

1 Time (50 sec/division)

!£) 25 CD

o 20

o 0) DC 10

O Control Muscimol Baclofen Muscimol / Baclofen

Figure 30. Muscimol-induced inhibition of 4AP-evoked glutamate release in the presence of baclofen. Synaptosomes were incubated in the presence of ImM CaCb and glutamate release evoked by the addition of ImM 4AP. Baclofen (50 |iM) was added 3 min prior to the addition of

4AP immediately followed by the addition of muscimol (200 pM). Mean ± S.E.M. release values were calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for statistical analysis. * Indicates significant differences at p<0.05 using One-way ANOVA followed by post-hoc LSD test (n = 6). a. On-line glutamate release in the absence or presence of muscimol, baclofen or muscimol/baclofen, b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, baclofen or musci mol/bac lofen.

- 1 3 6 - (24.6 ±1.0 nmol/mg/5 min, 98 % control). However, a significant reduction in 4AP- evoked glutamate release was detected in the presence of 10 jiM (19.3 ± 0.9 nmol/mg/5 min, 76.8 % control) and 30 |iM allopregnanolone (12.3 ±1.7 nmol/mg/5 min, 49 % control. Fig. 29). These results indicate that higher doses of allopregnanolone can activate presynaptic GABAa receptors, thus leading to an inhibition of glutamate release. Glutamate release was also measured when allopregnanolone (100 nM - 30 p-M) was added together with 200 pM muscimol. The addition of muscimol reduced release under control conditions (25.1 ± 0.6 nmol/mg/5 min) to 19.6 ±1.2 nmol/mg/5 min (78.1 % of control) as shown previously. In the presence of muscimol and increasing concentrations of allopregnanolone (Fig. 29), glutamate release evoked by 4AP was as follows: 24.5 ± 2.3 nmol/mg/5 min with 100 nM allopregnanolone (125 % of muscimol alone), 26.7 ± 1.1 nmol/mg/5 min with 300 nM allopregnanolone (136.2 % of muscimol alone), 22.7 ±1.2 nmol/mg/5 min with 1 p.M allopregnanolone (115.8 % of muscimol alone), 20.3 ± 2.2 nmol/mg/5 min with 3 p.M allopregnanolone (103.6 % of muscimol alone), 17.8 ± 0.9 nmol/mg/5 min with 10 p,M allopregnanolone (90.8 % of muscimol alone). Glutamate release was reduced to 7.9 ±1.3 nmol/mg/5 min with 30 p.M allopregnanolone in the presence of muscimol (40.3 % of muscimol alone), which was a statistically significant reduction. These results indicate that presynaptic GABAa receptors are sensitive to allopregnanolone acting as an allosteric modulator which enhances the agonist-dependent inhibition of glutamate release.

4.3.4 Functional cross-talk between presynaptic GABAa and GABAb receptors

Presynaptic GABAb receptors are well-established as inhibitors of glutamate release, operating primarily via a direct interaction between G-protein Py subunits and presynaptic VGCCs, thereby inhibiting the influx of Ca^"^ following depolarisation of nerve terminals

(Huston et al., 1995). To investigate if presynaptic GABAb receptors are present in the same population of glutamatergic nerve terminals as GABAa receptors we measured glutamate release in the absence or presence of 50 p,M baclofen, a specific agonist of these receptors. Glutamate release evoked by 4AP was 25.7 ± nmol/mg/5 min under control conditions. In the presence of baclofen, glutamate release was reduced to 17.7 ± nmol/mg/5 min (68.8 % of control). In the presence of muscimol, glutamate release was

- 137 - reduced to 21.7 ± nmol/mg/5 min (84.3 % of control). In the presence of baclofen and muscimol together, 4AP-evoked glutamate release was reduced to 17.6 ± nmol/mg/5 min (68.5 % of control values), which was at the level similar to the release measured in the presence of baclofen alone (Fig. 30). This indicates that GABAa and GABAb receptors may be present on the same population of glutamatergic nerve terminals.

- 138 4.4 Discussion

The presence of functional GABAa receptors on glutamatergic nerve terminals as suggested in Chapter 3 raises a question as to the role of these receptors in the control of glutamate release. The experimental data presented in this chapter demonstrates that the activation of presynaptic GABAa receptors leads to an inhibition of glutamate release, viz. in the presence of a specific GABAa receptor agonist (muscimol or isoguvacine), a dose- dependent inhibition of glutamate release was observed, which was abolished by blocking GAB A A receptors with a specific antagonist (picrotoxin).

The release of glutamate from synaptosomes can be evoked by a variety of biochemical means (secretagogues) which depolarise plasma membrane by different mechanisms leading to the activation of VGCCs and exocytosis of small synaptic vesicles. The regulation of glutamate release by a presynaptic receptor of either metabotropic or ionotropic nature often depends on the type of the mechanism activated by a secretagogue to trigger vesicle exocytosis (Perkinton and Sihra, 1998). In our experiments, GABAa receptor-mediated inhibition of glutamate release was observed when depolarisation of synaptosomes was mediated by 4AP, which blocks channels and destabilises the resting membrane potential, or in the presence of 10 mM KCl, which clamps the membrane potential by disrupting the external/internal [K^] gradient across the plasma membrane. In the first instance, destabilised membrane potential leads to a stochastic activation of tetrodotoxin-sensitive voltage-dependent Na^-channels similar to the activation caused by electrical stimulation (Tibbs et al., 1989). In the case of 10 mM KCl, the release is also inhibited by tetrodotoxin, which indicates that depolarisation involves voltage-dependent Na^-channels (Kidokoro and Ritchie, 1980). However, the inhibition of glutamate release by presynaptic GABAa receptors was not observed when the release was evoked by 30 mM KCl, which is likely to cause significantly stronger depolarisation and override any modulatory influences involving the activity of ion channels. Depolarisation caused by 30 mM KCl shows a lack of sensitivity to tetrodotoxin and, as such, is viewed as being unphysiological (Sihra, 1997).

The regulation of glutamate release could occur at the level of Ca^^-dependent exocytosis of glutamate-containing small synaptic vesicles or at the level of glutamate re-uptake

- 139- mechanism which involves Ca^^-independent glutamate transporter activity. One example of the later mechanisms is the regulation of glutamate release by kainate at high pM concentrations, which was shown to inhibit glutamate transporter activity, resulting in an apparent increase in glutamate release (Pocock and Nicholls, 1988). However, the lack of an effect of presynaptic GABAa receptor activation by muscimol on Ca^^-independent release of glutamate demonstrates that the underlying mechanism includes Ca^^-dependent release from synaptic vesicles. Conversely, the underlying mechanism is unlikely to include the regulation of synaptic vesicle trafficking, priming and fusion as indicated by the lack of this effect when the release is triggered by the Ca^^ ionophore ionomycin, bypassing voltage-dependent processes.

GAB A A receptors have been shown to be located presynaptically in the hippocampus, where the a2-containing receptors appear to depolarise mossy fibre terminals and regulate [Ca^^Ji (Ruiz et al., 2003). The inhibition of glutamate release by muscimol observed using hippocampal synaptosomes is consistent with this study. However, it would be of interest to determine if the inhibition of release correlates with changes in [Ca^"^];.

Although, the presynaptic GABAa autoreceptors have been demonstrated in the cerebellum

(Pouzat and Marty, 1999), there is currently no evidence that presynaptic GABAa receptors regulate the release of glutamate in this brain region. Here, we found that muscimol had no effect on glutamate release from cerebellar synaptosomes.

Although the exact subunit composition of presynaptically localised GABAa receptors is currently unknown, a variety of pharmacological reagents can be used to test the presence of specific subunits known to show sensitivity to these reagents. For example, benzodiazepines are a large group of allosteric modulators of GABAa receptor, which show specificity towards different a subunits (Korpi et al., 2002). Diazepam, one such modulator with a broad clinical relevance, is known to specifically regulate al-, a2-, a3- and a5-, but not a4- and a6-containing GABAa receptors (Wieland et al., 1992; Hadingham et al., 1996; Wafford et al., 1996; Davies et al., 1998). In our experiments, the inhibition of glutamate release by GABAa receptors was largely insensitive to diazepam suggesting that these receptors are likely to contain a4 and/or a6 subunits (Sigel et al., 1990). However, relatively high amounts of a l, a2, y2L and y2S subunits were detected in

- 140 - synaptosomal fractions by immunoblotting with specific antibodies as presented in Chapter 3. It would be of interest to test if higher doses of diazepam or other benzodiazepines can affect the regulation of glutamate release by presynaptic GABAa receptors.

Neurosteroids represent a large group of GABAa receptor allosteric modulators with a variety of effects on their function, acting as potent positive modulators and G ABA mimetics, as well as inverse agonists in some cases (Akk et al., 2001; Wang et al., 2002).

Whereas the subunit composition of GABAa receptors greatly influences the modulatory actions of benzodiazepines, the interaction of neurosteroids with GABAa receptors appears to display much less subunit specificity. Studies have demonstrated that the type of a subunit has little effect on the modulatory actions of allopregnanolone when co-expressed with pi and y2L subunits in Xenopus oocytes (Belelli et al., 2002). However, despite this, receptors which incorporate the a4 subunit or a6 subunit do appear less sensitive to allopregnanolone (Belelli et al., 2002). Additionally, receptors which contain the Ô subunit in conjuction with the a 1 or a4 subunit have been shown to have a higher selectivity for some neurosteroids, such as allopregnanolone and 3a,5a-THDOC (Belelli et al., 2002; Brown et al., 2002; Wohlfarth et al., 2002). The type of the p subunit has little effect on the sensitivity of GABA a receptors to neurosteroids (Hadingham et al., 1996; Belelli et al., 2002), while the y subunit appears to affect the potency of the steroid for the receptor but not its maximum response (Maitra and Reynolds, 1999; Belelli et al., 2002). In our experiments, high doses of allopregnanolone were found to cause an inhibition of glutamate release in the absence of muscimol, and to further enhance the inhibition of release caused by muscimol. Therefore, it appears that allopregnanolone can act as an activator of presynaptic GABAa receptors, as well as a weak positive allosteric modulator.

In our experiments activation of either GABAa or GABAb receptors inhibits glutamate release, although the effect of GABAb receptors is more potent. However, the effect of

GAB A A receptors is occluded by the activation of GABAb receptors suggesting that these two classes of GAB A receptor are present on the same population of glutamatergic nerve terminals. If the opposite was true, the activation of both types of receptors would result in an additive effect. It is also possible that activation of GABAb receptors immediately prior

- 141 - to the activation of GABAa receptors inactivates those VGCCs (Huston et al., 1995) which are integral to the mechanism of action of GABAa receptors.

In this chapter we have demonstrated that functional presynaptic GABAa receptors are present as heteroreceptors at glutamatergic nerve terminals. The activation of these receptors results in an inhibition of glutamate release. Furthermore, this population of

GABAa receptors is sensitive to the neurosteroid allopregnanolone, but insensitive to the benzodiazepine diazepam, and may be co-localised with GABAb receptors. Our results clearly demonstrate that the underlying mechanism of action of these receptors involves the activity of voltage-dependent ion channels involved in stimulus/secretion coupling rather than synaptic vesicle trafficking and exocytosis.

- 142 - 4.5 Conclusions

Activation of presynaptic GABAa receptors in the rat neocortex causes an inhibition of Ca^^-dependent glutamate release when elicited by 4AP and 10 mM KCl.

GABAa receptor-dependent inhibition of glutamate release is abolished when the exocytosis of synaptic vesicles is triggered directly by the Ca^"^ ionophore

ionomycin, indicating that the mechanism activated by presynaptic GABAa receptor is likely to involve modulation of voltage-gated ion channels.

GAB A A receptor-dependent inhibition of glutamate release occurs in the hippocampus, where a reduction in 4AP-evoked glutamate release is observed in the presence of muscimol and blocked by picrotoxin.

GAB AA-mediated regulation of glutamate release is not observed in the cerebellum.

GABA a receptor-dependent inhibition of glutamate release is insensitive to low concentrations of diazepam.

Presynaptic GABAa receptors are activated by high doses of the neurosteroid allopregnanolone, leading to an inhibition of glutamate release.

Although both GABA a and GABAb receptors cause an inhibition of glutamate

release from neocortical synaptosomes, the effect of GABAa receptors is occluded

by the prior activation of GABAb receptors suggesting that the two classes of receptors may be present on the same population of nerve terminals where they are dependent on a molecular mechanism(s) commonly impinging on VGCCs.

143 CHAPTER FIVE

-144 5. Modulation of Plasma Membrane Potential and Intraterminal Ca^^ Concentration by Presynaptic GABAa Receptors

5.1 Introduction

GABAa receptors are ligand-gated ion channels which are permeant to Cl and HCO 3 ions. Their activation generally leads to a plasma membrane hyperpolarisation due to the inwardly directed conductance of Cl' down its concentration gradient which dominates over the outwardly directed conductance for HCO 3 (Farrant and Kaila, 2007). The standard intracellular concentration of Cl' in mammalian neurones in the adult brain is approximately 7 - 9 mM, while the extracellular concentration is approximately 125 - 140 mM (Ben-Ari, 2002; Sipila et al., 2006). The direction of movement of Cl' ions through GAB A A channels depends not only on the concentration gradient formed across the plasma membrane but also on the plasma membrane potential. Together, these two parameters determine the plasma membrane potential at which the concentration-gradient force moving Cr in one direction is balanced by the electrical force moving Cl' in the opposite direction. This is also referred to as the equilibrium or reversal potential for Cl ion (Eci). The reversal potential for Cl can be estimated using the Nemst equation:

IS

Ec\ = chloride reversal potential R = gas constant (8.314 J.K'\mol'^) T = absolute temeperature (in Kelvins)

* = the valance of the ion (= 1 ) F = Faraday constant (0.0965 kJ.mol'\mV'^) (the amount of electrical charge contained in one mole of a univalent ion) [Cl']o= Concentration of extracellular Cl' [Cl']i= Concentration of intracellular Cl'

- 145 In the adult CNS, the anionic currents which flow through GABAa receptor channels have only a small driving force, meaning that the GABAa reversal potential (Eci or £^g a b a) is only a few millivolts more negative than -65 mV, the resting membrane potential. This means that changes in the intracellular C1‘ concentration (as small as 12 mM) can shift EcABA to above the resting membrane potential, switching the outcome of GABAa receptor activation from hyperpolarisation to depolarisation (Ganguly et al., 2001; Staley and Smith, 2001).

The activation of presynaptic GABAa receptors has been shown to cause a membrane depolarisation in some instances. For example, activation of GABAa receptors in the rat locus coruleus causes a membrane depolarisation and correlates with an increase in spontaneous glutamate release onto noradrenergic neurones (Koga et al., 2005). GABAa receptor-mediated depolarisation of nerve terminals also correlates with an increase in glutamate release in the auditory brainstem, ventromedial hypothalamus, CAl and CA3 pyramidal neurons (Turecek and Trussell, 2001; Turecek and Trussell, 2002; Jang et al., 2001; Jang et al., 2005; Jang et al., 2006). In agreement with these observations, the nerve terminal [Cl']i of approximately 21 mM in the calyx of Held giant terminal was found to be four to five times higher than in the parent cell body (Price and Trussell, 2006).

Several studies have demonstrated that the activation of GABAa receptors causes a membrane depolarisation and increased [Ca^^Ji in many areas of the developing brain, including the neocortex, hippocampus, hypothalamus and cerebellum (Leinekugel et al., 1999; Marie et al., 2001; Chen et al., 1996; Filers et al., 2001). These findings are in agreement with studies indicating that the intracellular concentration of Cl is 20 - 40 mM higher in immature neurones than in adult neurones (Fukuda et al., 1998; Kuner and Augustine, 2000). In neurones of the lateral superior olive, perforated patch-clamp recordings have shown a shift in Eci from -46 to -82 mV between birth and the postnatal day 10 (Kandler and Friauf, 1995). The high levels of intracellular Cl' in immature neurones are a direct consequence of the lack of the activity of cation-chloride cotransporter KCC2 which actively extrudes Cl' out of cell. Thus, the change in functional outcome of GABAa receptor activation correlates well with an increase in the levels of KCC2 mRNA expression in immature neurones (Rivera et al., 1999). Furthermore,

GABAa responses change from being excitatory to inhibitory following transfection of

- 146- KCC2 cDNA into immature hippocampal neurons (Yamada et al., 2004). In contrast, the NKCCl transporter, which actively transports Cl into the cell, was shown to contribute to the depolarising effects of GABAa receptor activation in dorsal root ganglia and neocortical neurons (Fukuda et al., 1998; Ganguly et al., 2001; Yamada et al., 2004; Dzhala et al., 2005). The transition in functional outcome of GABAa receptors from depolarisation to hyperpolarisation appears to be regulated by G ABA itself, because the inhibition of GABAa receptors slows down this transition, while activation of GABAa receptors accelerates it (Ganguly et al., 2001). This alteration in GABAa receptor signalling during neuronal development has also been shown to be regulated by a l nicotinic ACh receptors , which regulate the level of expression of chloride transporters (Liu et al., 2006).

Activation of GABAa receptors has also been reported to lead to a neuronal excitation in injured neurones. The switch from an inhibitory to an excitatory output was proposed to contribute to excitotoxicity in cultured hypothalamic neurons injured by scraping, where

GABAa receptor activation causes an increase in intracellular Ca^"^ (van den Pol et al., 1996). Axotomy of vagal motoneurones correlates with a decrease of KCC2 expression and elevation of intracellular Cl' (Nabekura et al., 2002). Under these circumstances, activation of GABAa receptors causes an increase in intracellular Ca^"^ levels and may play a role in neural survival and regeneration (Toyoda et al., 2003).

Following membrane depolarisation, VGCCs are thought to go through a series of conformational changes from non-conducting to conducting states which lead to a channel opening and influx of Ca^^(Papazian et al., 1991; Garcia et al., 1997). Ca^"^ is a ubiquitous intracellular messenger involved in the regulation of numerous cellular processes. Thus, VGCCs couple membrane depolarisation to a diverse set of neuronal functions. Alterations in [Ca^^ji can regulate the function of VGCCs (Budde et al., 2002), voltage-gated channels (Stocker, 2004) and voltage-gated Na"^ channels (Kobayashi et al., 2002). Various intracellular signalling molecules are also regulated by Ca^"^, including PKC (Tanaka and Nishizuka, 1994), calmodulin, Ca^Ycalmodulin dependent (CaM) kinases (Hook and Means, 2001) and phosphatases (PP2B, calcineurin) (Klee et al., 1979), and Ca^^-dependent proteases (calpain) (Croall and DeMartino, 1991). An altered Ca^"^ homeostasis has also been implicated in the ischaemic stroke, excitotoxicity (MacDonald et al., 2006), pathophysiology of Alzheimer’s disease (Mattson, 2002) and schizophrenia (Lidow, 2003).

- 147 - Additionally, changes in the expression of neuronal calcium sensor proteins (NCS) have been detected in Huntington’s disease (Luthi-Carter et al., 2000), epilepsy (Matsu-ura et al., 2002), chronic pain (Cheng and Penninger, 2004) and mental retardation (Bahi et al., 2003).

Calcium plays a critical role in neurotransmitter exocytosis. The physiological release of neurotransmitters from presynaptic nerve terminals occurs within 200 ps of the arrival of an action potential, which causes activation of VGCCs. These Ca^^ channels are in close apposition to the exocytotic release machinery, as indicated by the microdomains of high levels of intraterminal Ca^^ observed at the mouths of Ca^^ channels in the squid giant terminal (Llinas et al., 1992b), as well as in synaptosomes (Verhage et al., 1991; Sihra et al., 1992). The membrane depolarisation-dependent Ca^"^ influx through VGCCs can be measured in synaptosomes using a ratiometric assay which employs the Ca^"^ sensitive dye fura-2-AM (Komulainen and Bondy, 1987).

This chapter aims to characterise changes in the plasma membrane potential and intraterminal [Ca^^Ji, which may result from the activation of presynaptic GABAa receptors in synaptosomes. The contribution of these processes to the observed inhibition of glutamate release by these receptors will also be established.

- 148 5.2 Methods

5.2.1 Synaptosomal preparation

Synaptosomes were prepared as in Chapter 2 (section 2.1).

5.2.2 Measurement of synaptosomal membrane potential using DiSC3(5)

Changes in DiSC3(5) fluorescence were monitored as described in Chapter 2 (Section 2.4.2). Upon depolarisation, the release of DISC3(5) dye from the membrane bilayer was monitored as an increase in fluorescence due to sensitivity of the dye to intracellular and extracellular environments (Hargittai et al., 1991). DISC3(5) fluorescence is measured at an excitation wavelength of 651 nm and emission wavelength of 675 nm and data accumulated at 2 sec intervals. Results were analysed as described in Chapter 2 (section 2.4.2) using Lotus 1-2-3, MicroCal Origin and Microsoft Excel. Statistical analysis was carried out using One-way ANOVA followed by post-hoc LSD test.

5.2.3 Measurements of synaptosomal membrane potential - incubation protocols

Synaptosomes were kept at 4 °C after purification. Synaptosomes were resuspended to a final concentration of 0.07 mg/ml in 1.5 ml HBM containing 1 mg/ml BSA, transferred to a cuvette and placed in a Perkin-Elmer LS-5 spectrofluorimeter (Perkin-Elmer, Emeryville, CA) at 37 °C with stirring. After 1 min, 4 pM DiSC3(5) was added and allowed to equilibrate. This was followed by the addition of 1 mM CaC^ at 3 min and muscimol or isoguvacine at 7 min from the start. Synaptosomes were depolarised by the addition of 1 or 3 mM 4AP or 10 mM KCl at 10 min from the start (Protocol 1). In separate experiments, picrotoxin (100 pM) was added from the start of experiments and all other reagents added as described above (Protocol 2).

- 149- PROTOCOL 1 END + I— î - 4 ------î- 4 ' î î A 4 -C 37 “G 0150,(5) CaCI : MUSCIM0L7 4AP/ iSOGUVACINE KOI

PROTOCOL 2 END + 10 ' I - + - Î - 4 ------4 ' îîî A 4 °C y 37 °c PICROTOXIN DiSC,(5) CaCI ,

Schematic 3. Drug incubation protocols used to measure changes in synaptosomal membrane potential in Chapter 5.

5.2.4 Intraterminal Ca^^ measurements using fura-2-AM

Intraterminal Ca^"^ was measured as described in Chapter 2.4.3.

In our experiments the excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 505 nm were used and data collected at 3.5 sec intervals. Calibration procedures were carried out with 0.1 % (w/v) SDS and 5 mM Na-EGTA as described in

Chapter 2 (section 2.4.3). Results were analysed as described in Chapter 2 (section 2.4.3) using Lotus 1-2-3, MicroCal Origin and Microsoft Excel. Statistical analysis was carried out using One-way ANOVA followed by post-hoc LSD test.

5.2.5 Intraterminal Ca^^ measurements - incubation protocols

Synaptosomes were kept at 4 °C after purification. Synaptosomes were preincubated at 37

°C in HBM containing 5 pM fura-2-AM, 0.1 mM CaCL and 1 mg/ml BSA for 20 min.

Fura-loaded synaptosomes were centrifuged and the supernatant discarded. Synaptosomal pellets were resuspended in HBM containing 1 mg/ml BSA and transferred to a spectrofluorimeter with stirring (Perkin-Elmer, Emeryville, CA). After 3 min, 1 mM CaCb

- 150- was added. Muscimol was added at 7 min and 4AP added 10 min from the start (Protocol 3).

PROTOCOL 3 END

1 0 ’ 1 4 ’ I Î- Î t A 4 °C 37 °C MUSCIMOL 4AP

Schematic 4. Drug incubation protocol used to measure changes in intraterminal Ca^'*‘ concentration in Chapter 5.

- 151 - 5.3 Results

5.3.1 Fluorescent measurements of plasma membrane potential in response to agonists and antagonists of GABAa receptors

The experimental data presented in Chapters 3 and 4 indicates that the activation of presynaptic GABAa receptors in neocortical synaptosomes leads to a depolarisation of the plasma membrane and an increase in [Ca^^]i„possibly mediated by VGCCs. Plasma membrane potential changes can be monitored by voltage-sensitive dyes such as DiSCsCS).

In order to investigate the effects of presynaptic GABAa receptors on nerve terminal membrane potential more closely, we resuspended synaptosomes in HBM at 0.07 mg/ml concentration in the presence of 4 jiM DiSCsCS). After the steady-state fluorescence of DiSC3(5) was reached upon full translocation into the lipid bilayer, 10 mM KCl was added to depolarise synaptosomes. Depolarisation of synaptosomes resulted in an increase in fluorescence of 6.0 ± 0.4 fluorescence units/4 min (mean ± S.E.M.). When synaptosomes were incubated with either 50 pM or 100 pM picrotoxin followed by the addition of 10 mM KCl, no significant effect on the change in fluorescence was observed (5.8 ± 0.3; 6.4 ± 0.6 fluorescence units/4 min, respectively). In addition, no change was observed in the resting membrane potential prior to the addition of KCl (Fig. 31).

The effect of presynaptic GABAa receptor activation on plasma membrane potential was investigated in the presence of muscimol (1 - 500 pM). In control samples the addition of 10 mM KCl caused a change in fluorescence of 6.2 ± 0.3 fluorescence imits (mean ± S.E.M., n = 3). In the presence of increasing doses of muscimol added 3 min prior to the addition of 10 mM KCl, the following changes in fluorescence were detected: 7.0 ± 0.6 fluorescence units/4 min with 1 pM; 6.5 ± 0.9 fluorescence imits/4 min with 10 pM; 6.5 ± 0.3 fluorescence units/4 min with 50 pM; 5.4 ± 0.4 fluorescence units/4 min with 100 pM; 5.8 ± 0.5 fluorescence units/4 min with 200 pM; and 5.8 ± 0.5 fluorescence units/4 min with 500 pM (Fig. 32). When data was normalised with the appropriate control values, the following muscimol-induced

152 - Control 3c 7 50 |iM Picrotoxin 0) ü 6 mc 5 0i 4 KCl

3

£L1 2 0) C 1 n2 i 0

Time (50 sec/division)

c — Control D 7 — 100 |iM Picrotoxin 2 2 6 i 5 3o KCl LL

3

2

1

0

Time (50 sec/division)

Figure 31. Picrotoxin has no effect on KCl-triggered depolarisation of synaptosomes. Synaptosomes were incubated in the presence of 4 pM DiSC3(5) and in the absence (black) or presence (red) of 50 pM or 100 pM picrotoxin, for 10 min, prior to the addition of 10 mM KCl . Each trace represents mean ± S.E.M. of 3 independent experiments. Error bars are shown every 60 sec for clarity.

- 153- I 8 Control Control 3 1 nM Muscimol 10 jiM Muscimol $7

03

Muscimol KCl Muscimol KCl

I 0 1 Time (50 sec/division)

- Control Control - 20 |j M Muscimol 50 pM Muscimol

Muscimol KCl Muscimol KCl

1 Time (50 sec/division) Control Control 100 pM Muscimol 500 pM Muscimol

KCl Muscimol KCl Muscimol

n

1 Time (50 sec/division)

Figure 32. Muscimol has no effect on KCl-triggered depolarisation of synaptosomes. Synaptosomes were incubated in the presence of 4 pM DiSC3(5) and in the absence (black) or presence (red) of 1 pM - 500 pM muscimol, for 3 min, prior to the addition of 10 mM KCl. Each trace represents mean ± S.E.M. of 3 independent experiments. Error bars are shown every 60 sec for clarity.

- 154- 110

8 90

80

70

60

50 —-/ hr 0 1 10 100 [MUSCIMOL] (^iM)

Figure 33. Muscimol has no effect on KCl-triggered depolarisation of synaptosomes. Synaptosomes were incubated in the presence of 4 pM DiSCsCS) and in the absence or presence of 1 pM - 500 pM muscimol, for 3 min, prior to the addition of 10 mM KCl, Data was normalised with the appropriate control and expressed as a percentage of control values. Each value represents mean ± S.E.M. of 3 independent experiments, measured 4 min after the addition of KCl.

155 - changes in fluorescence were observed: 106.9 ± 5.3 % control with IjiM; 97.6 ± 9.7 % control with 10 |iM, 103.6 ± 4.5 % control with 50 |iM, 90.8 ± 4.3 % control with 100 |xM, 98.8 ± 10.8 % control with 200 |iM, and 96.7 ± 3.0 % control with 500 jiM muscimol (mean ± S.E.M., n = 3). Statistical analysis revealed that changes in fluorescence detected in the presence of muscimol were not significantly different from those obtained in control samples (Fig. 33).

Next, we examined the effect of the GABAa receptor specific agonist isoguvacine (1 - 500 pM) on the resting membrane potential and KCl-mediated depolarisation of synaptosomes. The addition of 10 mM KCl produced a fluorescence change of 6.7 ± 0.2 fluorescence units/4 min (mean ± S.E.M, n = 5) under control conditions. In the presence of increasing doses of isoguvacine added 3 min prior to the addition of 10 mM KCl, the following changes in fluorescence were detected: 6.2 ± 0.3 fluorescence units/4 min with 1 pM, 7.0 ± 0.3 fluorescence units/4 min with 10 pM; 6.3 ± 0.2 fluorescence units/4 min with 50 pM, 6.6 ± 0.4 fluorescence units/4 min with 100 pM, 6.7 ± 0.5 fluorescence units/4 min with 200 pM, and 6.4 ± 0.2 fluorescence units/4 min with 500 pM isoguvacine (mean ± S.E.M, n = 5; Fig. 34). These results demonstrate that no significant change in 10 mM KCl-evoked depolarisation occured in the presence of any concentration of isoguvacine tested. Isoguvacine had no detectable effect on the resting plasma membrane potential either (Fig. 34). When the values were normalised with the appropriate control values, the following isoguvacine-induced changes in fluorescence were obtained: 106.9 ± 5.3 % control with 1 pM, 97.6 ± 9.7 % control with 10 pM, 103.6 ± 4.5 % control with 50 pM, 90.8 ± 4.3 % control with 100 pM, 98.8 ± 10.8 % control with 200 pM, and 96.7 ± 3.0 % control with 500 pM isoguvacine (mean ± S.E.M, n = 5; Fig. 35).

While a high concentration of K^ ([K'^lo) triggers depolarisation by disrupting the electrochemical gradient across the plasma membrane, 4AP operates in a different manner by blocking K^ channels (Tibbs et al., 1989). It was therefore of interest to investigate changes in the membrane potential in response to 4AP,

156 - — Control — Control — 1 pM Isoguvacine — 10 nM Isoguvacine 1

Isoguvacine KCl Isoguvacine KCl

tim e (50 sec/division)

3 8 Control Control 0) 50 |iM Isoguvacine 100 |iM Isoguvacine Ë 7 CD g 6 I 5 LL Isoguvacine KCl Isoguvacine

I

JO IE 0 —I------1------1------r Time (50 sec/division) Control Control 200 |iM Isoguvacine 500 |iM Isoguvacine

Isoguvacine KCl Isoguvacine KCl

1 Time (50 sec/division)

Figure 34. Isoguvacine has no effect on KCl-triggered depolarisation of synaptosomes. Synaptosomes were incubated in the presence of 4 pM DiSC3(5) and in the absence (black) or presence (red) of 1 pM - 500 pM isoguvacine, for 3 min, prior to the addition of 10 mM KCl. Each trace represents mean ± S.E.M. of 5 independent experiments. Error bars are shown every 60 sec for clarity.

- 157- 120

g 110 ü 100

90

80

în 70

ü 60

50 l-V/r- I I I M I I 0 1 10 100 [ISOGUVACINE] (^M)

Figure 35. Isoguvacine has no effect on KCl-triggered depolarisation of synaptosomes.

Synaptosomes were incubated in the presence of 4 |liM DiSCsCS) and in the absence or presence of 1 ^iM - 500 )iM isoguvacine, for 3 min, prior to the addition of 10 mM KCl. Data was normalised with the appropriate control and expressed as % of control in the absence of isoguvacine. Each trace represents mean ± S.E.M. of 5 independent experiments, measured 4 min after the addition of KCl.

158 - - Control Control 3.0 - Muscimol Muscimol

Muscimol 1mM4AP Muscimol 3mM4AP

Cl 0.5

0.0

Time (50 sec/division)

Figure 36. Muscimol has no effect on 4AP-triggered depolarisation of synaptosomes. Synaptosomes were incubated in the presence of 4 pM DiSC3(5) and in the absence (black) or

presence (red) of 500 pM muscimol, for 3 min, prior to the addition of 1 mM or 3 mM 4AP. Each trace represents mean ± S.E.M. of 3 independent experiments. Error bars are shown every 60 sec for clarity.

- 159- and the effects that GABAa receptor agonists may have under these conditions. The addition of 1 mM 4AP caused an increase in fluorescence of 1.6 ± 0.1 fluorescence units/4 min (mean ± S.E.M., n = 3). Incubation of synaptosomes with 500 |iM muscimol followed by the addition of 1 mM 4AP produced no significant change in the resting plasma membrane potential. The 4AP-mediated increase of DiSCsCS) fluorescence due to depolarisation was also unaffected by the addition of muscimol (1.6 ± 0.2 fluorescence units/4 min; Fig. 36, left panel).

Depolarisation of synaptosomes with 3 mM 4AP caused an increase in DiSC3(5) fluorescence of 2.8 ± 0.2 fluorescence units/4 min (mean ± S.E.M., n = 3) under control conditions. In the presence of 500 p.M muscimol, the change observed after the addition of 4AP was 2.4 ± 0.4 fluorescence units/4 min (Fig. 36; right panel). Therefore, no significant effect of muscimol on the resting membrane potential or 4AP-evoked depolarisation was detected in these experiments. In summary, neither the activation of GABAa receptors by muscimol or isoguvacine, nor GABA a receptor channel inhibition by picrotoxin, resulted in any detectable changes in resting synaptosomal membrane potential or the level of synaptosomal depolarisation evoked by 4AP or KCl.

5.3.2 Fluorescent measurements of intraterminal Ca^^ concentration in response to the GABAa receptor agonist muscimol

In order to directly monitor changes in [Ca^^Ji in response to GABAa receptor agonists, we took advantage of an on-line fluorometric assay which is based on the fluorescent Ca^"^- sensitive indicator fura-2. Using this assay, we determined the [Ca^'^Ji at rest and following the depolarisation with 4AP.

Basal levels of [Ca^'^Ji, immediately prior to depolarisation with 4AP, were determined to be 155.1 ± 12.2 nM (mean ± S.E.M., n = 5) under control conditions. In the presence of muscimol, basal levels of [Ca^"^]i immediately prior to depolarisation with 4AP were: 139.3 ± 10.3 nM with 1 |iM, 149.0 ± 14.9 nM with 10 pM, 131.3 ± 11.4 nM with 50 pM, 135.8 ± 11.7 nM with 100 pM, 146.9 ± nM with 200 pM, and 137.2 ± 7.6 nM with 500 pM (mean

160 - 450 Control Control

400 1 |j M Muscimol 10 |iM Muscimol

350

300

250 Muscimol Muscimol CO 0 200

150

100

50

0 '------1------'------r "I '------1------'------1------1------1------1------'------r Time (50 sec / division)

— Control Control 450 50 |iM Muscimol 100 |iM Muscimol 400 4AP 4AP 350

^ 300 Muscimol Muscimol ^ 250 CC O 200

150

100

1 1 1 1 1 1 1—

Time (50 sec / division)

— Control Control 450 200 |iM Muscimol 4 /^p 500 ^iM Muscimol 4 /^p 400 T ■ 350

C 300 Muscimol ^ ■ - 250 Muscimol CO Ca2+ 0 200

150

100

50

0 1 ------'------1------'------1------«------r Time (50 sec / division)

Figure 37. Intrasynaptosomal Ca^^ concentration in the presence ofGABAa receptor agonist muscimol. Fura-2-labelled synaptosomes were incubated in the presence of 1 mM Ca^^ and in the

absence (black) or presence (red) of muscimol (1 - 500 pM), followed by the addition of 1 mM 4AP. Each trace represents a mean ± S.E.M. of 5 independent experiments. Error bars are shown every 60 sec for clarity.

- 161 - 100

2 90 c o Ü 80 6 + CO 70 Ü < 60

50 0 1 10 100 [Muscimol] (|iM)

100

2 90 c o Ü 80 0^ i 70 Ü <1 60

50 / / ] ------1------1------1— I I' I I q - - 1 1 1— I IT I I'l 0 1 10 100 [Muscimol] (p,M)

Figure 38. Regulation of intrasynaptosomal Ca^^ levels by the GABAa receptor agonist muscimol. Changes in intraterminal Ca^^ concentration ([Ca^’^jO due to 4AP-evoked depolarisation in the presence of increasing doses of muscimol were calculated and expressed as a percentage of values obtained in controls. Data points represent mean ± S.E.M. of 5 independent experiments. p<0.05 (in grey, One-way ANOVA followed by post-hoc LSD test). a. Data points obtained 20 sec after the addition of 4AP. b. Data points obtained 4 min after the addition of 4AP.

- 162- ± S.E.M., n = 5). These values were not significantly different from the control values (Fig. 37).

The addition of 1 mM 4AP leads to an activation of VGCCs and a rise in [Ca^'^Ji to 277.9 ± 24.3 nM (mean ± S.E.M) under control conditions (Fig. 37). In the presence of increasing doses of muscimol (1 - 500 |iM), the addition of 4AP resulted in the following [Ca^"^]i levels: 249.0 ± 22.1 nM with 1 jiM, 269.9 ± 29.6 nM with 10 p,M, 244.1 ± 22.1 nM with 50 |iM, 254.5 ± 22.6 nM with 100 |iM, 256.1 ± 23.0 nM with 200 jiM, and 237.7 ± 16.9 nM with 500 |iM, when measured 20 sec after the addition of 4AP. Intrasynaptosomal [Ca^"^]i measured 4 min after the addition of 4AP was 340.2 nM ± 39.1 nM (mean ± S.E.M) under control conditions (Fig. 37). In the presence of increasing doses of muscimol (1 - 500 jiM), the following concentrations of [Ca^^]i were measured: 311.9 ± 39.1 nM with 1 |iM, 325.0 ± 27.7 nM with 10 pM, 304.8 ± 26.0 nM with 50 pM, 311.4 ± 28.4 nM with 100 pM, 312.5 ± 29.5 nM with 200 pM, and 290.1 ± 19.6 nM with 500 pM (Fig. 38). Thus, the increasing doses of muscimol caused an apparent decrease in [Ca^^]i during depolarisation of synaptosomes with 4AP although these changes were not statistically significant.

When the changes in intraterminal Ca^'^ concentration (A[Ca^'^]i), evoked 20 sec after the addition of 4AP in the presence of muscimol were calculated and expressed as a percentage of A[Ca^"^]i measured under control conditions, the following normalized values were obtained: 86.1 ± 6.0 % control with 1 pM, 89.6 ± 8.3 % control with 10 pM, 85.7 ± 3.1 % control with 50 pM, 81.9 ± 1.2 % control with 100 pM, 83.5 ± 4.4 % control with 200 pM, and 85.0 ± 2.6 % control with 500 pM (mean ± S.E.M., n = 5; Fig. 38a). The same analysis was carried out with changes in intraterminal Ca^^ concentration (A[Ca^^]i) in the presence of muscimol 4 min after the addition of 4AP, and the following values obtained: 87.2 ± 4.0 % control with 1 pM, 90.2 ± 7.7 % control with 10 pM, 86.0 ± 5.1 % control with 50 pM, 83.6 ± 3.6 % control with 100 pM, 88.8 ± 3.6 % control with 200 pM, and 76.7 ± 6.4 % control with 500 pM (mean ± S.E.M., n = 5; Fig. 38b). This analysis revealed a statistically significant reduction in [Ca^^Ji evoked by 1 mM 4AP in the presence of muscimol (50 - 500 pM). The data indicates that the ability of nerve terminals to undergo depolarisation-

163 dependent activation of Ca^^ channels is significantly decreased by the activation of presynaptic GABAa receptors.

164 - 5.4 Discussion

The experimental data presented in Chapters 3 and 4 are consistent with the hypothesis that functional GABAa receptors are located on a population of glutamatergic nerve terminals where the activation of these receptors under resting conditions leads to increased [Ca^^]i as reflected in increased activity of presynaptic Ca^^-dependent signalling pathways.

However, the functional outcome of presynaptic GABAa receptor activity is a dose- dependent inhibition of glutamate release. To measure changes in plasma membrane potential and intraterminal [Ca^^ji directly, we employed on-line fluorimetry using voltage- and Ca^'^-sensitive dyes, DiSCsCS) and fura-2 respectively.

When changes in plasma membrane potential were monitored using DiSCsCS), no significant effects of GABAa receptor activation by muscimol or isoguvacine, or inactivation by picrotoxin, were observed under resting conditions. Upon the addition of 10 mM KCl, or 1 mM or 3 mM 4AP, an increase in fluorescence was detected due to depolarisation, but was unaffected by the activity of presynaptic GABAa receptors. The lack of any effect of GABAa receptors could be explained by the low sensitivity of this assay. Neocortical synaptosomes represent a mixture of many types of nerve terminals, the majority of which are glutamatergic and GABAergic. Our immunohistochemical data presented in Chapter 3 has indicated a specific localisation of presynaptic GABAa receptors to glutamatergic nerve terminals, which represent approximately 60 % of all terminals in the synaptosomal suspension (Beaulieu, 1993). However, GABAa receptors could be present only in a small population of glutamatergic terminals and therefore be below the level of detection of this assay. Additionally, the depolarisation caused by presynaptic GABAa receptors on membrane potential may be too small to be detected with this assay.

Using the Ca^^-sensitive fluorescent indicator fura-2, we observed a reduction in intraterminal Ca^"^ levels when synaptosomes were depolarised by 4AP in the presence of muscimol. Although contradictory to the observed increase in Ca^^-dependent phosphorylation of synapsin I in the presence of muscimol, these results are consistent with the decrease in glutamate release reported in Chapter 4. The decrease in both

- 165 - depolarisation-evoked Ca^^ influx and glutamate release in the presence of muscimol can be explained by inactivation of Na"^ and/or Ca^^ channels prior to the addition of 4AP, as observed in the posterior pituitary (Zhang and Jackson, 1995). In contrast, phosphorylation of synapsin occurs further downstream of these membrane delimited changes, and represents a consequence of a cascade of signalling events. It is also worth noting that changes in intraterminal Ca^"^ observed using fura-2 represent “global” changes within the whole nerve terminal, whilst any effects caused by presynaptic GABAa receptor activation are more likely to be localised to the immediate vicinity of the receptors and transmitter release sites they may be coupled to.

Inhibition of synaptic transmission between primary afferents and spinal neurons due to

GABAa receptor-mediated depolarisation has been reported (Eccles, 1964) but molecular mechanisms underlying this modulation are not known. GABAa receptor-mediated depolarisation was also found to inhibit synaptic transmission in the olfactory cortex (Pickles, 1979) and posterior pituitary (Dyball and Shaw, 1979; Saridaki et al., 1989; Zhang and Jackson, 1995). One possible mechanism for the observed regulation suggested by these authors involves the inactivation of Na"^ channels. This type of regulation was proposed to occur in the posterior pituitary (Zhang and Jackson, 1995) leading to a decrease in voltage-dependent activity of Ca^^ channels. Depolarisation caused by GABAa receptor activation could also be sufficient to open low-threshold Ca^"^ channels or to reduce Ca^^ extrusion. We hypothesise that an initial GABAa receptor-mediated depolarisation of nerve terminals could lead to an increase in [Ca^"^]i through activation of low-voltage activated Ca^^ channels and/or release of Ca^"^ from the intracellular stores. Although below detection levels in our assay, this initial rise in [Ca^'^]i could result in the Ca^'^- dependent inactivation of Ca^^ channels which are directly coupled to the neurotransmitter release (Imredy and Yue, 1994; Zuhlke et al., 1999; Lee et al., 1999). Alternatively, this regulation may involve dephosphorylation of Ca^"^ channels by Ca^^-dependent phosphatase PP2B/calcineurin (Kits and Mansvelder, 1996; Lukyanetz et al., 1998).

- 166 - 5.5 Conclusions

• Resting membrane potential in isolated nerve terminals is unaffected by GABAa receptor activation in the presence of muscimol or isoguvacine or inhibition in the presence of picrotoxin.

• Depolarisation of isolated nerve terminals evoked by 4AP or 10 mM KCl

concentration is unaffected by either activation or inhibition of GABAa receptors.

• Intraterminal [Ca^^]i is not significantly altered by muscimol under basal conditions.

• Depolarisation-dependent rise in [Ca^'^ji is significantly reduced following the

activation of presynaptic GABAa receptors by muscimol.

- 167 - CHAPTER SIX

-1 6 8 6. Functional coupling of the presynaptic GABAa receptors to the NKCCl cation co-transporter and voltage-dependent Ca^^ channels

6.1 Introduction

In the adult neocortex, activation of GABAa receptors (either by endogenous G ABA or exogenous GABAa receptor agonists) produces synaptic inhibition by causing a hyperpolarisation of the plasma membrane. In these instances, the chloride reversal potential (£'ci) is -60 to -70 mV, which is generally well below the threshold for action potential generation (-40 to -50 mV). However, when the concentration of intracellular chloride is elevated, the resting membrane potential becomes more negative than Ec\, which means that GABAa receptor activation results in Cl' efflux and consequent membrane depolarisation. Presynaptic GABAa receptors are known to be depolarising in some instances. These depolarising effects facilitate glutamate release in the auditory brainstem, ventromedial hypothalamus and locus coeruleus (Turecek and Trussell, 2001; Jang et al., 2001; Koga et al., 2005), and noradrenaline release from hippocampal synaptosomes (Fassio et al., 1999). In addition, the presynaptic depolarisation induced by GABAa receptors can increase spontaneous transmitter release in some hippocampal intemeurones (Jang et al., 2001; Kanematsu et al., 2002). In the posterior pituitary however, presynaptic GABAa receptor-mediated depolarisation inhibits neurosecretion by inactivating Na"^ channels (Zhang and Jackson, 1995). In the mossy fibre terminals, GABAa receptor activation causes a depolarisation followed by an increase in Ca^'^ influx (Ruiz et al., 2003). The inhibition of neurotransmitter release by presynaptic GABAa currents can occur due to “shunting” of the presynaptic membrane potential, as observed in the sensory afferent terminals (Stuart and Redman, 1992).

At rest, the concentration of intracellular Cl' is regulated by three groups of electroneutral cation-chloride co-transporters (CCCs): Na'^-Cl' co-transporters (NCCs), which are not found in nervous tissue (Clayton et al., 1998), Na^-K"^-Cr co-transporters (NKCCs) and K^- C r co-transporters (KCCs), which are found abundantly in neurones (Kaila, 1994). CCCs are described in greater detail in Section 1.9.

169 In some cases of brain trauma, GABAa receptors were found to cause membrane depolarisation which correlates with a reduction in KCC2 expression (Nabekura et al., 2002). The stimulation of NKCCl has been shown to be required for high extracellular K^- induced swelling, which contributes to glutamate release from astrocytes (Su et al., 2002). Bumetanide and furosemide are well established pharmacological agents which inhibit NKCCl and KCC2. Bumetanide has a -500 fold greater potency in inhibiting NKCCl than KCC2 which means that the low concentrations of bumetanide (2-10 pM) can be used to inhibit NKCC without greatly affecting the function of KCC2 (Payne et al., 2003). Furosemide has equal affinity and potency in inhibiting both transporters. Direct phosphorylation of NKCCl occurs in response to a reduced intracellular Cl concentration to increase the activity of this transporter and restore intracellular Cl levels (Lytle et al., 1992).

As a direct consequence of NKCC activity and an accumulation of CT inside the nerve terminals, activation of GABAa receptors could lead to a depolarisation of nerve terminal plasma membrane, activation of VGCCs and Ca^"^ influx (Tibbs et al., 1989; McMahon and Nicholls, 1991). Neurotransmitter release could then be triggered due to the high local Ca^^ concentration at the active zone triggering the exocytosis of small synaptic vesicles (Zucker and Fogelson, 1986). Depending on the type of VGCC activated by GABAa receptor- mediated depolarisation, these processes could lead to an increase in [Ca^'^Ji without directly effecting the neurotransmitter release. Ca^^ channels can be divided into low voltage-activated channels (LVA) and high voltage-activated channels (HVA). LVA channels consist only of the T-type Ca^^ channels. The HVA channels are comprised of L-, N-, P-, Q- and R-type Ca^'^ channels. Subtypes of VGCCs are spatially localised to different neuronal compartments where they regulate specific aspects of the neurotransmitter release and thereby play a major role in the control of synaptic transmission (Wu and Saggau, 1997; Reid et al., 2003). Expression of multiple VGCC subtypes could therefore contribute significantly to a large variation in the control of synaptic transmission by Ca^"^ (Dunlap et al., 1995). VGCCs are described in greater detail in Section 1.12.

Several subtypes of Ca^^ channels support the release of glutamate at excitatory synapses. The majority of VGCCs expressed in presynaptic nerve terminals are N, P and Q-type

- 170 - channels, whose activation is directly linked to the neurotransmitter release (Takahashi and Momiyama, 1993; Castillo et al., 1994). At glutamatergic synapses in the cerebral cortex, release is supported by P/Q-type Ca^^ channels to a larger extent than by N-type Ca^^ channels (Vazquez and Sanchez-Prieto, 1997). A prominent role of P/Q-type channels in comparison to N-type channels was also characterised in the entorhinal cortex, where the release of neurotransmitters is less sensitive to the block of N-type Ca^^ channels (Qian and Noebels, 2001). At granule cell to Purkinje cell synapse in rat cerebellar slices, the release was also shown to be triggered more effectively by P/Q-type channels, although multiple types of Ca^^ channels appear to co-operatively control individual release sites (Mintz et al., 1995). Co-localisation studies have shown that the mixture of N-type and P/Q-type channels can vary between the terminals of the same afferent in presynaptic terminals of cultured hippocampal neurones, suggesting that modulation of synaptic function may be terminal-specific (Reid et al., 1998). P-type channels play a more prominent role in glutamate release from hippocampal neurones in comparison with other channels involved (Luebke et al., 1993).

R-type channels can also play a role in neurotransmitter release, although not as much is understood about their specific role (Kamp et al., 2005). R-type channels have been shown to regulate neurotransmitter release in the calyx of Held synapse in the medial nucleus of the trapezoid body of the rat. The control of neurotransmitter release by these channels occurs with a lower efficacy than by the other types of Ca^"^ channels, contributing approximately 25% of the total presynaptic Ca^^ current (Wu et al., 1998). R-type VDCCs have also been shown to participate in glutamatergic transmission in the rat hippocampus (Gasparini et al., 2001), and oxytocin secretion in neurohypophysial terminals (Wang et al., 1999).

Although L-type channels are not generally thought to participate in neurotransmitter release at synapses mediating fast synaptic transmission (Home and Kemp, 1991; Ohno- Shosaku et al., 1994; Wheeler et al., 1994), they are known to reside presynaptically, where they regulate transmitter release from cultured hippocampal GABAergic nerve terminals (Jensen et al., 1999), GABAergic transmission in the supraoptic nucleus (Bhaukaurally et al., 2005) and excitatory amino acid release onto dopaminergic neurones of the ventral mesencephalon (Bonci et al., 1998). They have also been shown to play an important role

- 171 - in the release of excitatory amino acids from the retina (Tachibana et al., 1993; von Gerdsoff and Matthews, 1996), catecholamines from chromaffin cells (Lopez et al., 1994), and dynorphins from the dendrites of rat hippocampal granule cells (Simmons et al., 1995). L-type Ca^"^ channels also play a role in secretion of hormones or neuromodulators, including neuropeptides from the neurohypophysis (Pemey et al., 1986; Lemos and Nowycky, 1989). However, L-type Ca^"^ channels have been shown to have no direct role in the control of glutamate release from the cerebrocortical synaptosomes (Pocock et al., 1992).

While T-type Ca^"^ chaimels are located mainly on the soma and dendrites (Huguenard, 1996; Craig et al., 1999) of neurones in the brain, they have been shown to directly trigger the fast neurotransmitter release from retinal bipolar cells (Pan et al., 2001). Although synaptic transmission appears resistant to L-, N-, and P/Q-type channel antagonists in some brain regions (Mintz et al., 1995; Turner et al., 1995; Sabatini and Svoboda, 2000), there is no evidence to suggest that the release is regulated by T-type Ca^'*' channels.

The activities of several types of VGCCs can be facilitated or inhibited by Ca^"^, either via a direct interaction with calmodulin or by a direct phosphorylation/dephosphorylation of these channels. It is well documented that the inactivation kinetics of some types of VGCCs is increased by the rise in intracellular Ca^^, a process also referred to as the calcium-dependent inactivation (CDI). In the case of L-type Ca^^ channels, this entails a Ca^^ binding to calmodulin (CaM) (already associated with the channel at the C-terminal), causing a translocation of CaM to a different position on the C-terminus of the main a subunit (Soldatov, 2003). The EF-hand motifs (two helices approximately perpendicular to each other which pair to bind calcium) and CaM binding sites on the C-terminus of the Ca^"^ channel a-subunit which are necessary for CDI, also appear to be present in N, P/Q and R-type VGCCs (Chaudhuri et al., 2004). L-type Ca^"^ channels in the heart inactivate in response to Ca^'^ entry, displaying an important negative feedback property. In this case, Ca^"^ influx through one channel selectively contributes to the inactivation of the other adjacent channels, without a generalised elevation of [Ca^^ji. These negative interactions between Ca^^ chaimels can be eliminated in the presence of the intracellular Ca^"^ chelator BAPTA or by the local dispersal of the neighbouring channels (Imredy and Yue, 1992). Whereas the CDI of the L-type channels occurs by Ca^"^ binding to high affinity sites, and

- 172 - can be evoked by Cs?'^ influx through individual channels, CDI of other subtypes of VGCCs is dependent on the low affinity binding sites of CaM (Liang et al., 2003). They therefore require Ca^^ influx through a variety of channels and larger global rises in [Ca^^]i for CDI to occur. Thus CDI causes a reduction of Ca^"^ influx by acting as an activity- dependent negative feedback mechanism. Hence, this negative feedback could regulate the release of neurotransmitters following a global increase in [Ca^^]i (Lee et al., 2003). Ca^^- dependent dephosphorylation of N-type and L-type VGCCs by PP2B/calcineurin has also been suggested to result in a negative feedback regulation of Ca^"^ influx (Armstrong, 1989; Lukyanetz et al., 1998; Burley and Sihra, 2000).

In addition to CDI, VGCCs can also be positively modulated by Ca^"^. Ca^^-dependent facilitation (CDF) results in an increase in current amplitude due to co-operativity in the activation of the neighbouring Ca^"^ channels. CDF which is caused by a direct interaction with calmodulin, has been described for L-type (Zuhlke et al., 1999) and P/Q-type Ca^"^ channels (Meyer et al., 2000). In addition to this type of regulation, CaMK II has been shown to bind and phosphorylate the L-type Ca^^ channels in a Ca^^-dependent manner, thereby resulting in CDF (Hudmon et al., 2005). CaMK II has also been shown to positively modulate T-type Ca^^ channels, although it is unclear whether this regulation involves a direct phosphorylation of the channel (Welsby et al., 2003; Yao et al., 2006). Furthermore, while most studies have demonstrated that the dephosphorylation of VGCCs by calcineurin causes CDI, in certain neurones in the hippocampus an enhancement in L- type Ca^"^ channel activity was observed (Norris et al., 2002). The dual roles of some proteins in mediating both CDI and CDF could be due to a number of factors, including the presence of multiple protein binding sites, different splice variants, the type/location of the neurone within the neuronal circuit, or the functional state of the channel.

In this chapter, we aim to elucidate the mechanisms underlying GABAa receptor-mediared inhibition of glutamate release.

- 173 - 6.2 Methods

6.2.1 Synaptosomal preparation

Synaptosomes were prepared as in Chapter 2 (section 2.1). R-type VGCC knock-out mice lacking the a lE gene were a gift from Dr. G.J. Stephens, University of Reading (Perevesev et al., 2002).

6.2.2 Glutamate Release Assay

Glutamate release was measured as described in Chapter 4.2.2.

6.2.3 Drug-incubation protocols

Synaptosomes were kept at 4 °C after purification. Synaptosomal pellets were resuspended to a final concentration of 0.07 mg/ml in HBM and transferred to 37 °C at the start of each assay. CaCli (1 mM) was added 3 minutes, muscimol (200 pM) was added 7 minutes and the secretagogue 4AP (1 mM) was added 10 minutes after the start of incubation. Experiments were carried out in the absence and presence of bumetanide (10 pM) or W7 (20 pM) added from the start of the incubation (Protocol 1). Another set of experiments was carried out in the absence or presence of co-agatoxin IVA (100 nM), cù-conotoxin

GVIA (1 pM), NiCl 2 (50 pM), nifedipine (1 pM) or SNX-482 (100 nM), added 6 minutes after the start of incubation (Protocol 2). Additional experiments were performed using EGTA (200 pM) added 3 minutes and BaCU (ImM) added 7 minutes after the start of the incubation (Protocol 3).

174 - PROTOCOL 1 GLUTAMATE STANDARD

10’ i - h - S - î tî 4 “C y 3 7 - 0 BUMETANIDE/ CaCI MUSCIMOL W7

GLUTAMATE STANDARD PROTOCOL 2

6 ’ 7 ’ 1 0 ' I 1 - -I— I - 4 ' t t A A 4»C 3 7 °C

CaCI 2 (o-AGATOXIN IVA/ MUSCIMOL (D-CONOTOXIN GVIA/ N IC L /NIFEDIPINE/ SNX-482 GLUTAMATE STANDARD PROTOCOL 3 4 15 ' î t 4 °C 3 7 “C EGTA BaCI,

Schematic 5. Drug incubation protocols used for glutamate release experiments in Chapter

6 .

6.2.3 Analysis

Results were analysed as described in Chapter 2 (section 2.4.1.) using Lotus 1-2-3,

MicroCal Origin and Microsoft Excel. Statistical analysis was carried out using One-way

ANOVA followed by post-hoc LSD test.

- 175- 6.3 Results

6.3.1 Functional cross-talk between presynaptic GABAa receptors and the NKCCl co-transporter

Our experimental results described in Chapters 3, 4 and 5 are consistent with the hypothesis that the activation of GABAa receptors leads to a depolarisation of the nerve terminal membrane, which is sufficient to cause an increase in [Ca^^]i and enhance the activity of Ca^^-dependent signalling pathways, but insufficient to trigger the release of glutamate.

Presynaptic depolarisation caused by GABAa receptors would only occur due to an efflux of Cl through these channels as a consequence of high [Cl'Ji. To test if the inwardly directed Cl' transporter NKCCl is involved in maintaining the high [Cl']i, thereby contributing to the observed regulatory mechanisms, we incubated synaptosomes with 10 pM bumetanide, a selective NKCCl inhibitor. Glutamate release evoked by 1 mM 4AP was 26.1 ± 0.3 nmol/mg/5 min under control conditions. In the presence of 200 pM muscimol, release was significantly reduced to 21.9 ± 0.3 nmol/mg/5 min (83.9% of control release). In the presence of bumetanide (10 pM) from the start of the incubation, glutamate release evoked by ImM 4AP was potently reduced to 16.3 ±1.8 nmol/mg/5 min (62.6% of control release). However, muscimol-dependent inhibition of glutamate release was completely abolished in the presence of bumetanide. Under these conditions, 4AP-evoked release was 15.2 ± 1.0 nmol/mg/5 min, which was at the same level as the release measured in the presence of bumetanide alone (Fig. 39). Therefore, it appears that the activity of

NKCCl transporter, resulting in an increased intraterminal [Cl']i, is critical for GABAa receptor-dependent inhibition of glutamate release.

6.3.2 Ca^^ is required for muscimol-induced inhibition of glutamate release

As the partial depolarisation of glutamatergic nerve terminals at rest due to the presynaptic GAB A A receptors activity may affect the function of specific types of VGCCs and the Ca^"^ influx as demonstrated in Chapters 3 and 5, as well as by

- 176 - 30 Control — Bumetanide U) Muscimol — Bumetanide / Muscimol 20

Muscimol 4AP Muscimol 4AP

Time (50 sec/division)

o> 25

I 20

« 15 0) CC 10 o>

Ü Control Muscimol Bumetanide Muscimol / Bumetanide

Figure 39. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of bumetanide. Synaptosomes were incubated in the presence of 1 mM CaClz, and in the absence or presence of bumetanide (10 pM, blue and green) and/or muscimol (200 pM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, p<0.05, are presented in grey (One­ way ANOVA with post-hoc LSD test; n = 6). a. On-line glutamate release in the absence or presence of muscimol, bumetanide or muscimol/bumetanide. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, bumetanide or muscimol/bumetanide.

- 177- Control o) 30 Muscimol o 25

20

Muscimol 4AP

Time (50 sec/division)

c Ê 30 3) 25 Io E 20 Ï s 15 0) cc 0) 10 m E B 5 3 o 0 Control Muscimol

Figure 40. Muscimol has no effect on 4AP-evoked glutamate release in the absence of extrasynaptosomal Ca^\ Synaptosomes were incubated in the absence of Ca^"^ and in the absence (black) or presence (red) of muscimol (200 pM) prior to the addition of 1 mM 4AP. Glutamate release was triggered by the addition of 1 mM 4AP, immediately followed by the addition of ImM CaCb- Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis (n = 3). a. On­ line glutamate release in the absence or presence of muscimol, b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol.

- 178- Control Muscimol 25 I0 1 20

% 15 Muscimol 4AP Muscimol 4AP I 10 cc % 5 i 0 3 O

1 Time (50 sec/division)

30 c 1 25 I 0 20 E Ï 15 1 V cc 10

E 5 S 3 Ü 0 Control Muscimol BaCU + Muscimol

Figure 41. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of BaClz. Synaptosomes were incubated in the presence of 1 mM CaCh (black and red) or ImM BaCl 2 (green and blue), and in the absence or presence of muscimol (200 pM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, p<0.05, are presented in grey (One­ way ANOVA with post-hoc LSD test; n = 4). a. On-line glutamate release in the absence or presence of muscimol, BaCL or muscimol/BaClg. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, BaCl 2 or muscimol/BaC^.

- 179- other studies (Leinekugel et al., 1995), we next investigated the contribution of Ca^"^- dependence to GABAa receptor-mediated inhibition of glutamate release. First, we tested if the presence of extrasynaptosomal Ca^^ (1 mM) prior to the activation of GABAa receptors by muscimol was necessary for the inhibition of glutamate release to occur. Muscimol was therefore added in the absence of Ca^^ and glutamate release evoked by 1 mM 4AP which was immediately followed by the addition of 1 mM Ca^^ (McMahon and Nicholls, 1991). Using this paradigm, glutamate release in control experiments, in the absence of muscimol, was 29.1 ±1.6 nmol/mg/5 min, while in the presence of 200 jiM muscimol, the release was 27.9 ±1.8 nmol/mg/5 min. These results indicate that while the control release of glutamate under these experimental conditions was unaffected by the absence of extrasynaptosomal Ca^"^, prior to depolarisation by 4AP, muscimol-dependent inhibition of glutamate release was abolished, suggesting that the presence of Ca^"^ and probably the influx of Ca^"^' is essential for the observed inhibition of glutamate release by presynaptic GABAa receptors (Fig. 40).

In order to confirm that Ca^"^ is required for GABAa receptor-mediated inhibition of glutamate release, we used 1 mM BaClz in our incubations in place of CaCli. Ba^^ has been shown to enter nerve terminals through VGCCs (Nachshen and Blaustein, 1982; Augustine and Eckert, 1984) and acts as a substitute for Ca^"^ in triggering the release of glutamate following the nerve terminal depolarisation (Sihra et al., 1993). The release of glutamate can be elicited by the addition of Ba^^ alone, due to the inhibition of channels and depolarisation of the plasma membrane (Hagiwara et al., 1974). Glutamate release evoked by ImM 4AP was 25.6 ± 0.6 nmol/mg/5 min under control conditions. In the presence of 200 pM muscimol, release was decreased to 22.5 ± 0.7 nmol/mg/5 min (87.9% of control release). When Ba^^ was substituted for Ca^'^, glutamate release was 29.0 ± 0.9 nmol/mg/5 min (113.3% of control release) in the absence of muscimol, and 28.1 ± 0.8 nmol/mg/5 min in the presence of muscimol. Thus, the inhibition of glutamate release caused by muscimol was abolished when the Ca^'^ was substituted by Ba^^ (Fig. 41). These results validate the hypothesis that the influx of Ca^'^ into the nerve terminal is required for the GABAa receptor-mediated inhibition of glutamate release to occur.

- 180 6.3.3 GABA a receptor-mediated inhibition of glutamate release is dependent on L- and R-type VGCCs

The lack of muscimol-dependent inhibition of evoked glutamate release in the absence of extrasynaptosomal Ca^^ indicates that the underlying Ca^^-dependent molecular mechanism involves the influx of Ca^^ through VGCCs. Therefore, we applied selective VGCC inhibitors to characterise the role of individual Ca^"^ channel subtypes in GABAa receptor- mediated inhibition of glutamate release. We started by investigating the role of N-, P-, and Q-type VGCCs which are known to directly mediate Ca^"^ influx that triggers neurotransmitter release in synaptosomes (Vazquez and Sanchez-Prieto, 1997; Millan and Sanchez-Prieto, 2002). In order to assess the role of P/Q-type VGCCs in GABAa receptor- mediated inhibition of glutamate release, we preincubated synaptosomes with 100 nM co- Agatoxin IVA (Cù-Aga-IVA), a peptide toxin isolated from the funnel web spider Agelenopsis aperta (Mintz et al., 1991). Glutamate release evoked by 1 mM 4AP was 25.6 ±0.8 nmol/mg/5 min under control conditions. In the presence of 200 pM muscimol, glutamate release was reduced to 19.5 ±1.5 nmol/mg/5 min (76.2% of control release). Consistent with published observations, glutamate release in the presence of co-Aga IVA was decreased to 17.2 ± 1.5 nmol/mg/5 min (67.2% of control release). When 200 pM muscimol was added in the presence of co-Aga IVA, a further inhibition of glutamate release to 12.1 ±1.6 nmol/mg/5 min was observed (70.3% of co-Aga IVA release; Fig. 42). These results suggest that the mechanism by which presynaptic GABAa receptors mediate inhibition of glutamate release does not involve a direct regulation of P/Q-type VGCCs, although these channels contribute significantly to the 4AP-evoked glutamate release.

In order to block N-type VGCCs, synaptosomes were preincubated with co-conotoxin GVIA (co-CTx GVIA), a toxin isolated from the marine mollusc Conus geographus (Regan et al., 1991). Glutamate release caused by 1 mM 4AP was 25.6 ± 0.9 nmol/mg/5 min under control conditions. In the presence of 200 pM muscimol, glutamate release was reduced to 21.0 ± 1.9 nmol/mg/5 min (82.1% of control release). When synaptosomes were incubated with co-CTx GVIA, 4AP-evoked glutamate release was reduced to 21.4 ± 1.3 nmol/mg/5 min (84.0% of control).

181 - 30 Control (o-Agatoxin I VA Muscimol (o-Agatoxin IVA + I” Muscimol I 20 0) % 15 o Muscimol 4AP Muscimol 4AP CC0) 10 E

3 Ü

Time (50 sec/division)

3) 25

« 15

Control Muscimol co-AgatoxIn Muscimol + IVA o>Agatoxin IVA

Figure 42. Muscimol-induced inhibition of 4AP-evoked glutamate release is independent of the activity of P/Q type VGCCs. Synaptosomes were incubated in the presence of 1 mM CaClz, and in the absence or presence of co-agatoxin IVA (100 pM, blue and green) and/or muscimol (200 pM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, p<0.05, are presented in grey (One-way ANOVA with post-hoc LSD test; n = 7). a. On-line glutamate release in the absence or presence of muscimol, co-agatoxin IVA or muscimol/co-agatoxin IVA. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, co-agatoxin IVA or muscimol/co-agatoxin IVA.

- 182- 30 Control o>Conotoxln GVIA O) 25 Muscimol (o-Conotoxin GVIA + Muscimol 20

Muscimol 4AP Muscimol 4AP

Time (50 sec/division)

/Ts 30

CC 10

Control Muscimol o>Conotoxin Muscimol + GVIA oConotoxin GVIA Figure 43. Muscimol-induced inhibition of 4AP-evoked glutamate release is independent of the activity of N-type VGCCs. Synaptosomes were incubated in the presence of 1 mM CaCI], and in the absence or presence of (O-conotoxin GVIA (10 pM, blue and green) and/or muscimol (200 pM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, *p<0.05, are presented in grey (One-way ANOVA with post-hoc LSD test; n = 4). a. On-line glutamate release in the absence or presence of muscimol, co-conotoxin GVIA or muscimol/co-conotoxin GVIA. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, co-conotoxin GVIA or muscimol/co-conotoxin GVIA.

- 1 8 3 - 30 O) Control Muscimol I 25 E S 20 Ci (A 2 15 0) Muscimol 4AP Muscimol 4AP ce « 10 I s 3 C5 _

Time (50 sec/division)

o) 25

E 20

w 15

Ü Control Muscimol NiClg Muscimol + NiCIo

Figure 44. Muscimol-induced inhibition of 4AP-evoked glutamate release is independent of the activity of T-type VGCCs. Synaptosomes were incubated in the presence of 1 mM CaCl 2, and in the absence or presence of NiCl 2 (50 pM, blue and green) and/or muscimol (200 jaM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, " p<0.05, are presented in grey (One­ way ANOVA with post-hoc LSD test; n = 5). a. On-line glutamate release in the absence or presence of muscimol, NiC^ or muscimol/NiCL. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, NiCl 2 or muscimol/NiCL-

- 184- However, in the presence of both muscimol and co-CTx GVIA, 4AP-evoked glutamate release was further reduced to 17.1 ±1.4 nmol/mg/5 min (67.3% of co-CTx GVIA release; Fig. 43), indicating that the muscimol-mediated decrease in glutamate release is independent of N-type Ca^"^ channel activity, although these channels are directly involved in the regulation of neurotransmitter release.

A potential role of T-type Ca^"^ channels in muscimol-dependent inhibition of glutamate release was tested in the presence of 50 jiM NiCli, a specific blocker of these channels (Carbone and Swandulla, 1989; Toselli and Taglietti, 1992). Glutamate release evoked by 1 mM 4AP was 24.5 ± 0.5 nmol/mg/5 min under control conditions. In the presence of 200 pM muscimol, glutamate release was reduced to 18.7 ±1.9 nmol/mg/5 min (76.3% of control release). In the presence of NiCl 2 alone, glutamate release was 22.3 ± 1.8 nmol/mg/5 min. When synaptosomes were incubated with both 50 pM NiCli and 200 pM muscimol, glutamate release evoked by the addition of 1 mM 4AP was 18.1 ± 0.9 nmol/mg/5 min (81.2% of NiCl 2 release; Fig. 44), which indicates that the mechanism underlying the muscimol-dependent decrease in glutamate release does not include the activity of T-type Ca^^ channels.

The effect of L-type channel inhibition on muscimol-dependent inhibition of glutamate release was investigated in the presence of 1 mM nifedipine. Glutamate release triggered by 1 mM 4AP was 24.9 ±1.5 nmol/mg/5 min under control conditions. In the presence of 200 pM muscimol, release was reduced to 19.2 ±1.1 nmol/mg/5min (77.1% of control release). In the presence of nifedipine alone, 1 mM 4AP-evoked glutamate release was 27.9 ± 2.6 nmol/mg/5 min. When 200 pM muscimol was added in the presence of nifedipine, glutamate release was 25.0 ± 1.5 nmol/mg/5 min, which indicated that muscimol-induced inhibition of glutamate release was abolished in the absence of L-type Ca^"^ channel activity (Fig. 45).

The contribution of R-type Ca^"^ channel activity to the muscimol-dependent inhibition of glutamate release was investigated using SNX-482, a toxin from the tarantula Hysterocrates gigas that has been shovshown to be an antagonist of alE containing Ca^"^ channel subtypes (Newcomb et al., 1998).

185 O) Control Nifedipine Muscimol Nifedipine + Muscinnol

Muscimol 4AP Muscimol 4AP

1 Time (50 sec/division)

c 30 Ë % 25 I0 E 20 c Ï 15 3 1 10

5

0 Control Muscimol Nifedipine Muscimol + Nifedipine

Figure 45. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of L-type VGCC blocker nifedipine. Synaptosomes were incubated in the presence of 1 mM CaCI], and in the absence or presence of nifedipine (1 pM, blue and green) and/or muscimol

(200 pM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, ‘p<0.05, are presented in grey (One-way ANOVA with post-hoc LSD test; n = 4). a. On-line glutamate release in the absence or presence of muscimol, nifedipine or muscimol/nifedipine, b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, nifedipine or muscimol/nifedipine.

- 1 8 6 - 30 Control SNX-482 O) Muscimol SNX-482 + 25 Muscimol 20

Muscimol 4AP Muscimol 4AP

Time (50 sec/division)

O)

I 20

w 15

DC 10

O Control Muscimol SNX482 SNX-482 + Muscimol

Figure 46. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of R-type VGCC blocker SNX-482. Synaptosomes were incubated in the presence of 1 mM CaCl2, and in the absence or presence of SNX-482 (100 nM, blue and green) and/or muscimol (200 |iM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, p<0.05, are presented in grey (One-way ANOVA with post-hoc LSD test; n = 6). a. On-line glutamate release in the absence or presence of muscimol, SNX-482 or muscimol/SNX-482. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, SNX-482 or muscimol/SNX-482.

- 187- WT Control KO Control O) Muscimol KG Muscimol E 30

% 20

Muscimol 4AP Muscimol 4AP

Time (50 sec/division)

WT WT KO KO Control Muscimol Control Muscimol

Figure 47. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the R-type Ca^^ channel knock-out mice. Synaptosomes from wild-type (WT) and R-type Ca^^ channel knock-out (KO) mice were incubated in the presence of 1 mM CaCl 2, and in the absence (black and green) or presence of muscimol (200 pM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, p<0.05, are presented in grey (One-way ANOVA with post-hoc LSD test; n = 5). a. On-line glutamate release in the absence or presence of muscimol, in WT or R-type KO mice. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, in WT or R-type KO mice.

- 188- In concordance with the results presented previously, 4AP-evoked glutamate release (25.2 ±1.7 nmol/mg/5 min) was reduced to 20.4 ±1.4 nmol/mg/5 min in the presence of 200 jiM muscimol (81% of control release). Glutamate release following the addition of 100 nM SNX-482 was slightly reduced to 22.3 ±1.1 nmol/mg/5 min (88.5% of control release), although this was not statistically significant. When 200 |iM muscimol was added in the presence of 100 nM SNX-482, release was 22.9 ±1.1 nmol/mg/5 min, which indicated that no further inhibition of glutamate release by muscimol occurred in the presence of SNX- 482 (Fig. 46). These results suggest that the activity of R-type Ca^^ channels is an essential component of the mechanism that mediates muscimol-induced inhibition of glutamate release. Although SNX-482 has been shown to be highly effective at antagonising R-type Ca^"^ currents, it can affect other types of Ca^"^ channels at higher concentrations. Thus, high concentrations of SNX-482 were found to partially block the L-type Ca^"^ channels (Bourinet et al., 2001) and the P/Q-type Ca^^ channels (Arroyo et al., 2003). To further investigate the role of R-type Ca^"^ channels in the observed inhibition of glutamate release by muscimol, we carried out experiments utilising synaptosomes prepared from the neocortex of mice lacking the alE subunit of these channels (Pereverzev et al., 2002). In the wild-type mice, 1 mM 4AP-evoked glutamate release was 35.5 ±1.3 nmol/mg/5 min under control conditions. In the presence of 200 |iM muscimol, glutamate release was reduced to 31.6 ± 0.4 nmol/mg/5 min (89% of control release in WT mice), similar to experimental results obtained using rat neocortical synaptosomes. Glutamate release was reduced to 29.8 ±1.1 nmol/mg/5 min (83.9% of the release in WT mice) in mice which lacked the alE gene, suggesting that these channels may play an important role in control of glutamate release. In the presence of 200 |iM muscimol, 4AP-evoked release from synaptosomes lacking alE containing channels was 29.1 ± 1.3 nmol/mg/5 min, indicating that muscimol-dependent inhibition of glutamate release was abolished (Fig. 47). In agreement with our experimental data obtained using SNX-482, these experiments demonstrate a key role of R-type Ca^"^ channel activity in the presynaptic GABAa receptor- mediated inhibition of glutamate release.

- 189 - 6.3.4 GABA a receptor-induced Inhibition of glutamate release is mediated by calmodulin

Calmodulin is a ubiquitous Ca^^-binding protein which plays a key role in Ca^^-dependent regulation of Ca^"^ channels involved in neurotransmitter release (Hailing et al., 2006). To determine if calmodulin, as the main Ca^"^ binding protein in nerve terminals, plays a role in muscimol-dependent inhibition of glutamate release, we incubated synaptosomes in the presence of the calmodulin inhibitor W7. Glutamate release triggered by 1 mM 4AP was 26.8 ± 0.7 nmol/mg/5 min in control conditions. In the presence of 200 p.M muscimol, glutamate release was reduced to 21.4 ± 0.8 nmol/mg/5 min (79.9% of control release). In the presence of 20 pM W7 4AP-evoked glutamate release was inhibited to 16.7 ±1.7 nmol/mg/5 min (62.3 % of control release). When muscimol was added to synaptosomes in the presence of 20 pM W7, glutamate release was 17.9 ±1.6 nmol/mg/5 min, which indicates that inhibition of glutamate release by muscimol was abolished in the presence of W7 (Fig. 48). These results suggest that Ca^Vcalmodulin-dependent signalling pathways play a critical role in GABAa receptor-mediated inhibition of glutamate release.

190- 30 Control W7 O) W7 + Muscimol 25 Muscimol

20

Muscimol 4AP Muscimol 4AP 10

Time (50 sec/division)

3 30

% 25 I I 20

tl5 s

Figure 48. Muscimol-induced inhibition of 4AP-evoked glutamate release is abolished in the presence of W7. Synaptosomes were incubated in the presence of 1 mM CaCl2, and in the absence or presence of W7 (20 jiM, blue and green) and/or muscimol (200 |LtM, red and blue) prior to the addition of 1 mM 4AP. Mean ± S.E.M. release was calculated at each time point but cumulative glutamate release values following 5 min of incubation with 4AP were used for the statistical analysis. Statistically significant pairs of data sets, p<0.05, are presented in grey (One-way ANOVA with post-hoc LSD test; n = 3). a. On-line glutamate release in the absence or presence of muscimol, W7 or muscimol/W7. b. Cumulative glutamate release following 5 min of incubation with 4AP in the absence or presence of muscimol, W7 or muscimol/W7.

- 191 - 6.4 Discussion

Activation of GABAa receptors present on glutamatergic terminals in the rat neocortex correlates with an increase in intraterminal Ca^"^ concentration under resting conditions, as demonstrated in Chapter 3. Conversely, activation of GABAa receptors also correlates with a decrease in evoked Ca^"^ influx, that triggers glutamate release, as described in Chapter 5, and therefore with a reduction in glutamate release as described in Chapter 4. The key question we attempted to address in this chapter is focused on the molecular mechanism(s) underlying the presynaptic GABAa receptor-mediated regulation of glutamate release.

Our results are consistent with the hypothesis that the intraterminal Cl" concentration is maintained at a level higher than expected for inwardly directed Cl movement to occur upon the activation of presynaptic GABAa receptors. The intraterminal Cl concentration is regulated by the activity of the cation-chloride co-transporters, among which, NKCCl transports Cl" into the cell. To test if this transporter plays a role in determining the functional outcome of presynaptic GABAa receptors we employed bumetanide to specifically block its activity. We found that bumetanide obviated the effects of muscimol on glutamate release, suggesting that GABAa receptors located on glutamatergic terminals are functionally coupled to NKCCl. Presynaptic GABAa receptors have previously been shown to cause depolarisation by a mechanism linked to NKCCl activity. For example, in other preparations, inhibition of this transporter diminishes the GABAa receptor-mediated facilitation of glutamate release in the locus coeruleus (Koga et al., 2005), and has also been shown to attenuate the muscimol-induced facilitation of spontaneous EPSCs in the ventromedial hypothalamus (Jang et al., 2001).

Activation of presynaptic GABAa receptors is therefore likely to cause a depolarisation of the plasma membrane, which could be too small to be observed by DiSC3(5) fluorescence, as shown in Chapter 5. GABAa receptor-mediated depolarisation, although insufficient to trigger glutamate release, could potently affect the voltage-gated ion channels involved in depolarisation of the nerve terminals upon the arrival of an action potential. For example, inactivation of voltage-gated Na^ channels has been proposed as a mechanism underlying

- 192- the presynaptic GABAa receptor-mediated inhibition of nerve terminal excitability in the posterior pituitary (Zhang and Jackson, 1995; Jackson and Zhang, 1995). Presynaptic shunting effect, as a consequence of increased in/out ion flow upon the activation of

GABA a receptors, may also play a role in the observed inhibition of release as reported previously. However, our experiments indicate that the inhibition of the evoked glutamate release observed in the presence of muscimol is dependent on the presence of Ca^"^ in the extracellular milieu at the time muscimol is added. These results support the hypothesis that depolarisation mediated by the presynaptic GABA a receptors may lead to Ca^"^ influx through VGCCs and an increase in intraterminal Ca^^ concentration. GABAa receptor- induced depolarisation has already been demonstrated to elevate the intracellular Ca^^ concentration via VGCC activation (Leinekugel et al., 1995; Obrietan and van den Pol, 1995; Owens et al., 1996). In order to elucidate the types of VGCC involved in this mechanism, glutamate release was measured in the presence of muscimol and specific VGCCs antagonists. Muscimol caused a reduction in glutamate release when N-, P/Q- and

T-type Ca^"^ channels were antagonised with co-CTxGVIA, co-AgaTxIVA and NiCl 2, respectively, suggesting that these channels are not directly coupled to the presynaptic GAB A A receptors. However, when the R- and L-type Ca^"^ channel antagonists SNX-482 and nifedipine were used, the effect of muscimol on glutamate release was eliminated, pointing to the involvement of these two subtypes of VGCCs in the GABA a receptor- mediated inhibition of glutamate release. We then studied the effects of muscimol on glutamate release in R-type Ca^'^ channel knock-out mice (Pereverzev et al., 2002). Muscimol had no effect on glutamate release from synaptosomes isolated from these mice, giving further support to our results using SNX-482, and providing the additional evidence that GABAa receptor-mediated attenuation of glutamate release occurs via functional coupling to R-type Ca^"^ channels.

In order to further test the role of Ca^"^ in the GABAa receptor-mediated inhibition of glutamate release, experiments were performed whereby Ca^'^ was substituted by Ba^^. Activation of VGCCs following membrane depolarisation resulted in an influx of Ba^^ triggering the release of glutamate (Nachshen and Blaustein, 1982; Augustine and Eckert, 1984), but the effect of muscimol was abrogated, supporting further the main role of Ca^'^ in this regulation.

193- The key question therefore arose regarding the nature of the Ca^'^-dependent mechanism underlying GABAa receptor-mediated inhibition of glutamate release. We found that the effects of muscimol were ablated in the presence of W7, an inhibitor of the Ca^"^ binding protein calmodulin, suggesting that the activation of calmodulin following binding of Ca^"^ is one of the key components in the mechanism of action of presynaptic GABAa receptors.

The seemingly paradoxical observation that the activation of presynaptic GABAa receptors causes a depolarisation of the plasma membrane, yet also results in an inhibition of the evoked glutamate release, could potentially be explained by the Ca^^-dependent inactivation (GDI) of Ca^"^ channels. In order to effectively control Ca^"^ signalling, Ca^'^ channels can rapidly inactivate in response to an increase in the intracellular Ca^^ level. Calmodulin has been shown to be the main Ca^"^ sensor responsible for GDI of P/Q-type VGGGs (Lee et al., 1999; Meyer et al., 2000; Lee et al., 2003) and L-type VGGGs (Peterson et al., 1999). Ga^^-dependent activation or inactivation of VGGGs in various neuronal preparations has been well documented. Importantly, depression of R-type Ga^^ channels by Ga^"^ influx through L-type channels in dendritic spines has been demonstrated (Yasuda et al., 2003). Although these types of Ga^"^ channel regulation can have profound effects on neurotransmitter release, little is currently known about the physiological significance of this regulation within the presynaptic nerve terminal (Forsythe et al., 1998).

Our results therefore suggest a possible mechanism, whereby a relatively high intraterminal Gl" concentration is maintained by the NKGGl transporter, leading to an efflux of Gl' and membrane depolarisation when presynaptic GABAa receptors are activated. This results in an activation of L-type Ga^^ channels, which are localised away from the active zone and therefore have no direct effect on neurotransmitter release (Stanley, 1997). However, L- type chaimels have a profound effect on the activity of other Ga^"^ channel types which are directly involved in neurotransmitter release. In the case of glutamatergic nerve terminals expressing the presynaptic GABAa receptors, the main target for Ga^^-dependent inactivation appear to be R-type VGGGs, which also directly participate in the release of glutamate from these terminals.

-194 6.5 Conclusions

• Presynaptic GABAa receptors are functionally coupled to NKCCl co-transporter

activity which maintains a relatively high intraterminal Cl concentration. GABAa receptor-mediated decrease in glutamate release is sensitive to bumetanide, an

NKCCl-specific inhibitor, suggesting that presynaptic GABAa receptor activation affects plasma membrane depolarisation.

Influx of Ca^"^ as a consequence of GABAa receptor-mediated depolarisation of nerve terminals is essential for the inhibition of glutamate release.

• Inhibition of glutamate release by presynaptic GABA a receptors does not involve the activity of LVA T-type and HVA N- and P/Q-types of VGCCs, based on the lack of an effect of specific blockers of these channels.

• Inhibition of glutamate release by presynaptic GABAa receptors is dependent upon the activity of L-type and R-type VGCCs, based on the sensitivity of this regulation to specific channel blockers, nifedipine and SNX-482.

• The effects of muscimol are abolished in the R-type Ca^^ chaimel knock-out mice,

further demonstrating that the inhibition of glutamate release by GABAa receptors involves the activity of the R-type VGCCs.

• The Ca^^-binding protein calmodulin plays an integral role in the GABAa receptor- mediated inhibition of glutamate release, suggesting that the underlying mechanism involves the Ca^^-dependent inactivation of R-type Ca^"^ channels.

195- CHAPTER SEVEN

-196 7. Discussion

The overwhelming majority of studies of GABAa receptors have concentrated on the actions of these receptors located on postsynaptic nerve terminals. The work presented in this thesis aimed to identify the presence of functional GABAa receptors on presynaptic nerve terminals of the rat neocortex and to elucidate their cellular actions. Presynaptic

GABAa receptors could be expected to be involved in the modulation of nerve terminal excitability and neurotransmitter release, playing a key role in maintaining the balance between excitation and inhibition in the mammalian brain.

Purified isolated nerve terminals (synaptosomes) were employed to investigate the presence of functional GABAa receptors at presynaptic sites in the rat neocortex and examine their ability to modulate presynaptic signalling pathways and neurotransmitter release. Synaptosomes provide a useful model system and have been extensively used for the study of various presynaptic receptors and signalling pathways in the absence of functional postsynaptic components. Initial biochemical studies indicated that the activation of presynaptic GABAa receptors correlated with an increase in intraterminal [Ca^^h. These studies also showed that in the presence of a relatively high concentration of GAB A present in the synaptosomal preparation, presynaptic GABAa receptors were tonically active.

Additionally, agonists and antagonists of GABAa receptors produced bimodal effects in these assays. Immunohistochemical analysis of CaMK Il-phosphorylated synapsin I distribution in the rat neocortex demonstrated that this phospho-form of synapsin I is localised exclusively to glutamatergic nerve terminals, indicating that the biochemical changes observed in response to GABAa receptor ligands also occur specifically in glutamatergic nerve terminals. Following depolarisation of synaptosomes to trigger glutamate release in the presence of GABAa receptor-specific agonist muscimol, a decrease in intraterminal [Ca^^ji. was detected using fura-2 ratiometric assay. In correlation with these changes, GABAa receptor activation by muscimol caused a dose-dependent inhibition of glutamate release, which could be pharmacologically ablated using GABAa receptor antagonist picrotoxin. GABAa receptor-mediated inhibition of glutamate release was found to depend upon the activity of cation-chloride cotransporter NKCCl which actively transports chloride into the nerve terminals, suggesting that the activation of these

- 197 receptors leads to a plasma membrane depolarisation which is not sufficiently strong to evoke release. The effects of GABAa receptor activation on glutamate release also depend on the presence of Ca^"^ in the extrasynaptosomal milieu which suggested that activation of these receptors leads to a depolarisation-dependent influx of Ca^^. Using specific blockers of subtypes of voltage-gated Ca^"^ channels, muscimol-induced decrease in glutamate release was found to depend on the activity of L- and R-type Ca^"^ channels which play distinct roles in the regulation of glutamate release. Downstream of Ca^^ influx, the inhibition of glutamate release was dependent upon the activity of Ca^^ binding protein calmodulin, suggesting that Ca^^-dependent inhibition of Ca^"^ channels is an important component of the molecular mechanism underlying the observed effects of presynaptic

GABAa receptors.

7.1 Presynaptic GABAa receptors modulate intraterminal Ca^^

We initially identified the presence of GABAa receptor al, a2, P2, P3 and y2 subunits in purified synaptosomes, in addition to the presynaptic markers GAD and synapsin. However, it is quite possible that contamination of this preparation with non-functional postsynaptic elements could be responsible for some of these results. Despite this possibility, our results correlate well with the ultrastructural studies which reported the presence of presynaptic GABAa receptors on mossy fibre terminals containing the a2 subunit (Ruiz et al., 2003) and a l and P2/3 subunits on glutamatergic terminals in the olfactory bulb (Panzanelli et al., 2004).

Intraterminal Ca^"^ levels are known to be regulated by the presynaptic GABAa receptor activity in some brain regions (Ruiz et al., 2003, Turucek and Trussell, 2001}. The observed changes in [Ca^‘^]i in the presence of GABAa receptor agonists/ antagonists seen in our biochemical experiments, as manifested by changes in synapsin I P-site 3 phosphorylation, suggest that activation of GABAa receptors in neocortical synaptosomes causes a nerve terminal depolarisation and Ca^^ influx. These findings are further confirmed by the complementary changes in the phosphorylation state of the CaMK I/IV- dependent P-site 1 of synapsin I, and in phosphorylation of the a and P isoforms of CaMK II. Isoguvacine and bicuculline produced comparative results to muscimol and

198 - picrotoxin/GABase respectively confirming that the observed changes in presynaptic Ca^"^- dependent signalling pathways are the consequence of the activity of the presynaptic

GABAa receptors rather than the activity of presynaptic GAB Ac or GABAb receptors.

GABAa receptor-mediated depolarisation and modulation of [Ca^'^Ji are well documented. In developing and injured neurons, GABAa receptor activation leads to a depolarisation of the plasma membrane (Owens et al., 1996; Chen et al., 1996), which is also the case with presynaptic GABAa receptor activation in the auditory brainstem {Turucek and Trussell, 2001), ventromedial hypothalamus (Jang et al., 2001), locus coeruleus (Koga et al., 2005), posterior pituitary (Zhang and Jackson, 1995) and hippocampus (Fassio et al., 1999; Ruiz et al, 2003; Jang et al., 2005, Jang et al., 2006). In some studies, GABAA-induced depolarisation has been demonstrated to elevate intracellular [Ca^^i via VGCC activation (Leinekugel et al., 1995; Obrietan and van den Pol, 1995; Owens et al., 1996; Turucek and Trussell, 2001; Ruiz et al., 2003}.

Under basal conditions as well as during the release of neurotransmitters from synaptosomes, presynaptic GABAa receptors subtypes which have a relatively high affinity for agonists are expected to be in an active state. Hence, the addition of GABAa receptor agonists under these conditions could be expected to activate the subtypes of presynaptic

GABAa receptors which demonstrate a relatively low affinity for agonists. The subunit composition of GABAa receptors can considerably alter their functional properties as well as their cell surface distribution and dynamic regulation (Revers and Luddens, 1998). The apparent bimodal dose responses seen with both agonists and antagonists of GABAa receptors in our biochemical assays lead us to hypothesise that at least two populations of

GABAa receptors reside on presynaptic neocortical nerve terminals likely composed of different subunit combinations. In addition, the effects of GABA a receptor antagonists leading to a decrease in Ca^^-dependent phosphorylation of synapsin I under basal conditions indicate that presynaptic GABAa receptors may be tonically active. Tonic activity of GABAa receptors has been described in various brain regions (Farrant and Nusser, 2005) and is believed to play a critical role in network excitability and information transfer (Farrant and Nusser, 2005).

- 199- Following the release of GAB A into the synaptic cleft its concentration can reach millimolar levels (Nusser et al., 2001). Depending on the activity of GAB A uptake mechanisms, GAB A can freely diffuse over some distance and possibly reach receptors present on neighboring nerve terminals, resulting in a sufficient concentration to activate presynaptic GABAa auto- and heteroreceptors. GAB A present in the extracellular space may originate from a variety of sources, including release from astrocytes (Kozlov et al., 2006) and reversal of transporter activity (Richerson and Wu, 2003), in addition to action potential-mediated release (Attwell et al., 1993; Brickley et al., 1996). It is possible that presynaptic GABAa receptors are located at some distance from the synaptic cleft and therefore exposed to the spill-over of GABA released from the neighbouring terminals.

Although our biochemical assays strongly suggest that presynaptic GABAa receptor activity causes an increase in intraterminal [Ca^'^Ji, which is consistent with plasma membrane depolarisation, our attempts to directly monitor changes in membrane excitability using the voltage-sensitive dye DiSCsCS) showed no detectable effect of

GABAa receptor agonists. We postulate that the relatively low sensitivity of these assays was at least one of the reasons for these results. The activation of presynaptic GABAa receptors is likely to cause only a small depolarisation of the plasma membrane which is not sufficient to trigger neurotransmitter release but may be potent enough to modulate the activity of voltage-gated ion channels involved in this process. An alternative explanation is that GABAa receptors are present only on a small population of glutamatergic nerve terminals. Glutamatergic terminals correspond to roughly 60 % of terminals in the synaptosome preparation (Beaulieu, 1993). However, even if presynaptic GABAa receptors are also present on GABAergic nerve terminals, it is entirely possible that these receptors could have an opposite effect on the membrane excitability of those terminals. This could therefore mask the changes observed in the population of glutamatergic terminals. It would be of interest to develop a method which would allow us to separate various types of terminals according to their neurotransmitter content but currently no such procedure has been successfully applied.

Although the same limitations apply to measurement of [Ca^'^Ji using fura-2, the analysis carried out using values of intraterminal [Ca^^Ji changes obtained in the presence of muscimol normalised with the corresponding control values in the absence of muscimol,

- 200 - has revealed a muscimol-dependent inhibition of [Ca^^]i influx triggered by 4AP. The reduction of Ca?'^ influx in the presence of muscimol was also apparent but statistically insignificant when total levels of intraterminal were compared directly. We postulate that at least one of the reasons for this apparent discrepancy in analysis originates from a high level of the background noise observed in our experiments. Fura-2 based fluorescent assay allow us to measure global changes in intraterminal [Ca^^Ji within the whole nerve terminal, whereas VGCC activation and Ca^^ influx postulated to occur downstream of presynaptic GABAa receptor are likely to occur locally in the vicinity of the plasma membrane. We hypothesise that, based on these results, GABAa receptor activation leads to a small depolarisation of the plasma membrane sufficient to activate only a small proportion of VGCCs present in synaptosomes. This is consistent with our finding that the increase in Ca^^-dependent phosphorylation of synapsin I by CaMK II in response to muscimol or isoguvacine is relatively small in comparison with the increase caused by 4AP-triggered depolarisation of nerve terminals, as shown in Chapter 3.

7.2 Presynaptic GABAa receptors are localised to glutamatergic terminals where they inhibit glutamate release

Immunolocalisation studies presented in Chapter 3 showed that CaMK Il-dependent phosphorylation of synapsin I at P-site 3 occurs exclusively in glutamatergic nerve terminals. As mammals possess multiple synapsin genes, which are phosphorylated and dephosphorylated at multiple sites by several kinases and phosphatases, it is possible that synapsins are responsible for modulating synaptic transmission in excitatory and inhibitory nerve terminals in varying ways. Indeed, functional experiments using synapsin-depleted mice have revealed that synapsins are necessary for regulating the reserve pool of glutamatergic synaptic vesicles but modifying the size of the readily releasable pool of GABAergic synaptic vesicles (Gitler et al., 2004). The exclusive localisation of P-site 3 phospho-synapsin I to glutamatergic nerve terminals, lead us to investigate the effects

GABAa receptor activation on evoked glutamate release from synaptosomes.

Activation of presynaptic GABA a receptors by muscimol or isoguvacine caused a dose- dependent inhibition of glutamate release from neocortical synaptosomes. The lack of

-201 - GABAa receptor-mediated effects on basal levels of release indicates that GABAa receptor-mediated depolarisation may be relatively small. This finding, together with the observed decrease in intraterminal [Ca^'^Ji detected only following depolarisation with 4AP, suggests that the effects of presynaptic GABAa receptors are only apparent and therefore relevant in the presence of action potential-mediated depolarisation. While the inhibition of glutamate release by muscimol was blocked by the addition of picrotoxin, picrotoxin alone produced no effect on release suggesting that GABAa receptors are not tonically active under these experimental conditions. It is worth noting however that the concentration of synaptosomal suspension in these experiments was more than 10 times lower than the concentration of synaptosomal suspension used in biochemical experiments, and as such, contained proportionally lower concentration of ambient GABA.

Presynaptic GABAa receptors have already been shown to operate in some glutamatergic synapses in the hippocampus where they also cause a presynaptic depolarisation. GABAa receptors containing the a2 subunit cause a decrease in action potential-induced Ca^^ influx in mossy fibre terminals (Ruiz et al., 2003), while activation of presynaptic GABAa receptors in Schaffer collateral afferents and CAB pyramidal neurones facilitates glutamate release (Jang et al., 2005; Jang et al., 2006). Our studies showed that muscimol could produce an inhibition of glutamate release from hippocampal synaptosomes, which was blocked by picrotoxin. However, our experiments also indicate that muscimol had no effect on glutamate release from cerebellar synaptosomes, suggesting that presynaptic

GABAa receptors are not present on glutamatergic nerve terminals in this brain region.

Presynaptic GABAa autoreceptors have previously been described in the cerebellum, although their effect on release is unknown (Pouzat and Marty, 1999).

Although the activation of GABAa receptors by muscimol has consistently inhibited glutamate release evoked by 1 mM 4AP, differing effects were obtained when the release was evoked by 10 mM versus 30 mM KCl. In the presence of 10 mM KCl, the activation of GAB A A receptors caused a reduction to about 76 % of control levels of release which was consistent with the effect of these receptors on 4AP-evoked release. In contrast, muscimol had no effect on 30 mM KCl-evoked glutamate release. This discrepancy could be explained by the difference in mechanisms of release evoked by these compounds. In the case of 4AP-evoked depolarisation, the resultant destabilised membrane potential

- 202 - causes TTX-sensitive, stochastic activation of voltage-dependent Na'^ channels (Tibbs et al, 1989). Release evoked by 10 mM KCl is also partially TTX sensitive, suggesting that depolarisation by this compound also involves voltage-dependent Na^-channels (Kidokoro and Ritchie, 1980). However, release triggered by 30 mM KG is insensitive to TTX and therefore believed to be unphysiological. In addition, the effect on plasma membrane potential is substantially larger causing “clamped” activation of voltage-gated ion channels, and, as such, it is likely to supersede any modulatory effects which involve the activity of these channels. It has been suggested that glutamate release evoked by KCl may involve a complete fusion of synaptic vesicles, whereas glutamate release evoked by 4AP may have two components, one of which involves a complete synaptic vesicle fusion triggered by 1 0 mM KCl. The second phase, which is also PKC-dependent, may involve “kiss-and-run” type of fusion in addition to complete synaptic vesicle fusion (Cousin and Robinson, 2000). However, it has also been suggested that the release by both agents is biphasic, with a rapid phase representing the release of readily releasable vesicles at the active zone, followed by a more extensive slow phase representing the reserve pool of vesicles (McMahon and Nicholls, 1991).

7.3 Presynaptic GABAa receptors are acted upon by allopregnanolone but not diazepam

In our studies the inhibition of glutamate release by presynaptic GABAa receptor activation was found to be insensitive to low concentrations of diazepam. This may indicate that this population of presynaptic GABAa receptors is composed of a4 and/or a 6 subunits, as diazepam has no effect on these subunits (Wieland et al., 1992; Hadingham et al., 1996;

Wafford et al., 1996; Davies et al., 1998). However, a 6 -containing receptors are thought to be confined to the granule cell layer of the cerebellum (Nusser et al., 1996). Alternatively, it may be that higher concentrations of diazepam are required to modulate the activity of presynaptic GABAa receptors in the neocortex. It would be of interest to examine the effects of other benzodiazepines on muscimol-induced inhibition of glutamate release. As some of the benzodiazepines are more selective for specific subtypes of GABAa receptors (Korpi et al., 2002), the results of these experiments may provide us with a clue regarding the subunit composition of the receptor subtypes expressed presynaptically. In addition, if

203 - the modulation of glutamate release by any of these compounds could be established this would indicate that presynaptic GABAa receptors contain the y subunit, which is required for benzodiazepine binding.

We have investigated whether neurosteroids, which represent another class of GABAa receptor allosteric modulators, can regulate glutamate release either in the absence or presence of muscimol. Allopregnanolone is known to act as a positive allosteric modulator of GABAa receptors as well as a GABAa receptor agonist. High concentrations of allopregnanolone were found to inhibit glutamate release in the absence of GABAa receptor agonists and further decrease glutamate release in the presence of muscimol. Neurosteroids display considerably lower subunit specificity for GABAa receptors than benzodiazepines (Belelli et al., 2002). However, it has been suggested that allopregnanolone has a lower affinity for GABAa receptors which contain the a4 or a 6 subunit (Belelli et al., 2002). Additionally, allopregnanolone was suggested to affect more profoundly GABAa receptors which incorporate the 5 subunit in combination with a l or a4 subunits (Belelli et al., 2002; Brown et al., 2002; Wohlfarth et al., 2002). Our results suggest that allopregnanolone can act as both an agonist of presynaptic GABAa receptors, as well as an allosteric modulator of their function. .

7.4 Presynaptic GABAa receptors may co-localise with GABAb receptors

The lack of additive effects of presynaptic GABAa and GABAb receptors activation when specific agonists of these receptors were used in combination in our glutamate release experiments suggests that GABAa and GABAb receptors may be located on the same population of glutamatergic terminals. What would be the physiological significance of this co-localisation? GABAb receptor activation causes an inhibition of glutamate release by inactivating N- and P/Q-type Ca^^ chaimels which are directly involved in neurotransmitter release (Huston et al., 1995; Chen and van den Pol, 1998). Our experiments demonstrate that GABAa receptors inhibit glutamate release by regulating the activity of L- and R-type Ca^'*’ channels. Given that GABAb receptors are known to have a higher affinity for GABA than GABAa receptors, this suggests that GABAa receptor-

204 - mediated inhibition of glutamate release may occur during extremely high neuronal network activity, when GABAb receptors become desensitised due to prolonged activation. This desensitisation could be postulated to occur due to G-protein coupling or other downstream effector mechanisms of GABAb receptors (Kushner and Unterwald, 2001; Amantea and Bowery, 2004). If GABAa receptors regulate primarily the activity of R-type Ca^^ channels which contribute significantly less to the release mechanisms than N- and P/Q-type Ca^"^ channels, the activation of GABAb receptors immediately prior to the activation of GABAa receptors may preclude the detection of GABAa receptor-mediated effects. Alternatively, GABAa receptors may reside on a relatively small population of glutamatergic nerve terminals in comparison with GABAb receptors. If this was the case, the effects of GABAa receptors may be difficult to detect because of the prominent inhibitory influences mediated by GABAb receptors. It is also possible that GABAa and GABAb receptors are co-localised to the same population of terminals where they are involved in a direct protein-protein interaction. GABAa receptors have been shown to directly interact with dopamine D 5 receptors, modulating both GABAa and Dg-mediated actions independently of established G-protein coupled receptor activation and intracellular signalling pathways (Liu et al., 2 0 0 0 ). GABAa receptors have been shown to directly interact with GABAb receptors, possibly playing a role in regulation of GABAb receptor trafficking (Balasubramanian et al., 2004).

7.5 Presynaptic GABAa receptors are functionally coupled to NKCCl and L- and R-type VGCCs

The GABAa receptor-mediated depolarisation which was suggested to occur based on our biochemical experiments could mean that the [Cl']i of glutamatergic nerve terminals containing these receptors is maintained at a relatively high level. This would be expected to result in outwardly directed Cl' movement upon the activation of GABAa receptors. We hypothesised that elevated [Cl']i in these terminals was due to the activity of NKCCl cation-chloride cotransporter which actively transports Cl' into the cell. Inhibiting this transporter would therefore be expected to negate the effects of muscimol on glutamate release. Indeed, when glutamate release was measured in the presence of bumetanide, a specific inhibitor of NKCCl, the inhibition caused by muscimol was abrogated, confirming

- 205 - our hypothesis. Presynaptic GABAa receptors have previously been shown to cause depolarisation by a mechanism linked to NKCCl activity. Inhibition of this transporter diminishes the GABAa receptor-mediated facilitation of glutamate release in the locus coruleus (Koga et al., 2005), and has also been shown to attenuate muscimol-induced spontaneous EPSC facilitation in the ventromedial hypothalamus (Jang et al., 2001). Experimental evidence indicates that depolarisation of nerve terminals due to activation of presynaptic GABAa receptors can lead to either inhibition (Saridaki et al., 1989; Zhang and Jackson, 1993; Rudomin and Schmidt, 1999) or facilitation (Jang et al., 2001; Turecek and Trussell, 2001) of transmitter release, suggesting that this divergence of modulation depends on the presence of different molecular targets downstream of GABAa receptor activation and plasma membrane depolarisation in different types of terminals and/or different brain regions.

Addition of muscimol in the absence of extracellular Ca^^ abolishes GABAa receptor- mediated inhibition of glutamate release, suggesting that Ca^"^ influx through VGCCs is essential for this inhibition to occur. In order to confirm the role of Ca^^ in this regulation, experiments were performed whereby Ca^"^ was substituted by Ba^^, which can trigger neurotransmitter release but has no effect on Ca^^-dependent signalling in nerve terminals (Naschen and Bluastein, 1982; Augustine and Eckhart, 1984). Indeed, in the presence of

Ba^^, the activation of GABAa receptors had no effect on glutamate release. This experimental evidence indicates that Ca^"^ influx through VGCCs due to GABAa receptor- mediated depolarisation is a key component of the mechanism leading to inhibition of glutamate release.

Muscimol had no effect on glutamate release when the release was triggered by an increase in [Ca^^]i mediated by Ca^^ ionophore ionomycin. As ionomycin evokes glutamate release by a method which bypasses membrane depolarisation and ion channel activation, this indicates that an interaction downstream of ion channel activation, for example with the release machinery, is unlikely to provide the mechanism by which GABAa receptors inhibit release. Instead, they appear to modulate the activity of voltage-gated ion channels which play a role in stimulus/exocytosis coupling during neurotransmitter release.

-206 In order to elucidate the types of VGCC which mediate Ca^^ influx caused by GABAa receptor-depolarisation, glutamate release was measured in the presence of muscimol and specific antagonists of individual subtypes of VGCCs. Muscimol caused a reduction in glutamate release in the presence of co-CTxGVIA, co-AgalVA and NiCli respectively, suggesting that N-, P/Q- and T-type Ca^'^ channels are not directly affected by the activation of presynaptic GABAa receptors. However, muscimol had no effect on glutamate release in the presence of nifedipine and SNX-482, pointing to the involvement of L- and R-type Ca^"^ channels, respectively, in GABAa receptor-mediated inhibition of glutamate release.

It has been demonstrated that the influx of Ca^^ through L-type VGCCs during repetitive stimulation is involved in the enhancement of GABA release from cultured GABAergic neurones (Jensen et al., 1999). L-type VGCCs also appear to be present in large mossy fibre terminals where they mediate large Ca^^ currents during activation (Tokunaga et al., 2004). It is therefore feasible that L-type VGCCs are also able to take part in a presynaptic regulatory mechanism activated by presynaptic GABAa receptor at cortical glutamatergic synapses. Although L-type Ca^^ channels channels are mainly located at the soma and dendrites (Westenbroek et al., 1990), a small number of channels in the presynaptic nerve terminal could potentially cause a substantial increase in [Ca^^]i, due to the high surface area to volume ratio (Tokunaga et al., 2004).

The involvement of R-type Ca^^ channels in this process was further supported by the ablation of the muscimol-dependent inhibition of glutamate release from synaptosomes prepared from the cortex of R-type Ca^"^ channel knock-out mice (Pereverzev et al., 2002). In these mice glutamate release evoked by 4AP was reduced in comparison with the release from the wild-type mice, indicating that Ca^"^ influx through R-type VGCCs contributes directly to the release together with N- and P/Q-subtypes of VGCCs. R-type channels are known to regulate neurotransmitter release in the calyx of Held, although with lower efficacy than other types of Ca^^ channel. In this synapse, R-type Ca^"^ chaimels supply approximately 25% of the Ca^^ current (Wu et al., 1998). R-type VDCCs are also involved in glutamatergic transmission in the rat hippocampus (Gasparini et al., 2001) and in oxytocin secretion in neurohypophysial terminals (Wang et al., 1999). The contribution of both L- and R-type Ca^"^ channels to neurotransmitter release has been also shown in

-207 Meynert neurones. In these cells, L-type Ca^"^ channels play a role in spontaneous release of GABA, while evoked release requires the involvement of R-type VGCCs (Rhee et al., 1999). Unfortunately, direct electrophysiological recordings from either intact nerve terminals in the neocortex or from neocortical synaptosomes are impossible with the currently available techniques due to the small size of terminals. It is therefore impossible to unequivocally confirm the activity of these channels following GABAa receptor activation by directly measuring presynaptic Ca^^ currents.

7.6 Calmodulin is implicated in Ca^^-dependent inactivation of VGCCs following presynaptic GABAa receptor activation

An obvious question arising from our experimental data is in regard to the nature of the

Ca^^-dependent mechanism by which presynaptic GABAa receptors produce their inhibitory effects in glutamate release? We found that the inhibition of release caused by muscimol was ablated in the presence of W7, suggesting that activation of calmodulin by binding of Ca^^ is a key component of the mechanism of action of presynaptic GABAa receptors. The seemingly paradoxical observation that activation of presynaptic GABAa receptors causes depolarisation of the plasma membrane yet results in an inhibition of evoked glutamate release could potentially be explained by Ca^^-dependent inactivation of Ca^"^ channels. It is well established that in order to effectively control Ca^"^ signalling, Ca^"^ channels can rapidly inactivate in response to an increase in intracellular Ca^'^ levels. In the majority of cases this process is mediated by a direct binding of calmodulin to the voltage-gated Ca^"^ channels.

In the calyx of Held, small depolarisations have been shown to facilitate neurotransmitter release, whereas depolarisations greater than -60 mV produced varied effects, sometimes causing an inhibition in release (Awatramani et al., 2005). While larger depolarisations can inhibit release by inactivating Na"^ and/or Ca^^ channels (Graham and Redman, 1994; Zhang and Jackson, 1995), facilitation of released may occur due to a weak activation of VGCCs and the subsequent small increase in basal Ca^^ levels (Awatramani et al., 2005}. However, it is also possible that if this increase in Ca^"^ is large enough to result in Ca^"^- dependent facilitation of VGCCs, it is also large enough to instigate Ca^^-dependent

-208 inactivation of VGCCs depending on the composition of Ca^"^ channels present in various types of presynaptic nerve terminals.

Ca^^-dependent activation, deactivation, and inactivation of various subtypes of Ca^"^ channels have been well documented. Although these properties will affect overall Ca^"*" influx, their physiological significance at presynaptic terminals is currently unknown (Forsythe, 1998). Calmodulin has been shown to play a key role as a Ca^^ sensor responsible for Ca^^-dependent inactivation of the P/Q-type (Lee et al., 1999; Lee et al., 2000; Lee et al., 2003) and L-type VGCCs (Peterson et al., 1999). In has been demonstrated that the binding of Ca^^ to calmodulin, which is associated with VGCCs, initiates processes which lead to the channel closure. Ca^^-dependent inactivation of R- and N-type channels has also been reported. Inactivation of these channels occurs due to Ca^"^ binding to the N-terminal: lobe of calmodulin, which is associated with these channels (Liang et al., 2003).

Although it has been well documented that Ca^^-dependent inactivation of VGCCs occurs as a result of a direct interaction with calmodulin, Ca^Vcalmodulin-dependent phosphorylation and dephosphorylation of these channels also regulates their activity. Calcineurin/PP2B is known to be rapidly activated following Ca^'^ influx into the nerve terminal (Sihra et al., 1992). The major targets for calcineurin-mediated inhibition of Ca^"^ influx appear to be high voltage-gated types of Ca^"^ channels, apparently via a direct interaction (Burley and Sihra, 2000). However, the functional outcome of the calcineurin- mediated dephosphorylation of VGCCs is controversial. While some studies have suggested a negative feedback regulation of VGCC activity by calcineurin (Kalman et al., 1988; Lukyanetz et al., 1998: Burley and Sihra, 2000), this has been disputed by others (Victor et al., 1997; Zeilhofer et al., 2000).

Activity of R-type VGCCs has been shown to be depressed by L-type VGCC-mediated elevations of Ca^"^ in the hippocampus, despite L-type Ca^'^ channels supplying only a small proportion of Ca^"^ influx in these experiments (Yasuda et al., 2003). It was postulated that Ca^"^ could still reach sufficient levels in microdomains close to the channel mouth to trigger Ca^^-dependent reactions. Depression of R-type VGCCs was shown to be dependent on the activation of CaMK II, as a consequence of Ca^'^ influx through the L-

- 209 - type channels (Yasuda et al., 2003). Alternatively, the L-type channels can directly transduce voltage-dependent changes to the other types of Ca^^ channels. This has been observed in skeletal muscles, where the L-type Ca^^ channels enhance the activity of ryanodine receptors releasing Ca^"^ from the intracellular stores (Nikai et al., 1996). It is possible that L-type Ca^"^ channels and CaMK II together with calmodulin can exist as part of the same signalling complex (Yasuda et al., 2003).

7.7 Physiological implication for inhibition of glutamatergic transmission by presynaptic GABAa receptors

GABAa receptor-mediated inhibition of glutamate release presented in this thesis could have numerous physiological and pathophysiological implications. As the major inhibitory neurotransmitter in the brain, GABA maintains the inhibitory tone that counterbalances neuronal excitation.

One consequence of altering the balance between excitation and inhibition could be seizures. Therefore, activation of presynaptic GABAa receptors which cause inhibition of glutamate release could be influential in suppressing epileptogenic activity in the neocortex. Experimental and clinical evidence indicates that GABA plays an important role in the mechanism and treatment of epilepsy (Treiman, 2001).

Excitotoxicity due to a high activity of NMDA receptors contributes to the neuronal cell death in brain injuries, including stroke (Choi et al., 1985), leading to necrosis (Rothman 1985) or apoptosis (Lesort et al., 1997). Therefore the inhibition of glutamate release by activation of GABAa receptors could provide a mechanism which protects against this type of neuronal degeneration.

It is well established that alterations in GABAergic neurotransmission following chronic exposure to benzodiazepines and barbiturates can contribute to the symptoms of tolerance, dependence and withdrawal (Allison and Pratt, 2003). The mechanisms of these changes are believed to be closely linked to the dose of benzodiazepines and the duration of use. It may therefore be possible to produce neuronal inhibition by developing drugs that target

- 210 - different components of the signalling pathway described, ie NKCCl, R- and L-type VGCCs or calmodulin, in combination with GABA mimetic drugs which would potentially alleviate the problems associated with their prolonged application.

7.8 Conclusions

In summary, work presented in this thesis has demonstrated that GABAa heteroreceptors are present on a population of glutamatergic nerve terminals in the rat neocortex. The activation of these receptors causes an inhibition of glutamate release increasing further the tone of synaptic inhibition in the neocortex. We propose a model whereby activation of presynaptic GABAa receptors leads to an efflux of Cl" from the nerve terminals and depolarisation of the plasma membrane, as a consequence of a high [Cl"]i maintained by the NKCCl activity. Membrane depolarisation activates L-type VGCCs, elevates the intraterminal [Ca^^], and subsequently, via an interaction with calmodulin, inactivates R- type VGCCs which are directly coupled to the release of glutamate. We summarise the proposed molecular mechanism underlying the inhibition of glutamate release by presynaptic GABAa receptors in Figure 49.

-211 CaMKII-dependent NaVK Synapsin I Phosphorylation NKCCl ©-■ Cl Depolarisation Glutamate Release I

N P Q

Ca2+

Figure 49. Molecular mechanism underlying the presynaptic GABAa receptor-mediated inhibition of glutamate release. GABA a heteroreceptors are present on glutamatergic nerve terminals in the neocortex. Their activation results in Cl' efflux due to NKCCl activity, causing a depolarisation of the plasma membrane. This triggers opening of L-type VGCCs, activation of calmodulin and a consequent Ca^'^-dependent inactivation of R-type VGCCs. The resultant decrease in Ca^^ influx causes a decrease in evoked glutamate release.

-212- 7.9 Future Work

Do presynaptic GABAa receptors act as autoreceptors regulating the release of GABA in the rat neocortex? GABAa receptor modulation of GABA release has been demonstrated in the suprachiasmatic nucleus, hippocampus, cerebellum and substantia nigra. If GABAa receptors are indeed present on GABAergic terminals in the neocortex, they would conceivably be expected to facilitate release, as disinhibition appears to be counter-intuitive. The modulation of GABA release from neocortical synaptosomes can be investigated using assays based on high- pressure liquid chromatography.

What is the subunit composition of presynaptic GABAa receptors? This question could be addressed by measuring glutamate release and intraterminal [Ca^^]i in the

presence of various allosteric modulators of GABAa receptors which have been shown to selectively modulate specific subtypes of these receptors. For example, THIP could be used to ascertain the presence of 6 subunits (Adkins et al., 2001). A number of compounds show specificity towards various a subunit subtypes such as pCCt for the a l subunit (Cox et al., 1995), TPA 003 for the a3 subunit (Dias et al., 2005). Insensitivity to would however indicate the presence of o5 subunit in these receptors (Damgen and Luddens, 1999). Immunolocalisation experiments could be carried out to demonstrate the co-localization of pharmacologically identified subunits with glutamatergic/GABAergic markers and P-site 3 synapsin in neocortical brain slices.

Does the activity of presynaptic GABAa receptors regulate the trafficking and exocytosis of small synaptic vesicles? Styryl dyes such as FM I-43 could be used to

monitor GABAa receptor-mediated exocytosis of synaptic vesicles from glutamatergic nerve terminals. Using this methodology, we can also assess if the observed changes in intraterminal [Ca^'^Ji differentially affect various synapsin vesicle pool as well as synaptic vesicle endocytosis in presynaptic nerve terminals.

213 - Reference List

Adkins CE, Pillai GV, Kerby J, Bonneit TP, Haldon C, McKernan RM, Gonzalez JE, Oades K, Whiting PJ, Simpson PB (2001) a4p3Ô GABAa receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential. J Biol Chem 276:38934-38939.

Aguayo LG, Peoples RW, Yeh HH, Yevenes GE (2002) GABAa receptors as molecular sites of ethanol action. Direct or indirect actions? Curr Top Med Chem 2:869-885.

Akabas MH (2004) GABAa receptor structure-function studies: a re-examination in light of new acetylcholine receptor structures. Int Rev Neurobiol 62:1-43.

Akk G, Bracamontes J, Steinbach JH (2001) sulfate block of GABAa receptors: mechanism and involvement of a residue in the M2 region of the a subunit. J Physiol 532:673-684.

Albillos A, Neher E, Moser T (2000) R-Type Ca^^ channels are coupled to the rapid component of secretion in mouse adrenal slice chromaffin cells. J Neurosci 20:8323-8330.

All AB, Rossier J, Staiger JF, Audinat E (2001) Kainate receptors regulate unitary IPSCs elicited in pyramidal cells by fast-spiking intemeurons in the neocortex. J Neurosci 21:2992-2999.

Alldred MJ, Mulder-Rosi J, Lingenfelter SE, Chen G, Luscher B (2005) Distinct y2 subunit domains mediate clustering and synaptic function of postsynaptic GABA a receptors and gephyrin. J Neurosci 25:594-603.

Allgaier C, Wamke P, Stangl AP, Feuerstein TJ (1995) Effects of 5-HT receptor agonists on depolarization-induced [^H]-noradrenaline release in rabbit hippocampus and human neocortex. Br J Pharmacol 116:1769-1774.

Allison C, Pratt JA (2003) Neuroadaptive processes in GABAergic and glutamatergic systems in benzodiazepine dependence. Pharmacol Ther 98:171-195.

Amantea D, Tessari M, Bowery NG (2004) Reduced G-protein coupling to the GABAb receptor in the nucleus accumbens and the medial prefrontal cortex of the rat after chronic treatment with nicotine. Neurosci Lett 355:161-164.

Ambrosio AF, Malva JO, Carvalho AP, Carvalho CM (1997) Inhibition of N-,P/Q- and other types of Ca^"^ channels in rat hippocampal nerve terminals by the adenosine Al receptor. Eur J Pharmacol 340:301-310.

Anderson WM, Delinck DL, Benninger L, Wood JM, Smiley ST, Chen LB (1993) Cytotoxic effect of thiacarbocyanine dyes on human colon carcinoma cells and inhibition of bovine heart mitochondrial NADH-ubiquinone reductase activity via a rotenone-type mechanism by two of the dyes. Biochem Pharmacol 45:691-696.

Anggono V, Smillie KJ, Graham ME, Valova VA, Cousin MA, Robinson PJ (2006) Syndapin 1 is the phosphorylation-regulated dynamin 1 partner in synaptic vesicle endocytosis. Nat Neurosci 9:752-760.

Arbilla S, Kamal L, Langer SZ (1979) Presynaptic GABA autoreceptors on GABAergic nerve endings of the rat substantia nigra. Eur J Pharmacol 57:211-217.

-214 Armstrong DL (1989) Calcium channel regulation by calcineurin, a Ca^^-activated phosphatase in mammalian brain. Trends Neurosci 12:117-122.

Arrigoni E, Rainnie DG, McCarley RW, Greene RW (2001) Adenosine-mediated presynaptic modulation of glutamatergic transmission in the laterodorsal tegmentum. J Neurosci 21:1076-1085.

Arroyo G, Aldea M, Fuentealba J, Albillos A, Garcia AG (2003) SNX482 selectively blocks P/Q Ca^^ channels and delays the inactivation of Na"^ channels of chromaffin cells. Eur J Pharmacol 475:11-18.

Attwell D, Barbour B, Szatkowski M (1993) Nonvesicular release of neurotransmitter. Neuron 11:401-407.

Augustin I, Rosenmund C, Sudhof TC, Brose N (1999) Muncl3-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400:457-461.

Augustine GJ (2001) How does calcium trigger neurotransmitter release? Curr Opin Neurobiol 11:320-326.

Augustine GJ, Eckert R (1984) Divalent cations differentially support transmitter release at the squid giant synapse. J Physiol 346:257-271.

Awatramani GB, Price GD, Trussell LO (2005) Modulation of transmitter release by presynaptic resting potential and background calcium levels. Neuron 48:109-121.

Axmacher N, Draguhn A (2004) Inhibition of GABA release by presynaptic ionotropic GABA receptors in hippocampal CA3. Neuroreport 15:329-334.

Axmacher N, Winterer J, Stanton PK, Draguhn A, Muller W (2004) Two-photon imaging of spontaneous vesicular release in acute brain slices and its modulation by presynaptic GABAa receptors. Neuroimage 22:1014-1021.

Baer K, Essrich C, Benson JA, Benke D, Bluethmann H, Fritschy JM, Luscher B (1999) Postsynaptic clustering of y-aminobutyric acid type A receptors by the y3 subunit in vivo. Proc Natl Acad Sci U S A 96:12860-12865.

Bahi N, Friocourt G, Carrie A, Graham ME, Weiss JL, Chafey P, Fauchereau F, Burgoyne RD, Chelly J (2003) ILl receptor accessory protein like, a protein involved in X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and regulates exocytosis. Hum Mol Genet 12:1415-1425.

Bahler M, Greengard P (1987) Synapsin I bundles F-actin in a phosphorylation-dependent manner. Nature 326:704-707.

Balasubramanian S, Teissere JA, Raju DV, Hall RA (2004) Hetero-oligomerization between GABA a and GABA b receptors regulates GABA b receptor trafficking. J Biol Chem 279:18840- 18850.

Bali M, Akabas MH (2007) The location of a closed channel gate in the GABAa receptor channel. J Gen Physiol 129:145-159.

Bano D, Nicotera P (2007) Ca^^ signals and neuronal death in brain ischemia. Stroke 38:674-676.

215 - Barbaccia ML, Affricano D, Trabucchi M, Purdy RH, Colombo G, Agabio R, Gessa GL (1999) Ethanol markedly increases "GABAergic" neurosteroids in alcohol-preferring rats. Eur J Pharmacol 384:R1-R2.

Barbaccia ML, Roscetti G, Trabucchi M, Purdy RH, Mostallino MG, Concas A, Biggio G (1997) The effects of inhibitors of GABAergic transmission and stress on brain and plasma allopregnanolone concentrations, Br J Pharmacol 120:1582-1588.

Bardoni R, Torsney C, Tong CK, Prandini M, MacDermott AB (2004) Presynaptic NMDA receptors modulate glutamate release from primary sensory neurons in rat spinal cord dorsal horn. J Neurosci 24:2774-2781.

Bamekow A, Jahn R, Schartl M (1990) Synaptophysin: a substrate for the protein tyrosine kinase pp60c-src in intact synaptic vesicles. Oncogene 5:1019-1024.

Barnes EM, Jr. (2001) Assembly and intracellular trafficking of GABAa receptors. Int Rev Neurobiol 48:1-29.

Barnes JM, Dev KK, Henley JM (1994) unmasks AMPA-evoked stimulation of [^H]- L-glutamate release from rat hippocampal synaptosomes. Br J Pharmacol 113:339-341.

Bean BP (1985) Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology. J Gen Physiol 86:1-30.

Beaulieu C (1993) Numerical data on neocortical neurons in adult rat, with special reference to the GABA population. Brain Res 609:284-92.

Beck M, Brickley K, Wilkinson HL, Sharma S, Smith M, Chazot PL, Pollard S, Stephenson FA (2 0 0 2 ) Identification, molecular cloning, and characterization of a novel GABAa receptor- associated protein, GRIF-1. J Biol Chem 277:30079-30090.

Bedford FK, Kittler JT, Muller E, Thomas P, Uren JM, Merlo D, Wisden W, Triller A, Smart TG, Moss SJ (2001) GABA a receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1. Nat Neurosci 4:908-916.

Behar TN, Schaffner AE, Scott CA, O'Connell C, Barker JL (1998) Differential response of cortical plate and ventricular zone cells to GABA as a migration stimulus. J Neurosci 18:6378-6387.

Belelli D, Casula A, Ling A, Lambert J.J. (2002) The influence of subunit composition on the interaction of neurosteroids with GABAa receptors. Neuropharmacology 43:651-661.

Belenky MA, Sagiv N, Fritschy JM, Yarom Y (2003) Presynaptic and postsynaptic GABAa receptors in rat suprachiasmatic nucleus. Neuroscience 118:909-923.

Bell-Homer CL, Dohi A, Nguyen Q, Dillon GH, Singh M (2006) ERK/MAPK pathway regulates GABAa receptors. J Neurobiol 66:1467-1474.

Ben-Ari Y (2002) Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci 3:728-739.

Benfenati F, Neyroz P, Bahler M, Masotti L, Greengard P (1990) Time-resolved fluorescence study of the neuron-specific phosphoprotein synapsin I. Evidence for phosphorylation-dependent conformational changes. J Biol Chem 265:12584-12595.

216 - Benfenati F, Valtorta F, Rubenstein JL, Gorelick FS, Greengard P, Czemik AJ (1992) Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I. Nature 359:417-420.

Berezhnoy D, Nyfeler Y, Gonthier A, Schwob H, Goeldner M, Sigel E (2004) On the benzodiazepine binding pocket in GABAa receptors. Journal of Biological Chemistry 279:3160- 3168.

Berretta N, Jones RS (1996) Tonic facilitation of glutamate release by presynaptic N-methyl-D- aspartate autoreceptors in the entorhinal cortex. Neuroscience 75:339-344.

Berridge MJ, Bootman MD, Lipp P (1998) Calcium-a life and death signal. Nature 395:645-648.

Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1:11-21.

Bessman SP, Rossen J, Layne EC (1953) Gamma-Aminobutyric acid-glutamic acid transamination in brain, pp 385-391.

Betz A, Thakur P, Junge HJ, Ashery U, Rhee JS, Scheuss V, Rosenmund C, Rettig J, Brose N (2001) Functional interaction of the active zone proteins Muncl3-1 and RJMl in synaptic vesicle priming. Neuron 30:183-196.

Bhaukaurally K, Panatier A, Poulain DA, d iet SH (2005) Voltage-gated Ca^^ channel subtypes mediating GABAergic transmission in the rat supraoptic nucleus. Eur J Neurosci 21:2459-2466.

Blandina P, Goldfarb J, Craddock-Royal B, Green JP (1989) Release of endogenous dopamine by stimulation of 5-hydroxytryptamine 3 receptors in rat striatum. J Pharmacol Exp Ther 251:803-809.

Blau L, Weissmann G (1988) Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped. Biochemistry 27:5661-5666.

Blier P, Bouchard C (1993) Functional characterization of a 5 -HT3 receptor which modulates the release of 5-HT in the guinea-pig brain. Br J Pharmacol 108:13-22.

Bloom O, Evergren E, Tomilin N, Kjaerulff O, Low P, Brodin L, Pieribone VA, Greengard P, Shupliakov O (2003) Co-localization of synapsin and actin during synaptic vesicle recycling. J Cell Biol 161:737-747.

Bogdanov Y, Michels G, rmstrong-Gold C, Haydon PG, Lindstrom J, Pangalos M, Moss SJ (2006) Synaptic GABAa receptors are directly recruited from their extrasynaptic counterparts. EMBO J 25:4381-4389.

Boileau AJ, Evers AR, Davis AF, Czajkowski C (1999) Mapping the agonist binding site of the GABAa receptor: evidence for a ^-strand. J Neurosci 19:4847-4854.

Boileau AJ, Kucken AM, Evers AR, Czajkowski C (1998) Molecular dissection of benzodiazepine binding and allosteric coupling using chimeric y-aminobutyric acid A receptor subunits. Mol Pharmacol 53:295-303.

Boileau AJ, Newell JG, Czajkowski C (2002) GABAa receptor p2 Tyr97 and Leu99 line the GABA-binding site. Insights into mechanisms of agonist and antagonist actions. J Biol Chem 277:2931-2937.

- 217 - Boileau AJ, Pearce RA, Czajkowski C (2005) Tandem subunits effectively constrain GABAa receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAa receptor- associated protein. J Neurosci 25:11219-11230.

Bonanno G, Cavazzani P, Andrioli GC, Asaro D, Pellegrini G, Raiteri M (1989) Release-regulating autoreceptors of the GABAg-type in human cerebral cortex. Br J Pharmacol 96:341-346.

Bonanno G, Raiteri M (1992) Functional evidence for multiple y-aminobutyric acid B receptor subtypes in the rat cerebral cortex. J Pharmacol Exp Ther 262:114-118.

Bonanno G, Raiteri M (1993) y-Aminobutyric acid (GABA) autoreceptors in rat cerebral cortex and spinal cord represent pharmacologically distinct subtypes of the GABAg receptor. J Pharmacol Exp Ther 265:765-770.

Bonanno G, Raiteri M (1987) Release-regulating GABAa receptors are present on noradrenergic nerve terminals in selective areas of the rat brain. Synapse 1:254-257.

Bonci A, Grillner P, Mercuri NB, Bemardi G (1998) L-Type calcium channels mediate a slow excitatory synaptic transmission in rat midbrain dopaminergic neurons. J Neurosci 18:6693-6703.

Bonnert TP, McKeman RM, Farrar S, le BB, Heavens RP, Smith DW, Hewson L, Rigby MR, Sirinathsinghji DJ, Brown N, Wafford KA, Whiting PJ (1999) 0, a novel y-aminobutyric acid type A receptor subunit. Proc Natl Acad Sci U S A 96:9891-9896.

Bormann J (2000) The ABC of GABA receptors. Trends Pharmacol Sci 21:16-19.

Borodinsky LN, O'Leary D, Neale JH, Vicini S, Coso OA, Fiszman ML (2003) GABA-induced neurite outgrowth of cerebellar granule cells is mediated by GABAa receptor activation, calcium influx and CaMK II and erkl/2 pathways. J Neurochem 84:1411-1420.

Borst JG, Sakmann B (1998) Facilitation of presynaptic calcium currents in the rat brainstem. J Physiol 513 ( R 1):149-155.

Bourinet E, Soong TW, Sutton K, Slaymaker S, Mathews E, Monteil A, Zamponi GW, Nargeot J, Snutch TP (1999) Splicing of alA subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nat Neurosci 2:407-415.

Bourinet E, Stotz SC, Spaetgens RL, Dayanithi G, Lemos J, Nargeot J, Zamponi GW (2001) Interaction of SNX482 with domains III and IV inhibits activation gating of alE (Cav2.3) calcium channels. Biophys J 81:79-88.

Bowery NG, Enna SJ (2000) y-aminobutyric acid B receptors: first of the functional metabotropic heterodimers. J Pharmacol Exp Ther 292:2-7.

Bowery NG, Hill DR, Hudson AL, Doble A, Middlemiss DN, Shaw J, Turnbull M (1980) (- )Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor. Nature 283:92-94.

Bradford HE (1970a) Metabolic response of synaptosomes to electrical stimulation: release of amino acids. Brain Res 19:239-247.

Bradford HF (1970b) Responses of synaptosomes to electrical stimulation. Biochem J 117:36P.

-218 Bradford HF, Bennett GW, Thomas AJ (1973) Depolarizing stimuli and the release of physiologically active amino acids from suspensions of mammalian synaptosomes. J Neurochem 21:495-505.

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254.

Brandon NJ, Delmas P, Hill J, Smart TG, Moss SJ (2001) Constitutive tyrosine phosphorylation of the GABAa receptor y 2 subunit in rat brain. Neuropharmacology 41:745-752.

Brandon NJ, Delmas P, Kittler JT, McDonald BJ, Sieghart W, Brown DA, Smart TG, Moss SJ (2 0 0 0 ) GABAa receptor phosphorylation and functional modulation in cortical neurons by a protein kinase C-dependent pathway. J Biol Chem 275:38856-38862.

Brandon NJ, Jovanovic JN, Colledge M, Kittler JT, Brandon JM, Scott JD, Moss SJ (2003) A- kinase anchoring protein 79/150 facilitates the phosphorylation of GABAa receptors by cAMP- dependent protein kinase via selective interaction with receptor beta subunits. Mol Cell Neurosci 22:87-97.

Brandon NJ, Jovanovic JN, Smart TG, Moss SJ (2002) Receptor for activated C kinase-1 facilitates protein kinase C-dependent phosphorylation and functional modulation of GABA a receptors with the activation of G-protein-coupled receptors. J Neurosci 22:6353-6361.

Braun AP, Schulman H (1995) The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol 57:417-445.

Brennan MJ, Cantrill RC, Oldfield M, Krogsgaard-Larsen P (1981) Inhibition of y-aminobutyric acid release by y-aminobutyric acid agonist drugs. Pharmacology of the y-aminobutyric acid autoreceptor. Mol Pharmacol 19:27-30.

Brice NL, Berrow NS, Campbell V, Page KM, Brickley K, Tedder I, Dolphin AC (1997) Importance of the different beta subunits in the membrane expression of the alA and a2 calcium channel subunits: studies using a depolarization-sensitive alA antibody. Eur J Neurosci 9:749-759.

Brickley K, Smith MJ, Beck M, Stephenson FA (2005) GRIF-1 and OIP106, members of a novel gene family of coiled-coil domain proteins: association in vivo and in vitro with kinesin. J Biol Chem 280:14723-14732.

Brickley SG, Cull-Candy SG, Farrant M (1999) Single-channel properties of synaptic and extrasynaptic GABAa receptors suggest differential targeting of receptor subtypes. J Neurosci 19:2960-2973.

Brickley SG, Cull-Candy SG, Farrant M (1996) Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAa receptors. J Physiol 497 ( Pt 3):753-759.

Brose N, Rosenmund C, Rettig J (2000) Regulation of transmitter release by Une-13 and its homologues. Curr Opin Neurobiol 10:303-311.

Brown N, Kerby J, Bonnert TP, Whiting PJ, Wafford KA (2002) Pharmacological characterization of a novel cell line expressing human oc4(330 GABAa receptors. Br J Pharmacol 136:965-974.

219 - Brown RE, Haas HL (1999) On the mechanism of histaminergic inhibition of glutamate release in the rat dentate gyrus. J Physiol 515 ( Pt 3):777-786.

Brown RE, Reymann KG (1996) Histamine H 3 receptor-mediated depression of synaptic transmission in the dentate gyrus of the rat in vitro. J Physiol 496 ( Pt 1): 175-184.

Brunig I, Suter A, Knuesel I, Luscher B, Fritschy JM (2002) GABAergic terminals are required for postsynaptic clustering of dystrophin but not of GABAa receptors and gephyrin. J Neurosci 22:4805-4813.

Budde T, Meuth S, Pape HC (2002) Calcium-dependent inactivation of neuronal calcium channels. Nat Rev Neurosci 3:873-883.

Bureau I, Mulle C (1998) Potentiation of GABAergic synaptic transmission by AMPA receptors in mouse cerebellar stellate cells: changes during development. J Physiol 509 ( R 3):817-831.

Burley JR, Sihra TS (2000) A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca^^ entry. Eur J Neurosci 12:2881-2891.

Burnette WN (1981) "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195-203.

Caraiscos VB, Elliott EM, You-Ten KE, Cheng VY, Belelli D, Newell JG, Jackson ME, Lambert JJ, Rosahl TW, Wafford KA, MacDonald JF, Orser BA (2004) Tonic inhibition in mouse hippocampal CAl pyramidal neurons is mediated by a5 subunit-containing gamma-aminobutyric acid type A receptors. Proc Natl Acad Sci U S A 101:3662-3667.

Carbone E, Lux HD (1984) A low voltage-activated, fully inactivating Ca^^ channel in vertebrate sensory neurones. Nature 310:501-502.

Carbone E, Swandulla D (1989) Neuronal calcium channels: kinetics, blockade and modulation. Prog Biophys Mol Biol 54:31-58.

Carriedo SG, Yin HZ, Weiss JH (1996) Motor neurons are selectively vulnerable to AMPA/kainate receptor-mediated injury in vitro. J Neurosci 16:4069-4079.

Casagrande S, Cupello A, Pellistri F, Robello M (2007) Only high concentrations of ethanol affect GABAa receptors of rat cerebellum granule cells in culture. Neurosci Lett 414:273-276.

Castellano A, Wei X, Bimbaumer L, Perez-Reyes E (1993) Cloning and expression of a neuronal calcium channel beta subunit. J Biol Chem 268:12359-12366.

Castellano A, Wei X, Bimbaumer L, Perez-Reyes E (1993b) Cloning and expression of a third calcium channel (3 subunit. J Biol Chem. 268(5):3450-5.

Castillo PE, Weisskopf MG, Nicoll RA (1994) The role of Ca^"^ chaimels in hippocampal mossy fiber synaptic transmission and long-term potentiation. Neuron 12:261-269.

Catterall WA (1998) Structure and function of neuronal Ca^^ channels and their role in neurotransmitter release. Cell Calcium 24:307-323.

Catterall WA (1995) Structure and function of voltage-gated ion channels. Annu Rev Biochem 64:493-531.

- 220 - Chapman ER (2002) Synaptotagmin: a Ca^"^ sensor that triggers exocytosis? Nat Rev Mol Cell Biol 3:498-508.

Charych El, Yu W, Miralles CP, Serwanski DR, Li X, Rubio M, De Bias AL (2004) The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors. J Neurochem 90:173-189.

Chaudhuri D, Chang SY, DeMaria CD, Alvania RS, Soong TW, Yue DT (2004) Alternative splicing as a molecular switch for Ca^Ycalmodulin-dependent facilitation of P/Q-type Ca^^ channels. J Neurosci 24:6334-6342.

Chebib M, Johnston GA (1999) The ABC of GABA receptors: a brief review. Clin Exp Pharmacol Physiol 26:937-940.

Chen G, Trombley PQ, van den Pol AN (1996) Excitatory actions of GABA in developing rat hypothalamic neurones. J Physiol 494 (2):451-464.

Chen G, van den Pol AN (1998) Presynaptic GABAb autoreceptor modulation of P/Q-type calcium channels and GABA release in rat suprachiasmatic nucleus neurons. J Neurosci 18:1913-1922.

Chen TC, Law B, Kondratyuk T, Rossie S (1995) Identification of soluble protein phosphatases that dephosphorylate voltage-sensitive sodium channels in rat brain. J Biol Chem 270:7750-7756.

Chen ZW, Chang CS, Leil TA, Olcese R, Olsen RW (2005) GABAa receptor-associated protein regulates GABAa receptor cell-surface number in Xenopus laevis oocytes. Mol Pharmacol 68:152- 159.

Cheng HY, Penninger JM (2004) DREAMing about arthritic pain. Ann Rheum Dis 63 Suppl 2:ii72- Ü75.

Chi P, Greengard P, Ryan TA (2003) Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies. Neuron 38:69-78.

Chi P, Greengard P, Ryan TA (2001) Synapsin dispersion and reclustering during synaptic activity. Nat Neurosci 4:1187-1193.

Chittajallu R, Vignes M, Dev KK, Barnes JM, Collingridge GL, Henley JM (1996) Regulation of glutamate release by presynaptic kainate receptors in the hippocampus. Nature 379:78-81.

Choi DW (1987) Ionic dependence of glutamate neurotoxicity. J Neurosci 7:369-379.

Choi DW (1985) Glutamate neurotoxicity in cortical cell culture is calcium dependent. Neurosci Lett 58:293-297.

Churn SB, Rana A, Lee K, Parsons JT, De BA, Delorenzo RJ (2002) Calcium/calmodulin- dependent kinase 11 phosphorylation of the GABAa receptor a l subunit modulates benzodiazepine binding. J Neurochem 82:1065-1076.

Ciruela F, Ferre S, Casado V, Cortes A, Cunha RA, Lluis C, Franco R (2006) Heterodimeric adenosine receptors: a device to regulate neurotransmitter release. Cell Mol Life Sci 63:2427-2431.

Clarke VR, Ballyk BA, Hoo KH, Mandelzys A, Pellizzari A, Bath CP, Thomas J, Sharpe EF, Davies CH, Omstein PL, Schoepp DD, Kamboj RK, Collingridge GL, Lodge D, Bleakman D

-221 - (1997) A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission. Nature 389:599-603.

Clayton GH, Owens GC, Wolff JS, Smith RL (1998) Ontogeny of cation-Cf cotransporter expression in rat neocortex. Brain Res Dev Brain Res 109:281-292.

Cochilla AJ, Alford S (1999) NMDA receptor-mediated control of presynaptic calcium and neurotransmitter release. J Neurosci 19:193-205.

Collins GG (1972) GABA-2-oxoglutarate transaminase, glutamate decarboxylase and the half-life of GABA in different areas of rat brain, pp 2849-2858.

Connolly CN, Wafford KA (2004) The Cys-loop superfamily of ligand-gated ion channels: the impact of receptor structure on function. Biochem Soc Trans 32:529-534.

Cousin MA, Robinson PJ (2000) Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals. J Neurochem 75:1645-1653.

Cousin MA, Robinson PJ (2001) The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci 24:659-665.

Cox ED, az-Arauzo H, Huang Q, Reddy MS, Ma C, Harris B, McKeman R, Skolnick P, Cook JM (1998) Synthesis and evaluation of analogues of the partial agonist 6-(propyloxy)-4- (methoxymethyl)-p-carboline-3-carboxylic acid ethyl ester (6-PBC) and the full agonist 6- (benzyloxy)-4-(methoxymethyl)-p-carboline-3-carboxylic acid ethyl ester (7k 93423) at wild type and recombinant GABAa receptors. J Med Chem 41:2537-2552.

Craig PJ, Beattie RE, Folly EA, Banerjee MD, Reeves MB, Priestley JV, Carney SL, Sher E, Perez- Reyes E, Volsen SG (1999) Distribution of the voltage-dependent calcium channel alG subunit mRNA and protein throughout the mature rat brain. Eur J Neurosci 11:2949-2964.

Cremona O, Di PG, Wenk MR, Luthi A, Kim WT, Takei K, Daniell L, Nemoto Y, Shears SB, Flavell RA, McCormick DA, De CP (1999) Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99:179-188.

Crestani F, Keist R, Fritschy JM, Benke D, Vogt K, Pmt L, Bluthmann H, Mohler H, Rudolph U (2002) Trace fear conditioning involves hippocampal a5 GABAa receptors. Proc Natl Acad Sci U S A 99:8980-8985.

Crestani F, Low K, Keist R, Mandelli M, Mohler H, Rudolph U (2001) Molecular targets for the myorelaxant action of diazepam. Mol Pharmacol 59:442-445.

Croall DE, DeMartino GN (1991) Calcium-activated neutral protease (calpain) system: structure, function, and regulation. Physiol Rev 71:813-847.

Cuttle ME, Tsujimoto T, Forsythe ID, Takahashi T (1998) Facilitation of the presynaptic calcium current at an auditory synapse in rat brainstem. J Physiol 512 ( Pt 3):723-729.

Czemik AJ, Girault JA, Naim AC, Chen J, Snyder G, Kebabian J, Greengard P (1991) Production of phosphorylation state-specific antibodies. Methods Enzymol 201:264-283.

Czemik AJ, Pang DT, Greengard P (1987) Amino acid sequences surrounding the cAMP-dependent and calcium/calmodulin-dependent phosphorylation sites in rat and bovine synapsin I. Proc Natl Acad Sci U S A 84:7518-7522.

- 222 - Dajas-Bailador F, Wonnacott S (2004) Nicotinic acetylcholine receptors and the regulation of neuronal signalling. Trends Pharmacol Sci 25:317-324.

Damgen H, Liiddens H (1999) displays a selectivity to recombinant GABAa receptors different from zolipdem, and benzodiazepines, pp 139-148.

Darman RB, Forbush B (2002) A regulatory locus of phosphorylation in the N terminus of the Na- K-Cl cotransporter, NKCCl. J Biol Chem 277:37542-37550.

Davies M, Bateson AN, Dunn SM (1998) Structural requirements for ligand interactions at the benzodiazepine recognition site of the GABAa receptor. J Neurochem 70:2188-2194.

Davies PA, Hanna MC, Hales TG, Kirkness EF (1997) Insensitivity to anaesthetic agents conferred by a class of GABAa receptor subunit. Nature 385:820-823.

Davis BJ (1964) Disc electrophoresis. II. Method and application to human serum proteinS. Ann N Y Acad Sci 121:404-427.

De Camilli CP, Benfenati F, Valtorta F, Greengard P (1990) The synapsins. Annu Rev Cell Biol 6:433-460.

De Camilli CP, Cameron R, Greengard P (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections. J Cell Biol 96:1337-1354.

De Waard WM, Pragnell M, Campbell KP (1994) Ca^^ channel regulation by a conserved p subunit domain. Neuron 13:495-503.

DeFazio RA, Keros S, Quick MW, Hablitz JJ (2000) Potassium-coupled chloride cotransport controls intracellular chloride in rat neocortical pyramidal neurons. J Neurosci 20:8069-8076.

Del Castillo CJ, Katz B (1954) Quantal components of the end-plate potential. J Physiol 124:560- 573.

Delaney AJ, Jahr CE (2002) Kainate receptors differentially regulate release at two parallel fiber synapses. Neuron 36:475-482.

Delgado R, Maureira C, Oliva C, Kidokoro Y, Labarca P (2000) Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire. Neuron 28:941-953.

Dias R, et al. (2005) Evidence for a significant role of a3-containing GABAa receptors in mediating the anxiolytic effects of benzodiazepines. J Neurosci 25:10682-10688.

Dietrich D, Kirschstein T, Kukley M, Pereverzev A, von der BC, Schneider T, Beck H (2003) Functional specialization of presynaptic Cay2.3 Ca^^ channels. Neuron 39:483-496.

Dittman JS, Regehr WG (1996) Contributions of calcium-dependent and calcium-independent mechanisms to presynaptic inhibition at a cerebellar synapse. J Neurosci 16:1623-1633.

Donnelly JL, Macdonald RL (1996) Loreclezole enhances apparent desensitization of recombinant GABAa receptor currents. Neuropharmacology 35:1233-1241.

-223 Doreulee N, Yanovsky Y, Flagmeyer I, Stevens DR, Haas HL, Brown RE (2001) Histamine H 3 receptors depress synaptic transmission in the corticostriatal pathway. Neuropharmacology 40:106- 113.

Dudel J, Kuffler SW (1961) Presynaptic inhibition at the crayfish neuromuscular junction. J Physiol 155:543-562.

Dulubova I, Lou X, Lu J, Huryeva I, Alam A, Schneggenburger R, Sudhof TC, Rizo J (2005) A Munc 13/RIM/Rab3 tripartite complex: from priming to plasticity? EMBO J 24:2839-2850.

Dunkley PR, Heath JW, Harrison SM, Jarvie PE, Glenfield PJ, Rostas JA (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an SI fraction: homogeneity and morphology of subcellular fractions. Brain Res 441:59-71.

Dunkley PR, Jarvie PE, Heath JW, Kidd GJ, Rostas JA (1986) A rapid method for isolation of synaptosomes on Percoll gradients. Brain Res 372:115-129.

Dunlap K, Luebke JI, Turner TJ (1995a) Exocytotic Ca^^ channels in mammalian central neurons. Trends Neurosci 18:89-98.

Dyball RE, Shaw FD (1979) Inhibition by GABA of release from the neurohypophysis in the rat J Physiol 289:78-79.

Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE, Staley KJ (2005) NKCCl transporter facilitates seizures in the developing brain. Nat Med 11:1205- 1213.

Ebert V, Scholze P, Fuchs K, Sieghart W (1999) Identification of subunits mediating clustering of GABA a receptors by rapsyn. Neurochem Int 34:453-463.

Eccles JC (1964) Presynaptic inhibition in the spinal cord. Prog Brain Res 12:65-91.

Eccles JC (1982) The synapse: from electrical to chemical transmission. Annu Rev Neurosci 5:325- 339.

Eccles JC, Eccles RM, Magni F (1961) Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys. J Physiol 159:147-166.

Eccles JC, Magni F, Willis WD (1962) Depolarization of central terminals of Group I afferent fibres from muscle. J Physiol 160:62-93.

Eccles JC, Schmidt R, Willis WD (1963) Pharmacological studies on presynaptic inhibition. J Physiol 168:500-530.

Ehrlich I, Lohrke S, Friauf E (1999) Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl regulation. J Physiol 520 Pt 1:121-137.

Eilers J, Plant TD, Marandi N, Konnerth A (2001) GABA-mediated Ca^^ signalling in developing rat cerebellar Purkinje neurones. J Physiol 536:429-437.

Elmslie KS, Kammermeier PJ, Jones SW (1994) Re-evaluation of Ca^^ channel types and their modulation in bullfrog sympathetic neurons. Neuron 13:217-228.

224 - Endo T, Yanagawa Y, Obata K, Isa T (2005) Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus. J Neurophysiol 94:3893-3902.

Engelman HS, MacDermott AB (2004) Presynaptic ionotropic receptors and control of transmitter release. Nat Rev Neurosci 5:135-145.

Enna SJ, McCarson KE (2006) The role of GABA in the mediation and perception of pain. Adv Pharmacol 54:1-27.

Enz R, Brandstatter JH, Wassle H, Bormann J (1996) Immunocytochemical localization of the GABAc receptor rho subunits in the mammalian retina. J Neurosci 16:4479-4490.

Ertel EA, Campbell KP, Harpold MM, Hofmarm F, Mori Y, Perez-Reyes E, Schwartz A, Snutch TP, Tanabe T, Bimbaumer L, Tsien RW, Catterall WA (2000) Nomenclature of voltage-gated calcium channels. Neuron 25:533-535.

Essrich C, Lorez M, Benson JA, Fritschy JM, Luscher B (1998) Postsynaptic clustering of major GABAa receptor subtypes requires the y2 subunit and gephyrin. Nat Neurosci 1:563-571.

Evans RJ (1996) The molecular biology of P2X receptors. J Auton Pharmacol 16:309-310.

Fagni L, Chavis P, Ango F, Bockaert J (2000) Complex interactions between mGluRs, intracellular Ca^^ stores and ion channels in neurons. Trends Neurosci 23:80-88.

Fang C, Deng L, Keller CA, Fukata M, Fukata Y, Chen G, Luscher B (2006) GODZ-mediated palmitoylation of GABA a receptors is required for normal assembly and function of GABAergic inhibitory synapses. J Neurosci 26:12758-12768.

Farrant M, Kaila K (2007) The cellular, molecular and ionic basis of GABAa receptor signalling. Prog Brain Res 160:59-87.

Farrant M, Nusser Z (2005) Variations on an inhibitory theme: phasic and tonic activation of GABAa receptors. Nat Rev Neurosci 6:215-229.

Fassio A, Rossi F, Bonanno G, Raiteri M (1999) GABA induces norepinephrine exocytosis from hippocampal noradrenergic axon terminals by a dual mechanism involving different voltage- sensitive calcium channels. J Neurosci Res 57:324-331.

Fatt P, Katz B (1953) The effect of inhibitory nerve impulses on a crustacean muscle fibre.!. Physiol. 121(2):374-379.

Feigenspan A, Bormann J (1998) GABA-gated Cl' channels in the rat retina. Prog Retin Eye Res 17:99-126.

Feng J, Cai X, Zhao J, Yan Z (2001) Serotonin receptors modulate GABAa receptor channels through activation of anchored protein kinase C in prefrontal cortical neurons. J Neurosci 21:6502- 6511.

Fergestad T, Broadie K (2001) Interaction of stoned and synaptotagmin in synaptic vesicle endocytosis. J Neurosci 21:1218-1227.

Fergestad T, Davis WS, Broadie K (1999) The stoned proteins regulate synaptic vesicle recycling in the presynaptic terminal. J Neurosci 19:5847-5860.

- 225 - Femandez-Chacon R, Shin OH, Konigstoifer A, Matos MF, Meyer AC, Garcia J, Gerber SH, Rizo J, Sudhof TC, Rosenmund C (2002) Structure/function analysis of Ca^^ binding to the C2A domain of synaptotagmin 1. J Neurosci 22:8438-8446.

Ferreira A, Rapoport M (2002) The synapsins: beyond the regulation of neurotransmitter release. Cell Mol Life Sci 59:589-595.

Fesce R, Grohovaz F, Valtorta F, Meldolesi J (1994) Neurotransmitter release: fusion or 'kiss-and- run'? Trends Cell Biol 4:1-4.

Fischer A, Sananbenesi F, Spiess J, Radulovic J (2003) Cdk5: a novel role in learning and memory. Neurosignals 12:200-208.

Flemmer AW, Gimenez I, Dowd BF, Darman RB, Forbush B (2002) Activation of the Na-K-Cl otransporter NKCCl detected with a phospho-specific antibody. J Biol Chem 277:37551-37558.

Floran B, Silva I, Nava C, Aceves J (1988) Presynaptic modulation of the release of GABA by GABAa receptors in pars compacta and by GABAb receptors in pars reticulata of the rat substantia nigra. Eur J Pharmacol 150:277-286.

Fon EA, Edwards RH (2001) Molecular mechanisms of neurotransmitter release, pp 581-601.

Forsythe ID, Tsujimoto T, Bames-Davies M, Cuttle MF, Takahashi T (1998) Inactivation of presynaptic calcium current contributes to synaptic depression at a fast central synapse. Neuron 20:797-807.

Fox AP, Nowycky MC, Tsien RW (1987) Single-channel recordings of three types of calcium channels in chick sensory neurones. J Physiol 394:173-200.

Frassoni C, Inverardi F, Coco S, Ortino B, Grumelli C, Pozzi D, Verderio C, Matteoli M (2005) Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ. Neuroscience 131:813-823.

Friedrich P (2004) The intriguing Ca^^ requirement of calpain activation. Biochem Biophys Res Commun 323:1131-1133.

Friel DD, Tsien RW (1992) A caffeine- and ryanodine-sensitive Ca^^ store in bullfrog sympathetic neurones modulates effects of Ca^^ entry on [Ca^^].. J Physiol 450:217-246.

Fritschy JM, Brunig I (2003) Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications. Pharmacol Ther 98:299-323.

Fritschy JM, Mohler H (1995) GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol 359:154-194.

Fritschy JM, Schweizer C, Brunig I, Luscher B (2003) Pre- and post-synaptic mechanisms regulating the clustering of type A y-aminobutyric acid receptors (GABAa receptors). Biochem Soc Trans 31:889-892.

Fukuda A, Muramatsu K, Okabe A, Shimano Y, Hida H, Fujimoto I, Nishino H (1998a) Changes in intracellular Ca^"^ induced by GABA a receptor activation and reduction in Cl- gradient in neonatal rat neocortex. J Neurophysiol 79:439-446.

2 2 6 - Fukuda M, Moreira JE, Lewis FM, Sugimori M, Niinobe M, Mikoshiba K, Llinas R (1995) Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal. Proc Natl Acad Sci U S A 92:10708- 10712.

Fung SC, Fillenz M (1983) The role of pre-synaptic GABA and benzodiazepine receptors in the control of noradrenaline release in rat hippocampus. Neurosci Lett 42:61-66.

Gahwiler BH, Brown DA (1985) GABAg-receptor-activated current in voltage-clamped CA3 pyramidal cells in hippocampal cultures. Proc Natl Acad Sci U S A 82:1558-1562.

Galvez T, Parmentier ML, Joly C, Malitschek B, Kaupmann K, Kuhn R, Bittiger H, Froestl W, Bettler B, Pin JP (1999) Mutagenesis and modeling of the GABAg receptor extracellular domain support a venus flytrap mechanism for ligand binding. J Biol Chem 274:13362-13369.

Gamba G (2005) Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters. Physiol Rev 85:423-493.

Ganguly K, Schinder AF, Wong ST, Poo M (2001) GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 105:521-532.

Garcia J, Nakai J, Imoto K, Beam KG (1997) Role of S4 segments and the leucine heptad motif in the activation of an L-type calcium channel. Biophys J 72:2515-2523.

Garduno-Torres B, Trevino M, Gutierrez R, Rias-Montano JA (2007) Pre-synaptic histamine H 3 receptors regulate glutamate, but not GABA release in rat thalamus. Neuropharmacology 52:527- 535.

Gasparini S, Kasyanov AM, Pietrobon D, Voronin LL, Cherubini E (2001) Presynaptic R-type calcium channels contribute to fast excitatory synaptic transmission in the rat hippocampus. J Neurosci 21:8715-8721.

Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, Sudhof TC (1994) Synaptotagmin I: a major Ca^’*’ sensor for transmitter release at a central synapse. Cell 79:717-727.

Gillen CM, Brill S, Payne JA, Forbush B, III (1996a) Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family. J Biol Chem 271:16237-16244.

Gitler D, Takagishi Y, Feng J, Ren Y, Rodriguiz RM, Wetsel WC, Greengard P, Augustine GJ (2004) Different presynaptic roles of synapsins at excitatory and inhibitory synapses. J Neurosci 24:11368-11380.

Glitsch M, Marty A (1999) Presynaptic effects of NMDA in cerebellar Purkinje cells and intemeurons. J Neurosci 19:511-519.

Goda Y, Sudhof TC (1997) Calcium regulation of neurotransmitter release: reliably unreliable? Curr Opin Cell Biol 9:513-518.

Gorelick FS, Wang JK, Lai Y, Naim AC, Greengard P (1988) Autophosphorylation and activation of Ca^Vcalmodulin-dependent protein kinase II in intact nerve terminals. J Biol Chem 263:17209- 17212.

-227 Gotti C, Clementi, F (2004) Neuronal nicotinic receptors; from structure to pathology. Prog. Neurobiol 74:363-396.

Graham B, Redman S (1994) A simulation of action potentials in synaptic boutons during presynaptic inhibition. J Neurophysiol 71:538-549.

Granseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51:773-786.

Gray EG, Whittaker VP (1962) The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat 96:79-88.

Gray R, Rajan AS, Radcliffe KA, Yakehiro M, Dani JA (1996) Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature 383:713-716.

Greengard P, Valtorta F, Czemik AJ, Benfenati F (1993) Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 259:780-785.

Grillner P, Bonci A, Svensson TH, Bemardi G, Mercuri NB (1999) Presynaptic muscarinic M 3 receptors reduce excitatory transmission in dopamine neurons of the rat mesencephalon. Neuroscience 91:557-565.

Grossfeld RM, Yancey SW, Baxter CF (1984) Inhibitors of crayfish glutamic acid decarboxylase, pp 947-963.

Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca^^ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450.

Gu JG, MacDermott AB (1997) Activation of ATP P2X receptors elicits glutamate release from sensory neuron synapses. Nature 389:749-753.

Guo J, Dceda SR (2005) Coupling of metabotropic 8 to N-type Ca^^ channels in rat sympathetic neurons. Mol Pharmacol 67:1840-1851.

Haddjeri N, Blier P (1995) Pre- and post-synaptic effects of the 5-HTg agonist 2-methyl-5-HT on the 5-HT system in the rat brain. Synapse 20:54-67.

Hadingham KL, Garrett EM, Wafford KA, Bain C, Heavens RP, Sirinathsinghji DJ, Whiting PJ (1996) Cloning of cDNAs encoding the human y-aminobutyric acid type A receptor a 6 subunit and characterization of the pharmacology of a 6 -containing receptors. Mol Pharmacol 49:253-259.

Hall ZW, Kravitz EA (1967) The metabolism of y-aminobutyric acid (GABA) in the lobster nervous system. II. Succinic semialdehyde dehydrogenase, pp 55-61.

Hagiwara S, Fukuda J, Eaton D (1974) Membrane currents carried by Ca, Sr and Ba in barnacle muscle fibre during voltage clamp. J. Gen. Physiol. 63:564-578.

Hailing DB, Racena-Parks P, Hamilton SL (2006) Regulation of voltage-gated Ca^^ channels by calmodulin. Sci STKE 2006:erl.

Harata N, Ryan TA, Smith SJ, Buchanan J, Tsien RW (2001) Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion. Proc Natl Acad Sci U S A 98:12748-12753.

-228- Harata NC, Choi S, Pyle IL, Aravanis AM, Tsien RW (2006) Frequency-dependent kinetics and prevalence of kiss-and-run and reuse at hippocampal synapses studied with novel quenching methods. Neuron 49:243-256.

Hargittai FT, Youmans SJ, Lieberman EM (1991) Determination of the membrane potential of cultured mammalian Schwann cells and its sensitivity to potassium using a thiocarbocyanine fluorescent dye. Glia 4:611-616.

Hatzipetros T, Yamamoto BK (2006) Dopaminergic and GABAergic modulation of glutamate release from rat subthalamic nucleus efferents to the substantia nigra. Brain Res 1076:60-67.

Haydar TF, Wang F, Schwartz ML, Rakic P (2000) Differential modulation of proliferation in the neocortical ventricular and subventricular zones. J Neurosci 20:5764-5774.

Heist EK, Schulman H (1998) The role of Ca^Vcalmodulin-dependent protein kinases within the nucleus. Cell Calcium 23:103-114.

Hendry SH, Schwark HD, Jones EG, Yan J (1987) Numbers and proportions of GABA- immunoreactive neurons in different areas of monkey cerebral cortex, pp 1503-1519.

Henneberger C, Juttner R, Rothe T, Grantyn R (2002) Postsynaptic action of BDNF on GABAergic synaptic transmission in the superficial layers of the mouse superior colliculus. J Neurophysiol 88:595-603.

Herrero I, Miras-Portugal MT, Sanchez-Prieto J (1998) Functional switch from facilitation to inhibition in the control of glutamate release by metabotropic glutamate receptors. J Biol Chem 273:1951-1958.

Herring D, Huang R, Singh M, Robinson LC, Dillon GH, Leidenheimer NJ (2003) Constitutive GABAa receptor endocytosis is dynamin-mediated and dependent on a dileucine AP2 adaptin- binding motif within the beta 2 subunit of the receptor. J Biol Chem 278:24046-24052.

Heuser J (1989) The role of coated vesicles in recycling of synaptic vesicle membrane. Cell Biol Int Rep 13:1063-1076.

Hevers W, Luddens H (1998) The diversity of GABA a receptors. Pharmacological and electrophysiological properties of GABA a channel subtypes. Mol Neurobiol 18:35-86.

Hilfiker S, Pieribone VA, Czemik AJ, Kao HT, Augustine GJ, Greengard P (1999) Synapsins as regulators of neurotransmitter release. Philos Trans R Soc Lond B Biol Sci 354:269-279.

Hill DR (1985) GABAb receptor modulation of adenylate cyclase activity in rat brain slices. Br J Pharmacol 84:249-257.

Hill DR, Bowery NG (1981) ^H-baclofen and ^H-GABA bind to bicuculline-insensitive GABA B sites in rat brain. Nature 290:149-152.

Hodgkin AL, Huxley AF (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117:500-544.

Holden JH, Czajkowski C (2002) Different residues in the GABA a receptor alT60-alK70 region mediate GABA and SR-95531 actions. J Biol Chem 277:18785-18792.

Hollmann M, Heinemann S (1994) Cloned glutamate receptors. Annu Rev Neurosci 17:31-108.

- 229 - Hook SS, Means AR (2001) Ca^VCaM-dependent kinases: from activation to function. Annu Rev Pharmacol Toxicol 41:471-505.

Hopkins MH, Bichler KA, Su T, Chamberlain CL, Silverman RB (1992) Inactivation of y- aminobutyric acid aminotransferase by various amine buffers. J Enzyme Inhib 6:195-199.

Horenstein J, Wagner DA, Czajkowski C, Akabas MH (2001) Protein mobility and GABA-induced conformational changes in GABAa receptor pore-lining M2 segment. Nat Neurosci 4:477-485.

Home AL, Kemp JA (1991) The effect of co-conotoxin GVIA on synaptic transmission within the nucleus accumbens and hippocampus of the rat in vitro. Br J Pharmacol 103:1733-1739.

Hosaka M, Hammer RE, Sudhof TC (1999) A phospho-switch controls the dynamic association of synapsins with synaptic vesicles. Neuron 24:377-387.

Hosie AM, Dunne EL, Harvey RJ, Smart TG (2003) Zinc-mediated inhibition of GABAa receptors: discrete binding sites underlie subtype specificity. Nat Neurosci 6:362-369.

Hosie AM, Wilkins ME, da Silva HM, Smart TG (2006) Endogenous neurosteroids regulate GABAa receptors through two discrete transmembrane sites. Nature 444:486-489.

Houston CM, Lee HH, Hosie AM, Moss SJ, Smart TG (2007) Identification of the Sites for CaMK- Il-dependent Phosphorylation of GABAa Receptors. J Biol Chem 282:17855-17865.

Houston CM, Smart TG (2006) CaMK-II modulation of GABA a receptors expressed in HEK293, NG108-15 and rat cerebellar granule neurons. Eur J Neurosci 24:2504-2514.

Huang Q, Zhou D, Sapp E, Aizawa H, Ge P, Bird ED, Vonsattel JP, DiPiglia M (1995) -induced increases in calbindin D28k immunoreactivity in rat striatal neurons in vivo and in vitro mimic the pattern seen in Huntington's disease. Neuroscience 65:397-407.

Huang SM, Bisogno T, Trevisani M, Al-Hayani A, De PL, Fezza F, Tognetto M, Petros TJ, Krey JF, Chu CJ, Miller JD, Davies SN, Geppetti P, Walker JM, Di M, V (2002) An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VRl receptors. Proc Natl Acad Sci U S A 99:8400-8405.

Hudmon A, Schulman H, Kim J, Maltez JM, Tsien RW, Pitt GS (2005) CaMKII tethers to L-type Ca^^ channels, establishing a local and dedicated integrator of Ca^^ signals for facilitation. J Cell Biol 171:537-547.

Hugel S, Schlichter R (2000) Presynaptic P2X receptors facilitate inhibitory GABAergic transmission between cultured rat spinal cord dorsal horn neurons. J Neurosci 20:2121-2130.

Hughes DI, Bannister AP, Pawelzik H, Thomson AM (2000) Double immunofluorescence, peroxidase labelling and ultrastructural analysis of intemeurones following prolonged electrophysiological recordings in vitro. J Neurosci Methods 101:107-116.

Huguenard JR (1996) Low-threshold calcium currents in central nervous system neurons. Annu Rev Physiol 58:329-348.

Hussy N, Bres V, Rochette M, Duvoid A, Alonso G, Dayanithi G, Moos FC (2001) Osmoregulation of vasopressin secretion via activation of neurohypophysial nerve terminals glycine receptors by glial . J Neurosci 21:7110-7116.

230 - Huston E, Cullen GP, Burley JR, Dolphin AC (1995) The involvement of multiple calcium channel sub-types in glutamate release from cerebellar granule cells and its modulation by GABAb receptor activation. Neuroscience 68:465-478.

Huttner WB, Greengard P (1979) Multiple phosphorylation sites in protein I and their differential regulation by cyclic AMP and calcium. Proc Natl Acad Sci U S A 76:5402-5406.

Ilouz N, Branski L, Pamis J, Pamas H, Linial M (1999) Depolarization affects the binding properties of muscarinic acetylcholine receptors and their interaction with proteins of the exocytic apparatus. J Biol Chem 274:29519-29528.

Imredy JP, Yue DT (1992) Submicroscopic Ca^"^ diffusion mediates inhibitory coupling between individual Ca^^ channels. Neuron 9:197-207.

Imredy JP, Yue DT (1994) Mechanism of Ca^^-sensitive inactivation of L-type Ca^^ channels. Neuron 12:1301-1318.

Jackson MB, Zhang SJ (1995) Action potential propagation and propagation block by GABA in rat posterior pituitary nerve terminals. J Physiol 483 (3):597-611.

Jacob TC, Bogdanov YD, Magnus C, Saliba RS, Kittler JT, Haydon PG, Moss SJ (2005) Gephyrin regulates the cell surface dynamics of synaptic GABA a receptors. J Neurosci 25:10469-10478.

Jang IS, Ito Y, Akaike N (2005) Feed-forward facilitation of glutamate release by presynaptic GABAa receptors. Neuroscience 135:737-748.

Jang IS, Jeong HJ, Akaike N (2001) Contribution of the Na-K-Cl cotransporter on GABA a receptor-mediated presynaptic depolarization in excitatory nerve terminals. J Neurosci 21:5962- 5972.

Jang IS, Nakamura M, Ito Y, Akaike N (2006) Presynaptic GABAa receptors facilitate spontaneous glutamate release from presynaptic terminals on mechanically dissociated rat CA3 pyramidal neurons. Neuroscience 138(1 ):25-35.

Jensen K, Jensen MS, Lambert JD (1999) Role of presynaptic L-type Ca^^ channels in GABAergic synaptic transmission in cultured hippocampal neurons. J Neurophysiol 81:1225-1230.

Jensen ML, Timmermann DB, Johansen TH, Schousboe A, Vanning T, Ahring PK (2002) The beta subunit determines the ion selectivity of the GABAa receptor. J Biol Chem 277:41438-41447.

Jeong HJ, Jang IS, Nabekura J, Akaike N (2003a) Adenosine Ai receptor-mediated presynaptic inhibition of GABAergic transmission in immature rat hippocampal CAI neurons. J Neurophysiol 89:1214-1222.

Jeong HJ, Jang IS, Moorhouse AJ, Akaike N (2003b) Activation of presynaptic glycine receptors facilitates glycine release from presynaptic terminals synapsing onto rat spinal sacral dorsal commissural nucleus neurons. J Physiol 550:373-383.

Johnson EM, Maeno H, Greengard P (1971) Phosphorylation of endogenous protein of rat brain by cyclic adenosine 3',5'-monophosphate-dependent protein kinase. J Biol Chem 246:7731-7739.

Jones-Davis DM, Song L, Gallagher MJ, Macdonald RL (2005) Structural determinants of benzodiazepine allosteric regulation of GABAa receptor currents. J Neurosci 25:8056-8065.

-231 Jovanovic JN, Benfenati F, Slow YL, Sihra TS, Sanghera JS, Pelech SL, Greengard P, Czemik AJ (1996) Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions. Proceedings of the National Academy of Science USA 93:3679-3683,

Jovanovic JN, Sihra TS, Naim AC, Hemmings HC, Jr., Greengard P, Czemik AJ (2001) Opposing changes in phosphorylation of specific sites in synapsin I during Ca^^-dependent glutamate release in isolated nerve terminals. J Neurosci 21:7944-7953.

Jovanovic JN, Thomas P, Kittler JT, Smart TG, Moss SJ (2004) Brain-derived neurotrophic factor modulates fast synaptic inhibition by regulating GABAa receptor phosphorylation, activity, and cell-surface stability. J Neurosci 24:522-530.

Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F, Zaugg M, Vogt KE, Ledermann B, Antkowiak B, Rudolph U (2003) General anesthetic actions in vivo strongly attenuated by a point mutation in the GABAa receptor (33 subunit. FASEB J 17:250-252.

Kaila K (1994) Ionic basis of GABAa receptor channel function in the nervous system. Prog Neurobiol 42:489-537.

Kalman D, O'Lague PH, Erxleben C, Armstrong DL (1988) Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells. J Gen Physiol 92:531-548.

Kamiya H, Ozawa S (2000) Kainate receptor-mediated presynaptic inhibition at the mouse hippocampal mossy fibre synapse. J Physiol 523 Pt 3:653-665.

Kamp MA, Krieger A, Henry M, Hescheler J, Weiergraber M, Schneider T (2005) Presynaptic 'Cav2.3-containing' E-type Ca channels share dual roles during neurotransmitter release. Eur J Neurosci 21:1617-1625.

Kandler K, Friauf E (1995) Development of electrical membrane properties and discharge characteristics of superior olivary complex neurons in fetal and postnatal rats. Eur J Neurosci 7:1773-1790.

Kanematsu T, Fujii M, Mizokami A, Kittler JT, Nabekura J, Moss SJ, Hirata M (2007) Phospholipase C-related inactive protein is implicated in the constitutive intemalization of GABAa receptors mediated by clathrin and AP2 adaptor complex. J Neurochem 101:898-905.

Kanematsu T, Jang IS, Yamaguchi T, Nagahama H, Yoshimura K, Hidaka K, Matsuda M, Takeuchi H, Misumi Y, Nakayama K, Yamamoto T, Akaike N, Hirata M, Nakayama K (2002) Role of the PLC-related, catalytically inactive protein p i30 in GABAa receptor function. EMBO J 21:1004- 1011.

Kanematsu T, Yasunaga A, Mizoguchi Y, Kuratani A, Kittler JT, Jovanovic JN, Takenaka K, Nakayama KI, Fukami K, Takenawa T, Moss SJ, Nabekura J, Hirata M (2006) Modulation of GABAa receptor phosphorylation and membrane trafficking by phospholipase C-related inactive protein/protein phosphatase 1 and 2A signaling complex underlying brain-derived neurotrophic foctor-dependent regulation of GABAergic inhibition. J Biol Chem 281:22180-22189.

Kao HT, Porton B, Hilfiker S, Stefani G, Pieribone VA, DeSalle R, Greengard P (1999) Molecular evolution of the synapsin gene family. J Exp Zool 285:360-377.

Karlsson U, Sundgren-Andersson AK, Johansson S, Krupp JJ (2005) Capsaicin augments synaptic transmission in the rat medial preoptic nucleus. Brain Res 1043:1-11.

- 232 - Kash TL, Trudell JR, Harrison NL (2004) Structural elements involved in activation of the y- aminobutyric acid type A (GABAa) receptor. Biochem Soc Trans 32:540-546.

Katsurabayashi S, Kubota H, Tokutomi N, Akaike N (2003) A distinct distribution of functional presynaptic 5-HT receptor subtypes on GABAergic nerve terminals projecting to single hippocampal CAI pyramidal neurons. Neuropharmacology 44:1022-1030.

Kaupmann K, Huggel K, Held J, Flor PJ, Bischoff S, Mickel SJ, McMaster G, Angst C, Bittiger H, Froestl W, Bettler B (1997) Expression cloning of GABAb receptors uncovers similarity to metabotropic glutamate receptors. Nature 386:239-246.

Kavanaugh MP, Arriza JL, North RA, Amara SG (1992) Electrogenic uptake of y-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes, pp 22007-22009.

Kawa K (2003) Glycine facilitates transmitter release at developing synapses: a patch clamp study from Purkinje neurons of the newborn rat. Brain Res Dev Brain Res 144:57-71.

Kee Y, Scheller RH (1996) Localization of synaptotagmin-binding domains on syntaxin. J Neurosci 16:1975-1981.

Keller CA, Yuan X, Panzanelli P, Martin ML, Alldred M, Sassoe-Pognetto M, Luscher B (2004) The y2 subunit of GABAa receptors is a substrate for palmitoylation by GODZ. J Neurosci 24:5881-5891.

Kelly PT, McGuinness TL, Greengard P (1984) Evidence that the major postsynaptic density protein is a component of a Ca^Vcalmodulin-dependent protein kinase. Proc Natl Acad Sci U S A 81:945-949.

Kerchner GA, Wang GD, Qiu CS, Huettner JE, Zhuo M (2001) Direct presynaptic regulation of GAB A/glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism. Neuron 32:477-488.

Keynan S, Kanner BI (1988) y-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes, pp 12-17.

Kidokoro Y, Ritchie AK (1980) Chromaffin cell action potentials and their possible role in adrenaline secretion from rat adrenal medulla. J Physiol 307:199-216.

Kilb W, Ikeda M, Uchida K, Okabe A, Fukuda A, Luhmann HJ (2002) Depolarizing glycine responses in Cajal-Retzius cells of neonatal rat cerebral cortex. Neuroscience 112:299-307.

Kimura F, Baughman RW (1997) Distinct muscarinic receptor subtypes suppress excitatory and inhibitory synaptic responses in cortical neurons. J Neurophysiol 77:709-716.

Kitaichi K, Day JC, Quirion R (1999) A novel muscarinic M 4 receptor antagonist provides further evidence of an autoreceptor role for the muscarinic M% receptor sub-type. Eur J Pharmacol 383:53- 56.

Kits KS, Mansvelder HD (1996) Voltage gated calcium channels in molluscs: classification, Ca^^ dependent inactivation, modulation and functional roles. Invert Neurosci 2:9-34.

Kittler JT, Delmas P, Jovanovic JN, Brown DA, Smart TG, Moss SJ (2000) Constitutive endocytosis of GABAa receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons. J Neurosci 20:7972-7977.

-233 - Kittler JT, Moss SJ (2003) Modulation of GABAa receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition. Curr Opin Neurobiol 13:341-347.

Kittler JT, Rostaing P, Schiavo G, Fritschy JM, Olsen R, Triller A, Moss SJ (2001) The subcellular distribution of GABARAP and its ability to interact with NSF suggest a role for this protein in the intracellular transport of GABAa receptors. Mol Cell Neurosci 18:13-25.

Kittler JT, Thomas P, Tretter V, Bogdanov YD, Haucke V, Smart TG, Moss SJ (2004) Huntingtin- associated protein 1 regulates inhibitory synaptic transmission by modulating y-aminobutyric acid type A receptor membrane trafficking. Proc Natl Acad Sci U S A 101:12736-12741.

Klee CB, Crouch TH, Krinks MH (1979) Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Natl Acad Sci U S A 76:6270-6273.

Klee CB, Haiech J (1980) Concerted role of calmodulin and calcineurin in calcium regulation. Ann N Y Acad Sci 356:43-54.

Kloda JH, Czajkowski C (2007) Agonist-, antagonist-, and benzodiazepine-induced structural changes in the a l Metl 13-Leul32 region of the GABA a receptor. Mol Pharmacol 71:483-493.

Kneussel M, Brandstatter JH, Gasnier B, Feng G, Sanes JR, Betz H (2001) Gephyrin-independent clustering of postsynaptic GABAa receptor subtypes. Mol Cell Neurosci 17:973-982.

Kobayashi H, Shiraishi S, Yanagita T, Yokoo H, Yamamoto R, Minami S, Saitoh T, Wada A (2002) Regulation of voltage-dependent sodium channel expression in adrenal chromaffin cells: involvement of multiple calcium signaling pathways. Ann N Y Acad Sci 971:127-134.

Koenig JH, Ikeda K (1989) Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J Neurosci 9:3844- 3860.

Kofalvi A, Oliveira CR, Cunha RA (2006) Lack of evidence for functional TRPVl vanilloid receptors in rat hippocampal nerve terminals. Neurosci Lett 403:151-156.

Koga H, Ishibashi H, Shimada H, Jang IS, Nakamura TY, Nabekura J (2005) Activation of presynaptic GABAa receptors increases spontaneous glutamate release onto noradrenergic neurons of the rat locus coeruleus. Brain Res 1046:24-31.

Komulainen H, Bondy SC (1987) Transient elevation of intrasynaptosomal free calcium by . Brain Res 401:50-54.

Korpi ER, Grunder G, Luddens H (2002) Drug interactions at GABAa receptors. Prog Neurobiol 67:113-159.

Korpi ER, Mattila MJ, Wisden W, Luddens H (1997) GABAA-receptor subtypes: clinical efficacy and selectivity of benzodiazepine site ligands. Arm Med 29:275-282.

Koschak A, Reimer D, Huber I, Grabner M, Glossmaim H, Engel J, Striessnig J (2001) alD (Cay 1.3) subunits can form L-type Ca^'^ chamiels activating at negative voltages. J Biol Chem 276:22100-22106.

Koulen P, Brandstatter JH, Enz R, Bormaim J, Wassle H (1998) Synaptic clustering of GABAc receptor rho-subunits in the rat retina. Eur J Neurosci 10:115-127.

- 234 - Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM, Nonet ML (2001) A post­ docking role for active zone protein Rim. Nat Neurosci 4:997-1005.

Koyama S, Matsumoto N, Kubo C, Akaike N (2000) Presynaptic 5-HT] receptor-mediated modulation of synaptic GABA release in the mechanically dissociated rat amygdala neurons. J Physiol 529 Pt 2:373-383.

Kozlov AS, Angulo MC, Audinat E, Charpak S (2006) Target cell-specific modulation of neuronal activity by astrocytes. Proc Natl Acad Sci U S A 103:10058-10063.

Kmjevic K, Schwartz S (1966) Is y-aminobutyric acid an inhibitory transmitter? pp 1372-1374.

Kubota H, Katsurabayashi S, Moorhouse AJ, Murakami N, Koga H, Akaike N (2003) GABAb receptor transduction mechanisms, and cross-talk between protein kinases A and C, in GABAergic terminals synapsing onto neurons of the rat nucleus basalis of Meynert. J Physiol 551:263-276.

Kuffler SW, Edwards C (1958) Mechanism of y-aminobutyric acid (GABA) action and its relation to synaptic inhibition. J. Neurophysiol. 21(6):589-610.

Kuhse J, Betz H, Kirsch J (1995) The inhibitory glycine receptor: architecture, synaptic localization and molecular pathology of a postsynaptic ion-channel complex. Curr Opin Neurobiol 5:318-323.

Kulak JM, McIntosh JM, Yoshikami D, Clivera BM (2001) Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels. J Neurochem 77:1581-1589.

Kullmann DM (2000) Spillover and synaptic cross talk mediated by glutamate and GABA in the mammalian brain. Prog Brain Res 125:339-351.

Kumar S, Fleming RL, Morrow AL (2004) Ethanol regulation of y-aminobutyric acid A receptors: genomic and nongenomic mechanisms. Pharmacol Ther 101:211-226.

Kuner T, Augustine GJ (2000) A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 27:447-459.

Kuriyama K, Haber B, Sisken B, Roberts E (1966) The gamma-aminobutyric acid system in rabbit cerebellum, pp 846-852.

Kuromi H, Kidokoro Y (2000) Tetanic stimulation recruits vesicles from reserve pool via a cAMP- mediated process in Drosophila synapses. Neuron 27:133-143.

Kushner SA, Unterwald EM (2001) Chronic cocaine administration decreases the functional coupling of GABAb receptors in the rat ventral tegmental area as measured by baclofen-stimulated 35S-GTPgammaS binding. Life Sci 69:1093-1102.

Lafon-Cazal M, Viennois G, Kuhn R, Malitschek B, Pin JP, Shigemoto R, Bockaert J (1999) mGluR7-like receptor and GABAb receptor activation enhance neurotoxic effects of N-methyl-D- aspartate in cultured mouse striatal GABAergic neurones. Neuropharmacology 38:1631-1640.

Lambert JJ, Belelli D, Peden DR, Vardy AW, Peters JA (2003) Neurosteroid modulation of GABAa receptors. Progress in Neurobiology 71:67-80.

Lambert JJ, Harney SC, Belelli D, Peters JA (2001) Neurosteroid modulation of recombinant and synaptic GABAa receptors. Int Rev Neurobiol 46:177-205.

-235 - Lambert NA, Wilson WA (1993) Discrimination of post- and presynaptic GABAb receptor- mediated responses by tetrahydroaminoacridine in area CA3 of the rat hippocampus. J Neurophysiol 69:630-635.

LaPorte DC, Wierman BM, Storm DR (1980) Calcium-induced exposure of a hydrophobic surface on calmodulin. Biochemistry 19:3814-3819.

Lauri SE, Delany C, VR JC, Bortolotto ZA, Omstein PL, Isaac TR, Collingridge GL (2001) Synaptic activation of a presynaptic kainate receptor facilitates AMPA receptor-mediated synaptic transmission at hippocampal mossy fibre synapses. Neuropharmacology 41:907-915.

Laurie DJ, Seeburg PH, Wisden W (1992) The distribution of 13 GABAa receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J Neurosci 12:1063-1076.

Le KT, Villeneuve P, Ramjaun AR, McPherson PS, Beaudet A, Seguela P (1998) Sensory presynaptic and widespread somatodendritic immunolocalization of central ionotropic P2X ATP receptors. Neuroscience 83:177-190.

Lee A, Wong ST, Gallagher D, Li B, Storm DR, Scheuer T, Catterall WA (1999) Ca^Vcalmodulin binds to and modulates P/Q-type calcium channels. Nature 399:155-159.

Lee A, Zhou H, Scheuer T, Catterall WA (2003b) Molecular determinants of Ca^Vcalmodulin- dependent regulation of Ca^2.1 channels. Proc Natl Acad Sci U SA 100:16059-16064.

Leil TA, Chen ZW, Chang CS, Olsen RW (2004) GABAa receptor-associated protein traffics GABAa receptors to the plasma membrane in neurons. J Neurosci 24:11429-11438.

Leinekugel X, Khalilov I, McLean H, Gaillard O, Gaiarsa JL, Ben-Ari Y, Khazipov R (1999) GABA is the principal fast-acting excitatory transmitter in the neonatal brain. Adv Neurol 79:189- 201.

Leinekugel X, Tseeb V, Ben-Ari Y, Bregestovski P (1995) Synaptic GABAa activation induces Ca^^ rise in pyramidal cells and intemeurons from rat neonatal hippocampal slices. J Physiol 487 (2:319-329.

Lemos JR, Nowycky MC (1989) Two types of calcium channels co-exist in peptide-releasing vertebrate nerve terminals. Neuron 2:1419-1426.

Lesort M, Esclaire F, Yardin C, Hugon J (1997) NMDA induces apoptosis and necrosis in neuronal cultures. Increased APP immunoreactivity is linked to apoptotic cells. Neurosci Lett 221:213-216.

Letts VA, Felix R, Biddlecome GH, Arikkath J, Mahaffey CL, Valenzuela A, Bartlett FS, Mori Y, Campbell KP, Frankel WN (1998) The mouse stargazer gene encodes a neuronal Ca^^-channel gamma subunit. Nat Genet 19:340-347.

Li C, Davletov BA, Sudhof TC (1995a) Distinct Ca^^ and Sr^'*' binding properties of synaptotagmins. Definition of candidate Ca^"^ sensors for the fast and slow components of neurotransmitter release. J Biol Chem 270:24898-24902.

Li GD, Chiara DC, Sawyer GW, Husain SS, Olsen RW, Cohen JB (2006) Identification of a GABA a receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. J Neurosci 26:11599-11605.

-236 Li L, Chin LS, Shupliakov O, Brodin L, Sihra TS, Hvaiby O, Jensen V, Zheng D, McNamara JO, Greengard P .(1995) Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice. Proc Natl Acad Sci U S A 92:9235-9239.

Li YH, Han TZ (2007) Glycine binding sites of presynaptic NMDA receptors may tonically regulate glutamate release in the rat visual cortex. J Neurophysiol 97:817-823.

Liang H, DeMaria CD, Erickson MG, Mori MX, Alseikhan BA, Yue DT (2003) Unified mechanisms of Ca^'^ regulation across the Ca^^ channel family. Neuron 39:951-960.

Lidow MS (2003) Calcium signaling dysfunction in schizophrenia: a unifying approach. Brain Res Brain Res Rev 43:70-84.

Lievens JC, Woodman B, Mahal A, Bates GP (2002) Abnormal phosphorylation of synapsin I predicts a neuronal transmission impairment in the R6/2 Huntington's disease transgenic mice. Mol Cell Neurosci 20:638-648.

Linial M, Ilouz N, Pamas H (1997) Voltage-dependent interaction between the muscarinic ACh receptor and proteins of the exocytic machinery. J Physiol 504 ( Pt 2):251-258.

Lipscombe D, Pan JQ, Gray AC (2002) Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of Ca^al. Mol Neurobiol 26:21-44.

Lisman J, Schulman H, Cline H (2002) The molecular basis of CaMK II function in synaptic and behavioural memory. Nat Rev Neurosci 3:175-190.

Liu F, Wan Q, Pristupa ZB, Yu XM, Wang YT, Niznik HB (2000) Direct protein-protein coupling enables cross-talk between dopamine Dg and y-aminobutyric acid A receptors. Nature 403:274-280.

Liu G, Tsien RW (1995) Properties of synaptic transmission at single hippocampal synaptic boutons. Nature 375:404-408.

Liu QS, Patrylo PR, Gao XB, van den Pol AN (1999) Kainate acts at presynaptic receptors to increase GABA release from hypothalamic neurons. J Neurophysiol 82:1059-1062.

Liu Z, Neff RA, Berg DK (2006a) Sequential interplay of nicotinic and GABAergic signaling guides neuronal development. Science 314:1610-1613.

Liu ZW, Yang S, Zhang YX, Liu CH (2003) Presynaptic a-7 nicotinic acetylcholine receptors modulate excitatory synaptic transmission in hippocampal neurons. Sheng Li Xue Bao 55:731-735.

Llinas R, Gruner JA, Sugimori M, McGuinness TL, Greengard P (1991) Regulation by synapsin I and Ca^^-calmodulin-dependent protein kinase II of the transmitter release in squid giant synapse. J Physiol 436:257-282.

Llinas R, Sugimori M, Hillman DF, Cherksey B (1992a) Distribution and functional significance of the P-type, voltage-dependent Ca^"^ channels in the mammalian central nervous system. Trends Neurosci 15:351-355.

Llinas R, Sugimori M, Silver RB (1992b) Microdomains of high calcium concentration in a presynaptic terminal. Science 256:677-679.

Loebrich S, Bahring R, Katsuno T, Tsukita S, Kneussel M (2006) Activated radixin is essential for GABAa receptor a5 subunit anchoring at the actin cytoskeleton. FMBO J 25:987-999.

-237 - Loo DD, Eskandari S, Boorer KJ, Sarkar HK, Wright EM (2000) Role of Cl' in electrogenic Na"^- coupled cotransporters GATl and SGLTl. pp 37414-37422.

Lopes LV, Cunha RA, Kull B, Fredholm BB, Ribeiro JA (2002) Adenosine Aia receptor facilitation of hippocampal synaptic transmission is dependent on tonic A(l) receptor inhibition. Neuroscience 112:319-329.

Lopez MG, Albillos A, de la Puente MT, Borges R, Gandia L, Carbone E, Garcia AG, Artalejo AR (1994) Localized L-type calcium channels control exocytosis in cat chromaffin cells. Pflugers Arch 427:348-354.

Low K, Crestani F, Keist R, Benke D, Brunig I, Benson JA, Fritschy JM, Rulicke T, Bluethmann H, Mohler H, Rudolph U (2000) Molecular and neuronal substrate for the selective attenuation of anxiety. Science 290:131-134.

Lu J, Karadsheh M, Delpire E (1999) Developmental regulation of the neuronal-specific isoform of K-Cl cotransporter KCC2 in postnatal rat brains. J Neurobiol 39:558-568.

Lu KT, Wu CY, Cheng NC, Wo YY, Yang JT, Yen HH, Yang YL (2006) Inhibition of the Na+ -K+ -2Cr -cotransporter in choroid plexus attenuates traumatic brain injury-induced brain edema and neuronal damage. Eur J Pharmacol 548:99-105.

Lubin M, Erisir A, Aoki C (1999) Ultrastructural immunolocalization of the a l nAChR subunit in guinea pig medial prefrontal cortex. Ann N Y Acad Sci 868:628-632.

Luebke JI, Dunlap K, Turner TJ (1993) Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus. Neuron 11:895-902.

Lukyanetz EA, Piper TP, Sihra TS (1998) Calcineurin involvement in the regulation of high- threshold Ca^^ channels in NG108-15 (rodent neuroblastoma x glioma hybrid) cells. J Physiol 510 (2):371-385.

Luthi-Carter R, Strand A, Peters NL, Solano SM, Hollingsworth ZR, Menon AS, Frey AS, Spektor BS, Penney EB, Schilling G, Ross CA, Borchelt DR, Tapscott SJ, Young AB, Cha JH, Olson JM (2000) Decreased expression of striatal signaling genes in a mouse model of Huntington's disease. Hum Mol Genet 9:1259-1271.

Luu T, Gage PW, Tierney ML (2006) GABA increases both the conductance and mean open time of recombinant GABAa channels co-expressed with GABARAP. J Biol Chem 281:35699-35708.

Lydiard RB (2003) The role of GABA in anxiety disorders. J Clin Psychiatry 64 Suppl 3:21-27.

Lytle C, Xu JC, Biemesderfer D, Haas M, Forbush B, III (1992) The Na-K-Cl cotransport protein of shark rectal gland. I. Development of monoclonal antibodies, immunoaffinity purification, and partial biochemical characterization. J Biol Chem 267:25428-25437.

MacDermott AB, Role LW, Siegelbaum SA (1999) Presynaptic ionotropic receptors and the control of transmitter release. Annu Rev Neurosci 22:443-485.

MacDonald JF, Xiong ZG, Jackson MF (2006) Paradox of Ca^^ signaling, cell death and stroke. Trends Neurosci 29:75-81.

Macdonald RL, Olsen RW (1994) GABAa receptor channels. Annu Rev Neurosci 17:569-602.

-238- Maitra R, Reynolds JN (1999) Subunit dependent modulation of GABAa receptor function by neuroactive steroids. Brain Res 819:75-82.

Margeta-Mitrovic M, Jan YN, Jan LY (2001) Function of GBl and GB2 subunits in G protein coupling of GABA(B) receptors. Proc Natl Acad Sci U S A 98:14649-14654.

Marie D, Liu QY, Marie I, Chaudry S, Chang YH, Smith SV, Sieghart W, Fritschy JM, Barker JL (2001) GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA(A) autoreceptor/Cl- channels. J Neurosci 21:2343- 2360.

Martens S, Kozlov MM, McMahon HT (2007) How synaptotagmin promotes membrane fusion. Science 316:1205-1208.

Martin KF, Hannon S, Phillips I, Heal DJ (1992) Opposing roles for 5-HTjb and 5 -HT3 receptors in the control of 5-HT release in rat hippocampus in vivo. Br J Pharmacol 106:139-142.

Martin LJ, Furuta A, Blackstone CD (1998) AMPA receptor protein in developing rat brain: glutamate receptor - 1 expression and localization change at regional, cellular, and subcellular levels with maturation. Neuroscience 83:917-928.

Matsu-ura T, Konishi Y, Aoki T, Naranjo JR, Mikoshiba K, Tamura TA (2002) Seizure-mediated neuronal activation induces DREAM gene expression in the mouse brain. Brain Res Mol Brain Res 109:198-206.

Matsubara M, Kusubata M, Ishiguro K, Uchida T, Titani K, Taniguchi H (1996) Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions. J Biol Chem 271:21108-21113.

Mattson MP (2002) Oxidative stress, perturbed calcium homeostasis, and immune dysfunction in Alzheimer's disease. J Neurovirol 8:539-550.

Maycox PR, Jahn R (1990) Reconstitution of neurotransmitter carriers from synaptic vesicles. Biochem Soc Trans 18:381-384.

Mayer ML, Westbrook GL, Guthrie PB (1984) Voltage-dependent block by Mg^"^ of NMDA responses in spinal cord neurones. Nature 309:261-263.

McDonald BJ, Amato A, Connolly CN, Benke D, Moss SJ, Smart TG (1998) Adjacent phosphorylation sites on GABA a receptor p subunits determine regulation by cAMP-dependent protein kinase. Nat Neurosci 1:23-28.

McDonald BJ, Moss SJ (1994) Differential phosphorylation of intracellular domains of gamma- aminobutyric acid type A receptor subunits by calcium/calmodulin type 2-dependent protein kinase and cGMP-dependent protein kinase. J Biol Chem 269:18111-18117.

McDonald BJ, Moss SJ (1997) Conserved phosphorylation of the intracellular domains of GABAA receptor P2 and P3 subunits by cAMP-dependent protein kinase, cGMP-dependent protein kinase protein kinase C and Ca^Vcalmodulin type Il-dependent protein kinase. Neuropharmacology 36:1377-1385.

McDonough SI, Swartz KJ, Mintz IM, Boland LM, Bean BP (1996) Inhibition of calcium channels in rat central and peripheral neurons by œ-conotoxin MVIIC. J Neurosci 16:2612-2623.

239 - McGehee DS, Heath MJ, Gelber S, Devay P, Role LW (1995) Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science 269:1692-1696.

McGlade-McCulloh E, Yamamoto H, Tan SB, Brickey DA, Soderling TR (1993) Phosphorylation and regulation of glutamate receptors by calcium/calmodulin-dependent protein kinase II. Nature 362:640-642.

McKeman RM, et al. (2000) Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABAa receptor a l subtype. Nat Neurosci 3:587-592.

McMahon HT, Nicholls DG (1991h) Transmitter glutamate release from isolated nerve terminals: evidence for biphasic release and triggering by localized Ca^^. J Neurochem 56:86-94.

Mellon SH, Griffin LD, Compagnone NA (2001) Biosynthesis and action of neurosteroids. Brain Res Brain Res Rev 37:3-12.

Menegon A, Bonanomi D, Albertinazzi C, Lotti F, Ferrari G, Kao HT, Benfenati F, Baldelli P, Valtorta F (2006) Protein kinase A-mediated synapsin I phosphorylation is a central modulator of Ca^^-dependent synaptic activity. J Neurosci 26:11670-11681.

Meyer DK, Olenik C, Hofmann F, Barth H, Leemhuis J, Brunig I, Aktories K, Norenberg W (2000) Regulation of somatodendritic GABAa receptor channels in rat hippocampal neurons: evidence for a role of the small GTPase Racl. J Neurosci 20:6743-6751.

Meyer T, Hanson PI, Stryer L, Schulman H (1992) Calmodulin trapping by calcium-calmodulin- dependent protein kinase. Science 256:1199-1202.

Micheau J, Riedel G (1999) Protein kinases: which one is the memory molecule? Cell Mol Life Sci 55:534-548.

Mihalek RM, Banerjee PK, Korpi ER, Quinlan JJ, Firestone LL, Mi ZP, Lagenaur C, Tretter V, Sieghart W, Anagnostaras SG, Sage JR, Fanselow MS, Guidotti A, Spigelman I, Li Z, DeLorey TM, Olsen RW, Homanics GE (1999) Attenuated sensitivity to neuroactive steroids in y- aminobutyrate type A receptor Ô subunit knockout mice. Proc Natl Acad Sci U S A 96:12905- 12910.

Mikami A, Imoto K, Tanabe T, Niidome T, Mori Y, Takeshima H, Narumiya S, Numa S (1989) Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature 340:230-233.

Millan C, Sanchez-Prieto J (2002) Differential coupling of N- and P/Q-type calcium channels to glutamate exocytosis in the rat cerebral cortex. Neurosci Lett 330:29-32.

Miller LP, Martin DL (1973) An artifact in the radiochemical assay of brain mitochondrial glutamate decarboxylase, pp 1023-1032.

Milligan CJ, Buckley NJ, Garret M, Deuchars J, Deuchars SA (2004) Evidence for inhibition mediated by coassembly of GABAa and G ABAc receptor subunits in native central neurons. J Neurosci 24:7241-7250.

Mintz IM, Sabatini BL, Regehr WG (1995) Calcium control of transmitter release at a cerebellar synapse. Neuron 15:675-688.

240 Mintz IM, Venema VJ, Swiderek KM, Lee TD, Bean BP, Adams ME (1992) P-type calcium channels blocked by the spider toxin co-Aga-IVA. Nature 355:827-829.

Misgeld U, Muller W, Brunner H (1989) Effects of (-)baclofen on inhibitory neurons in the guinea pig hippocampal slice. Pflugers Arch 414:139-144.

Mi sura KM, May AP, Weis W1 (2000) Protein-protein interactions in intracellular membrane fusion. Curr Opin Struct Biol 10:662-671.

Mitchell PR, Martin, IL (1978) Is GABA release modulated by presynaptic receptors? Nature 274:904.

Miyazawa A, Fujiyoshi Y, Unwin N (2003) Structure and gating mechanism of the acetylcholine receptor pore. Nature 423:949-955.

Mochida S, Westenbroek RE, Yokoyama CT, Itoh K, Catterall WA (2003) Subtype-selective reconstitution of synaptic transmission in sympathetic ganglion neurons by expression of exogenous calcium channels. Proc Natl Acad Sci U S A 100:2813-2818.

Molina-Hemandez A, Nunez A, Sierra JJ, Rias-Montano JA (2001) Histamine H 3 receptor activation inhibits glutamate release from rat striatal synaptosomes. Neuropharmacology 41:928- 934.

Moss FJ, Viard P, Davies A, Bertaso F, Page KM, Graham A, Canti C, Plumpton M, Plumpton C, Clare JJ, Dolphin AC (2002) The novel product of a five-exon stargazin-related gene abolishes Cay2.2 calcium channel expression. EMBO J 21:1514-1523.

Moss SJ, Doherty CA, Huganir RL (1992a) Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the pl, y2S, and y2L subunits of the gamma-aminobutyric acid type A receptor. J Biol Chem 267:14470-14476.

Moss SJ, Smart TG, Blackstone CD, Huganir RL (1992b) Functional modulation of GABAa receptors by cAMP-dependent protein phosphorylation. Science 257:661-665.

Moss SJ, Gorrie GH, Amato A, Smart TG (1995) Modulation of GABAa receptors by tyrosine phosphorylation. Nature 377:344-348.

Moss SJ, Smart TG (1996) Modulation of amino acid-gated ion channels by protein phosphorylation. Int Rev Neurobiol 39:1-52.

Moss SJ, Smart TG (2001) Constructing inhibitory synapses. Nature Reviews Neuroscience 2:240- 250.

Munoz A, Mendez P, DeFelipe J, Alvarez-Leefmans FJ (2007) Cation-chloride cotransporters and GABA-ergic innervation in the human epileptic hippocampus. Epilepsia 48:663-673.

Murthy VN, De Camilli CP (2003) Cell biology of the presynaptic terminal. Annu Rev Neurosci 26:701-728.

Nabekura J, Ueno T, Okabe A, Furuta A, Iwaki T, Shimizu-Okabe C, Fukuda A, Akaike N (2002) Reduction of KCC2 expression and GABAa receptor-mediated excitation after in vivo axonal injury. J Neurosci 22:4412-4417.

241 - Nachshen DA, Blaustein MP (1982) Influx of calcium, strontium, and barium in presynaptic nerve endings. J Gen Physiol 79:1065-1087.

Nakai J, Sekiguchi N, Rando TA, Allen PD, Beam KG (1998) Two regions of the ryanodine receptor involved in coupling with L-type Ca^^ channels. J Biol Chem 273:13403-13406.

Nakanishi K, Yamada J, Takayama C, Oohira A, Fukuda A (2007) NKCCl activity modulates formation of functional inhibitory synapses in cultured neocortical neurons. Synapse 61:138-149.

Nakanishi S (1992) Molecular diversity of glutamate receptors and implications for brain function. Science 258:597-603.

Nakatsuka T, Gu JG (2001) ATP P2X receptor-mediated enhancement of glutamate release and evoked EPSCs in dorsal horn neurons of the rat spinal cord. J Neurosci 21:6522-6531.

Nayak SV, Ronde P, Spier AD, Lummis SC, Nichols RA (1999) Calcium changes induced by presynaptic 5-hydroxytryptamine-3 serotonin receptors on isolated terminals from various regions of the rat brain. Neuroscience 91:107-117.

Nerbonne JM, Gurney AM (1987) Blockade of Ca^^ and K^ currents in bag cell neurons of Aplysia califomica by dihydropyridine Ca^^ antagonists. J Neurosci 7:882-893.

Newcomb R, Szoke B, Palma A, Wang G, Chen X, Hopkins W, Cong R, Miller J, Urge L, Tarczy- Homoch K, Loo JA, Dooley DJ, Nadasdi L, Tsien RW, Lemos J, Miljanich G (1998) Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry 37:15353-15362.

Newell JG, Czajkowski C (2003) The GABAa receptor a l subunit Prol74-Aspl91 segment is involved in GABA binding and channel gating. J Biol Chem 278:13166-13172.

Newell JG, McDevitt RA, Czajkowski C (2004) Mutation of glutamate 155 of the GABAa receptor p2 subunit produces a spontaneously open channel: a trigger for channel activation. J Neurosci 24:11226-11235.

Newton AJ, Kirchhausen T, Murthy VN (2006) Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis. Proc Natl Acad Sci U S A 103:17955-17960.

Nguyen L, Malgrange B, Breuskin I, Bettendorff L, Moonen G, Belachew S, Rigo JM (2003) Autocrine/paracrine activation of the GABA a receptor inhibits the proliferation of neurogenic polysialylated neural cell adhesion molecule-positive (PSA-NCAM4-) precursor cells from postnatal striatum. J Neurosci 23:3278-3294.

Nguyen PV, Woo NH (2003) Regulation of hippocampal synaptic plasticity by cyclic AMP- dependent protein kinases. Prog Neurobiol 71:401-437.

Nicholls DG (2003) Bioenergetics and transmitter release in the isolated nerve terminal. Neurochem Res 28:1433-1441.

Nicholls DG, Coffey ET (1994) Glutamate exocytosis from isolated nerve terminals. Adv Second Messenger Phosphoprotein Res 29:189-203.

Nicholls DG, Sihra TS (1986) Synaptosomes posses an exocytotic pool of glutamate. Nature 321:772-773.

- 242 - Nicholls DG, Sihra TS, Sanchez-Prieto J (1987) The role of the plasma membrane and intracellular organelles in synaptosomal calcium regulation. Soc Gen Physiol Ser 42:31-43.

Nichols RA, Sihra TS, Czernik AJ, Naim AC, Greengard P (1990) Calcium/calmodulin-dependent protein kinase II increases glutamate and noradrenaline release from synaptosomes. Nature 343:647-651.

Nilius B, Hess P, Lansman JB, Tsien RW (1985) A novel type of cardiac calcium channel in ventricular cells. Nature 316:443-446.

Norris CM, Blalock EM, Chen KC, Porter NM, Landfield PW (2002) Calcineurin enhances L-type Ca^^ channel activity in hippocampal neurons: increased effect with age in culture. Neuroscience 110:213-225.

North RA, Barnard EA (1997) Nucleotide receptors. Curr Opin Neurobiol 7:346-357.

Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A (1984) gates glutamate- activated channels in mouse central neurones. Nature 307:462-465.

Nowycky MC, Fox AP, Tsien RW (1985a) Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644. Proc Natl Acad Sci U SA 82:2178-2182.

Nusser Z, Mody I (2002) Selective modulation of tonic and phasic inhibitions in dentate gyms granule cells. J Neurophysiol 87:2624-2628.

Nusser Z, Naylor D, Mody I (2001) Synapse-specific contribution of the variation of transmitter concentration to the decay of inhibitory postsynaptic currents. Biophys J 80:1251-1261.

Nusser Z, Sieghart W, Benke D, Fritschy JM, Somogyi P (1996) Differential synaptic localization of two major y-aminobutyric acid type A receptor a subunits on hippocampal pyramidal cells. Proc Natl Acad Sci U S A 93:11939-11944.

Nymann-Andersen J, Wang H, Chen L, Kittler JT, Moss SJ, Olsen RW (2002a) Subunit specificity and interaction domain between GABA a receptor-associated protein (GABARAP) and GABA a receptors. J Neurochem 80:815-823.

Nymann-Andersen J, Wang H, Olsen RW (2002b) Biochemical identification of the binding domain in the GABAa receptor-associated protein (GABARAP) mediating dimer formation. Neuropharmacology 43:476-481.

O'Sullivan GA, Kneussel M, Elazar Z, Betz H (2005) GABARAP is not essential for GABA receptor targeting to the synapse. Eur J Neurosci 22:2644-2648.

Obrietan K, van den Pol AN (1995) GABA neurotransmission in the hypothalamus: developmental reversal from Ca^^ elevating to depressing. J Neurosci 15:5065-5077.

Ohno-Shosaku T, Hirata K, Sawada S, Yamamoto C (1994) Contributions of multiple calcium channel types to GABAergic transmission in rat cultured hippocampal neurons. Neurosci Lett 181:145-148.

Okamoto N, Hori S, Akazawa C, Hayashi Y, Shigemoto R, Mizuno N, Nakanishi S (1994) Molecular characterization of a new metabotropic glutamate receptor mGluR7 coupled to inhibitory cyclic AMP signal transduction. J Biol Chem 269:1231-1236.

-243 - Ong J, Kerr DI (2000) Recent advances in GABAb receptors: from pharmacology to molecular biology. Acta Pharmacol Sin 21:111-123.

Omstein L (1964) Disc electrophoresis. 1. Background and theory. Ann N Y Acad Sci 121:321-349.

Ouimet CC, McGuinness TL, Greengard P (1984) Immunocytochemical localization of calcium/calmodulin-dependent protein kinase 11 in rat brain. Proc Natl Acad Sci U S A 81:5604- 5608.

Owens DF, Boyce LH, Davis MB, Kriegstein AR (1996) Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci 16:6414-6423.

Pagani R, Song M, McEnery M, Qin N, Tsien RW, Toro L, Stefani E, Uchitel OD (2004) Differential expression of alpha 1 and beta subunits of voltage dependent Ca^^ channel at the neuromuscular junction of normal and P/Q Ca^^ channel knockout mouse. Neuroscience 123:75-85.

Palma E, Amici M, Sobrero F, Spinelli G, Di AS, Ragozzino D, Mascia A, Scoppetta C, Esposito V, Miledi R, Eusebi F (2006) Anomalous levels of Cl' transporters in the hippocampal subiculum from temporal lobe epilepsy patients make GABA excitatory. Proc Natl Acad Sci U S A 103:8465- 8468.

Pan ZH, Hu HJ, Perring P, Andrade R (2001) T-type Ca^^ channels mediate neurotransmitter release in retinal bipolar cells. Neuron 32:89-98.

Panzanelli P, Homanics GE, Ottersen OP, Fritschy JM, Sassoe-Pognetto M (2004) Pre- and postsynaptic GABA receptors at reciprocal dendrodendritic synapses in the olfactory bulb. Eur J Neurosci 20:2945-2952.

Papazian DM, Timpe LC, Jan YN, Jan LY (1991) Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence. Nature 349:305-310.

Parfitt KD, Madison DV (1993) Phorbol esters enhance synaptic transmission by a presynaptic, calcium-dependent mechanism in rat hippocampus. J Physiol 471:245-268.

Payne JA, Rivera C, Voipio J, Kaila K (2003) Cation-chloride co-transporters in neuronal communication, development and trauma. Trends Neurosci 26:199-206.

Payne JA, Stevenson TJ, Donaldson LF (1996) Molecular characterization of a putative K-Cl cotransporter in rat brain. A neuronal-specific isoform. J Biol Chem 271:16245-16252.

Pearce BR, Freedman EG, Dutton GR (1982) Autoreceptors modify the evoked release of [^HjGABA from cerebellar neurons in dissociated cell culture. Eur J Pharmacol 82:131-135.

Pende M, Lanza M, Bonanno G, Raiteri M (1993) Release of endogenous glutamic and aspartic acids from cerebrocortex synaptosomes and its modulation through activation of a y-aminobutyric acid B (GABAb) receptor subtype. Brain Res 604:325-330.

Pereverzev A, Klockner U, Henry M, Grabsch H, Vajna R, Olyschlager S, Viatchenko-Karpinski S, Schroder R, Hescheler J, Schneider T (1998) Stmctural diversity of the voltage-dependent Ca 2+ channel a 1 E-subunit. Eur J Neurosci 10:916-925.

Perez-Reyes E (2003) Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev 83:117-161.

- 244 - Perkinton MS, Sihra TS (1998) Presynaptic GABA b receptor modulation of glutamate exocytosis from rat cerebrocortical nerve terminals: receptor decoupling by protein kinase C. J Neurochem 70:1513-1522.

Perkinton MS, Sihra TS (1999) A high-affinity presynaptic kainate-type glutamate receptor facilitates glutamate exocytosis from cerebral cortex nerve terminals (synaptosomes). Neuroscience 90:1281-1292.

Pemey TM, Himing LD, Leeman SE, Miller RJ (1986) Multiple calcium channels mediate neurotransmitter release from peripheral neurons. Proc Natl Acad Sci U S A 83:6656-6659.

Peterson BZ, DeMaria CD, Adelman JP, Yue DT (1999) Calmodulin is the Ca^^ sensor for Ca^^ - dependent inactivation of L-type calcium channels. Neuron 22:549-558.

Pevsner J, Hsu SC, Braun JE, Calakos N, Ting AE, Bennett MK, Scheller RH (1994) Specificity and regulation of a synaptic vesicle docking complex. Neuron 13:353-361.

Pickles HO (1979) Presynaptic y-aminobutyric acid responses in the olfactory cortex. Br J Pharmacol 65:223-228.

Pin JP, Duvoisin R (1995) The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34:1-26.

Plotkin MD, Kaplan MR, Peterson LN, Gullans SR, Hebert SC, Delpire E (1997) Expression of the Na^-K^-2Cr cotransporter BSC2 in the nervous system. Am J Physiol 272:C173-C183.

Plummer MR, Logothetis DE, Hess P (1989) Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons. Neuron 2:1453-1463.

Pocock JM, Murphie HM, Nicholls DG (1988) inhibits the synaptosomal plasma membrane glutamate carrier and allows glutamate leakage from the cytoplasm but does not affect glutamate exocytosis J Neurochem 50(3):745-51.

Pocock JM, Venema VJ, Adams ME (1992) co-agatoxins differentially block calcium channels in locust, chick and rat synaptosomes. Neurochem Int 20:263-270.

Poisik O, Raju DV, Verreault M, Rodriguez A, Abeniyi GA, Conn PJ, Smith Y (2005) Metabotropic glutamate receptor 2 modulates excitatory synaptic transmission in the rat globus pallidus. Neuropharmacology 49 Suppl 1:57-69.

Porton B, Ferreira A, DeLisi LE, Kao HT (2004) A rare polymorphism affects a mitogen-activated protein kinase site in synapsin III: possible relationship to schizophrenia. Biol Psychiatry 55:118- 125.

Pouzat C, Marty A (1999) Somatic recording of GABAergic autoreceptor current in cerebellar stellate and basket cells. J Neurosci 19:1675-1690.

Price CJ, Karayannis T, Pal BZ, Capogna M (2005) Group II and III mGluRs-mediated presynaptic inhibition of EPSCs recorded from hippocampal intemeurons of CAI stratum lacunosum moleculare. Neuropharmacology 49 Suppl 1:45-56.

Price GD, Tmssell LG (2006) Estimate of the chloride concentration in a central glutamatergic terminal: a gramicidin perforated-patch study on the calyx of Held. J Neurosci 26:11432-11436.

245 - Prince DA, Stevens CF (1992) Adenosine decreases neurotransmitter release at central synapses. Proc Natl Acad Sci U S A 89:8586-8590.

Pritchett DB, Sontheimer H, Gorman CM, Kettenmann H, Seeburg PH, Schofield PR (1988) Transient expression shows ligand gating and allosteric potentiation of GABAa receptor subunits. Science 242:1306-1308.

Qian J, Noebels JL (2001) Presynaptic Ca2-f- channels and neurotransmitter release at the terminal of a mouse cortical neuron. J Neurosci 21:3721-3728.

Qin S, Hu XY, Xu H, Zhou JN (2004) Regional alteration of synapsin I in the hippocampal formation of Alzheimer's disease patients. Acta Neuropathol (Berl) 107:209-215.

Randall A, Tsien RW (1995) Pharmacological dissection of multiple types of Ca^^ channel currents in rat cerebellar granule neurons. J Neurosci 15:2995-3012.

Randall AD, Tsien RW (1997) Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels. Neuropharmacology 36:879-893.

Rathenberg J, Kittler JT, Moss SJ (2004) Palmitoylation regulates the clustering and cell surface stability of GABA a receptors. Mol Cell Neurosci 26:251-257.

Raymond S, Weintraub L (1959) Acrylamide gel as a supporting medium for zone electrophoresis. Science 130:711.

Regan U (1991) Voltage-dependent calcium currents in Purkinje cells from rat cerebellar vermis. J Neurosci 11:2259-2269.

Regan LJ, Sah DW, Bean BP (1991) Ca^^ channels in rat central and peripheral neurons: high- threshold current resistant to dihydropyridine blockers and omega-conotoxin. Neuron 6:269-280.

Reid CA, Bekkers JM, Clements JD (1998) N- and P/Q-type Ca^^ channels mediate transmitter release with a similar cooperativity at rat hippocampal autapses. J Neurosci 18:2849-2855.

Reid CA, Bekkers JM, Clements JD (2003) Presynaptic Ca^^ channels: a functional patchwork. Trends Neurosci 26:683-687.

Reshkin SJ, Lee SI, George JN, Turner RJ (1993) Identification, characterization and purification of a 160 kD bumetanide-binding glycoprotein from the rabbit parotid. J Membr Biol 136:243-251.

Rhee JS, Ishibashi H, Akaike N (1999) Calcium channels in the GABAergic presynaptic nerve terminals projecting to meynert neurons of the rat. J Neurochem 72:800-807.

Rhee SG, Bae YS (1997) Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem 272:15045-15048.

Richards DA, Guatimosim C, Rizzoli SO, Betz WJ (2003) Synaptic vesicle pools at the frog neuromuscular junction. Neuron 39:529-541.

Richards JG, Schoch P, Haring P, Takacs B, Mohler H (1987) Resolving GABAA/benzodiazepine receptors: cellular and subcellular localization in the CNS with monoclonal antibodies. J Neurosci 7:1866-1886.

246 Richerson GB, Wu Y (2003) Dynamic equilibrium of neurotransmitter transporters: not just for reuptake anymore. J Neurophysiol 90:1363-1374.

Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K (1999) The KVCl' co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397:251-255.

Roberts E, Frankel S (1950) y-Aminobutyric acid in brain: its formation from glutamic acid, pp 55- 63.

Robinson PJ, Dunkley PR (1983) Depolarisation-dependent protein phosphorylation in rat cortical synaptosomes: factors determining the magnitude of the response. J Neurochem 41:909-918.

Rodriguez FJ, Lluch M, Dot J, Blanco I, Rodriguez-Alvarez J (1997) Histamine modulation of glutamate release from hippocampal synaptosomes. Eur J Pharmacol 323:283-286.

Rodriguez-Moreno A, Herreras O, Lerma J (1997) Kainate receptors presynaptically downregulate GABAergic inhibition in the rat hippocampus. Neuron 19:893-901.

Rodriguez-Moreno A, Sihra TS (2004) Presynaptic kainate receptor facilitation of glutamate release involves protein kinase A in the rat hippocampus. J Physiol 557:733-745.

Roettger VR, Amara SG (1999) GABA and glutamate transporters: therapeutic and etiologic implications for epilepsy. Adv Neurol 79:551-560.

Ronde P, Nichols RA (1998) High calcium permeability of serotonin 5 -HT3 receptors on presynaptic nerve terminals from rat striatum. J Neurochem 70:1094-1103.

Ropert N, Guy N (1991) Serotonin facilitates GABAergic transmission in the CAI region of rat hippocampus in vitro. J Physiol 441:121-136.

Rosen A, Bali M, Horenstein J, Akabas MH (2007) Channel opening by anesthetics and GABA induces similar changes in the GABAa receptor M2 segment. Biophys J 92:3130-3139.

Rothman JE (1994) Mechanisms of intracellular protein transport. Nature 372:55-63.

Rothman SM (1985) The neurotoxicity of excitatory amino acids is produced by passive chloride influx. J Neurosci 5:1483-1489.

Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, Martin JR, Bluethmann H, Mohler H (1999) Benzodiazepine actions mediated by specific y-aminobutyric acid (A) receptor subtypes. Nature 401:796-800.

Rudomin P, Schmidt RF (1999) Presynaptic inhibition in the vertebrate spinal cord revisited. Exp Brain Res 129:1-37.

Ruiz A, Fabian-Fine R, Scott R, Walker MC, Rusakov DA, Kullmann DM (2003) GABAa receptors at hippocampal mossy fibers. Neuron 39:961-973.

Russell JM (2000) Sodium-potassium-chloride cotransport. Physiol Rev 80:211-276.

Ryan TA, Reuter H, Wendland B, Schweizer FE, Tsien RW, Smith SJ (1993) The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron 11:713-724.

- 247 - Saalmann YB, Kirkcaldie MT, Waldron S, Calford MB (2007) Cellular distribution of the GABAa receptor-modulating 3a-hydroxy, 5a-reduced pregnane steroids in the adult rat brain. J Neuroendocrinol 19:272-284.

Sabatini BL, Svoboda K (2000) Analysis of calcium channels in single spines using optical fluctuation analysis. Nature 408:589-593.

Sakaba T, Neher E (2003) Direct modulation of synaptic vesicle priming by GABAb receptor activation at a glutamatergic synapse. Nature 424:775-778.

Sanchez-Prieto J, Sihra TS, Nicholls DG (1987) Characterization of the exocytotic release of glutamate from guinea-pig cerebral cortical synaptosomes. J Neurochem 49:58-64.

Sanna E, Murgia A, Casula A, Biggio G (1997) Differential subunit dependence of the actions of the general anesthetics alphaxalone and etomidate at y-aminobutyric acid type A receptors expressed in Xenopus laevis oocytes. Mol Pharmacol 51:484-490.

Saridaki E, Carter DA, Lightman SL (1989) y-Aminobutyric acid regulation of neurohypophysial hormone secretion in male and female rats. J Endocrinol 121:343-349.

Sassoe-Pognetto M, Fritschy JM (2000) Mini-review: gephyrin, a major postsynaptic protein of GABAergic synapses. Eur J Neurosci 12:2205-2210.

Satake S, Saitow F, Rusakov D, Konishi S (2004) AMPA receptor-mediated presynaptic inhibition at cerebellar GABAergic synapses: a characterization of molecular mechanisms. Eur J Neurosci 19:2464-2474.

Satake S, Saitow F, Yamada J, Konishi S (2000) Synaptic activation of AMPA receptors inhibits GABA release from cerebellar intemeurons. Nat Neurosci 3:551-558.

Scanziani M, Capogna M, Gahwiler BH, Thompson SM (1992) Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus. Neuron 9:919-927.

Schaerer MT, Buhr A, Baur R, Sigel E (1998) Amino acid residue 200 on the a l subunit of GABAa receptors affects the interaction with selected benzodiazepine binding site ligands. Eur J Pharmacol 354:283-287.

Scheffer IE, Berkovic SF (2003) The genetics of human epilepsy. Trends Pharmacol Sci 24:428- 433.

Schiavo G, Stenbeck G, Rothman JE, Sollner TH (1997) Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses. Proc Natl Acad Sci U S A 94:997-1001.

Schiebler W, Jahn R, Doucet JP, Rothlein J, Greengard P (1986) Characterization of synapsin I binding to small synaptic vesicles. J Biol Chem 261:8383-8390.

Schmitz D, Mellor J, Frerking M, Nicoll RA (2001) Presynaptic kainate receptors at hippocampal mossy fiber synapses. Proc Natl Acad Sci U S A 98:11003-11008.

Schoch S, Gundelfinger ED (2006) Molecular organization of the presynaptic active zone. Cell Tissue Res 326:379-391.

-248 Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H, Rhee LM, Ramachandran J, Reale V, Glencorse TA, . (1987) Sequence and functional expression of the GABAa receptor shows a ligand-gated receptor super-family. Nature 328:221-227.

Schousboe A (2003) Role of astrocytes in the maintenance and modulation of glutamatergic and GABAergic neurotransmission, pp 347-352.

Schuler V, Luscher C, Blanchet C, Klix N, Sansig G, Klebs K, Schmutz M, Heid J, Gentry C, Urban L, Fox A, Spooren W, Jaton AL, Vigouret J, Pozza M, Kelly PH, Mosbacher J, Froestl W, Kaslin E, Korn R, Bischoff S, Kaupmann K, van der Putten H, Bettler B. (2001) Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABAb responses in mice lacking GABAei. Neuron 31:47-58.

Semyanov A, Walker MC, Kullmann DM, Silver RA (2004) Tonically active GABAa receptors: modulating gain and maintaining the tone. Trends Neurosci 27:262-269.

Sen A, Martinian L, Nikolic M, Walker MC, Thom M, Sisodiya SM (2007) Increased NKCCl expression in refractory human epilepsy. Epilepsy Res 74:220-227.

Sharma G, Vijayaraghavan S (2003) Modulation of presynaptic store calcium induces release of glutamate and postsynaptic firing. Neuron 38:929-939.

Sheehan JP, Swerdlow RH, Parker WD, Miller SW, Davis RE, Tuttle JB (1997) Altered calcium homeostasis in cells transformed by mitochondria from individuals with Parkinson's disease. J Neurochem 68:1221-1233.

Sherman AD, Hegwood TS, Baruah S, Waziri R (1992) Presynaptic modulation of amino acid release from synaptosomes. Neurochem Res 17:125-128.

Shimizu-Okabe C, Yokokura M, Okabe A, Ikeda M, Sato K, Kilb W, Luhmann HJ, Fukuda A (2002) Layer-specific expression of Cl" transporters and differential [Cl'], in newborn rat cortex. Neuroreport 13:2433-2437.

Shirataki H, Kaibuchi K, Sakoda T, Kishida S, Yamaguchi T, Wada K, Miyazaki M, Takai Y (1993) Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin. Mol Cell Biol 13:2061-2068.

Shivers BD, Killisch I, Sprengel R, Sontheimer H, Kohler M, Schofield PR, Seeburg PH (1989) Two novel GABAa receptor subunits exist in distinct neuronal subpopulations. Neuron 3:327-337.

Sieghart W (1995) Structure and pharmacology of y-aminobutyric acid A receptor subtypes. Pharmacol Rev 47:181-234.

Sieghart W, Sperk G (2002) Subunit composition, distribution and function of GABAa receptor subtypes. Curr Top Med Chem 2:795-816.

Sigel E (2002) Mapping of the benzodiazepine recognition site on GABAa receptors. Curr Top Med Chem 2:833-839.

Sigel E, Baur R (1988) Allosteric modulation by benzodiazepine receptor ligands of the GABAa receptor channel expressed in Xenopus oocytes. J Neurosci 8:289-295.

Sigel E, Baur R, Kellenberger S, Malherbe P (1992) Point mutations affecting antagonist affinity and agonist dependent gating of GABAa receptor channels. EMBO J 11:2017-2023.

- 249 - Sigel E, Baur R, Trube G, Mohler H, Malherbe P (1990) The effect of subunit composition of rat brain GABAareceptors on channel function. Neuron 5:703-711.

Sigel E, Buhr A (1997) The benzodiazepine binding site of GABAa receptors. Trends Pharmacol Sci 18:425-429.

Sihra, TS (1997) Protein phosphorylation and dephosphorylation in isolated nerve terminals (synaptosomes). In: Neuromethods (30): Regulatory protein modification: Techniques and protocols. Ed: HC Jennings Jr. Humana Press Inc.

Sihra TS, Bogonez E, Nicholls DG (1992) Localized Ca^"^ entry preferentially effects protein dephosphorylation, phosphorylation, and glutamate release. J Biol Chem 267:1983-1989.

Sihra TS, Naim AC, Kloppenburg P, Lin Z, Pouzat C (1995) A role for calcineurin (protein phosphatase-2B) in the regulation of glutamate release. Biochem Biophys Res Commun 212:609- 616.

Sihra TS, Nicholls DG (1987) 4-Aminobutyrate can be released exocytotically from guinea-pig cerebral cortical synaptosomes. J Neurochem 49:261-267.

Sihra TS, Piomelli D, Nichols RA (1993) Barium evokes glutamate release from rat brain synaptosomes by membrane depolarization: involvement of K^, Na"^, and Ca^^ channels. J Neurochem 61:1220-1230.

Sihra TS, Wang JK, Gorelick FS, Greengard P (1989) Translocation of synapsin I in response to depolarization of isolated nerve terminals. Proc Natl Acad Sci U S A 86:8108-8112.

Simmonds MA (1981) Distinction between the effects of barbiturates, benzodiazepines and on responses to y-aminobutyric acid receptor activation and antagonism by bicuculline and picrotoxin. Br J Pharmacol 73:739-747.

Simmons ML, Terman GW, Gibbs SM, Chavkin C (1995) L-type calcium channels mediate dynorphin neuropeptide release from dendrites but not axons of hippocampal granule cells. Neuron 14:1265-1272.

Singer D, Biel M, Lotan I, Flockerzi V, Hofmann F, Dascal N (1991) The roles of the subunits in the function of the calcium channel. Science 253:1553-1557.

Sipila ST, Schuchmann S, Voipio J, Yamada J, Kaila K (2006) The cation-chloride cotransporter NKCCl promotes sharp waves in the neonatal rat hippocampus. J Physiol 573:765-773.

Sjostrom PJ, Turrigiano GG, Nelson SB (2003) Neocortical LTD via coincident activation of presynaptic NMD A and cannabinoid receptors. Neuron 39:641-654.

Smith GB, Olsen RW (1994) Identification of a [^H]muscimol photoaffinity substrate in the bovine y-aminobutyric acid A receptor a subunit. J Biol Chem 269:20380-20387.

Smith TC, Herlihy JT, Robinson SC (1981) The effect of the fluorescent probe, 3,3'- dipropylthiadicarbocyanine iodide, on the energy metabolism of Ehrlich ascites tumor cells. J Biol Chem 256:1108-1110.

Snodgrass SR (1978) Use of ^H-muscimol for GABA receptor studies. Nature 273:392-394.

-250 Soldatov NM (2003) channel moving tail: link between Ca^^-induced inactivation and Ca^^ signal transduction. Trends Pharmacol Sci 24:167-171.

Soliakov L, Wonnacott S (1996) Voltage-sensitive Ca^"^ channels involved in nicotinic receptor- mediated [^HJdopamine release from rat striatal synaptosomes. J Neurochem 67:163-170.

Soliakov L, Wonnacott S (2001) Involvement of protein kinase C in the presynaptic nicotinic modulation of -dopamine release from rat striatal synaptosomes. Br J Pharmacol 132:785-791.

Song M, Messing RO (2005) Protein kinase C regulation of GABAa receptors. Cell Mol Life Sci 62:119-127.

Spoerri PE (1988) Neurotrophic effects of GABA in cultures of embryonic chick brain and retina. Synapse 2:11-22.

Staley K, Smith R (2001) A new form of feedback at the GABA a receptor. Nat Neurosci 4:674-676.

Stanley EF (1997) The calcium channel and the organization of the presynaptic transmitter release face. Trends Neurosci 20:404-409.

Stell BM, Brickley SG, Tang CY, Farrant M, Mody I (2003) Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABAa receptors. Proc Natl Acad Sci U S A 100:14439-14444.

Stimson DT, Estes PS, Rao S, Krishnan KS, Kelly LE, Ramaswami M (2001) Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization. J Neurosci 21:3034-3044.

Stocker M (2004) Ca^^-activated channels: molecular determinants and function of the SK family. Nat Rev Neurosci 5:758-770.

Stoffel-Wagner B (2003) Neurosteroid biosynthesis in the human brain and its clinical implications. Ann N Y Acad Sci 1007:64-78.

Stuart GJ, Redman SJ (1992) The role of GABAa and GABAb receptors in presynaptic inhibition of la EPSPs in cat spinal motoneurones. J Physiol 447:675-692.

Stutzmann GE (2005) Calcium dysregulation, IP 3 signaling, and Alzheimer's disease. Neuroscientist 11:110-115.

Su G, Kintner DB, Sun D (2002) Contribution of Na^-K^-Cl' cotransporter to high-[K^]o- induced swelling and EAA release in astrocytes. Am J Physiol Cell Physiol 282:C1136-C1146.

Sudhof TC, Jahn R (1991) Proteins of synaptic vesicles involved in exocytosis and membrane recycling, pp 665-677.

Sugita S, Uchimura N, Jiang ZG, North RA (1991) Distinct muscarinic receptors inhibit release of y-aminobutyric acid and excitatory amino acids in mammalian brain. Proc Natl Acad Sci U S A 88:2608-2611.

Szabadics J, Varga C, Molnar G, Olah S, Barzo P, Tamas G (2006) Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits. Science 311:233-235.

Tachibana M, Okada T, Arimura T, Kobayashi K (1993) Dihydropyridine-sensitive calcium current mediates neurotransmitter release from retinal bipolar cells. Ann N Y Acad Sci 707:359-361.

-251 - Tafoya LC, Mameli M, Miyashita T, Guzowski JF, Valenzuela CF, Wilson MC (2006) Expression and function of SNAP-25 as a universal SNARE component in GABAergic neurons, J Neurosci 26:7826-7838.

Takago H, Nakamura Y, Takahashi T (2005) G protein-dependent presynaptic inhibition mediated by AMPA receptors at the calyx of Held. Proc Natl Acad Sci U S A 102:7368-7373.

Takahashi M, Seagar MJ, Jones JF, Reber BF, Catterall WA (1987) Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle. Proc Natl Acad Sci U S A 84:5478-5482.

Takahashi T, Momiyama A (1993) Different types of calcium channels mediate central synaptic transmission. Nature 366:156-158.

Takeshita Y, Watanabe T, Sakata T, Munakata M, Ishibashi H, Akaike N (1998) Histamine modulates high-voltage-activated calcium channels in neurons dissociated from the rat tuberomammillary nucleus. Neuroscience 87:797-805.

Tanabe T, Beam KG, Powell JA, Numa S (1988) Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature 336:134-139.

Tanabe T, Takeshima H, Mikami A, Flockerzi V, Takahashi H, Kangawa K, Kojima M, Matsuo H, Hirose T, Numa S (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328:313-318.

Tanaka C, Nishizuka Y (1994) The protein kinase C family for neuronal signaling. Annu Rev Neurosci 17:551-567.

Tanaka T, Saito H, Matsuki N (1997) Inhibition of GABAa synaptic responses by brain-derived neurotrophic factor (BDNF) in rat hippocampus. J Neurosci 17:2959-2966.

Teissere JA, Czajkowski C (2001) A ^-strand in the y2 subunit lines the benzodiazepine binding site of the GABAA receptor: structural rearrangements detected during channel gating. J Neurosci 21:4977-4986.

Terunuma M, Jang IS, Ha SH, Kittler JT, Kanematsu T, Jovanovic JN, Nakayama KI, Akaike N, Ryu SH, Moss SJ, Hirata M (2004) GABAA receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein. J Neurosci 24:7074-7084.

Thomas GM, Huganir RL (2004) MAPK cascade signalling and synaptic plasticity. Nat Rev Neurosci 5:173-183.

Thomas P, Mortensen M, Hosie AM, Smart TG (2005) Dynamic mobility of functional GABAa receptors at inhibitory synapses. Nat Neurosci 8:889-897.

Thompson SM, Gahwiler BH (1992) Comparison of the actions of baclofen at pre- and postsynaptic receptors in the rat hippocampus in vitro. J Physiol 451:329-345.

Tibbs GR, Barrie AP, Van Mieghem FJ, McMahon HT, Nicholls DG (1989a) Repetitive action potentials in isolated nerve terminals in the presence of 4-aminopyridine: effects on cytosolic free Ca^^ and glutamate release. J Neurochem 53:1693-1699.

252 - Tokunaga T, Miyazaki K, Koseki M, Mobarakeh JI, Ishizuka T, Yawo H (2004) Pharmacological dissection of calcium channel subtype-related components of strontium inflow in large mossy fiber boutons of mouse hippocampus. Hippocampus 14:570-585.

Torri TF, Bossi M, Fesce R, Greengard P, Valtorta F (1992) Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation. Neuron 9:1143-1153.

Toselli M, Taglietti V (1992) Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons. Pflugers Arch 421:59-66.

Toyoda H, Ohno K, Yamada J, Ikeda M, Okabe A, Sato K, Hashimoto K, Fukuda A (2003) Induction of NMDA and GABAa receptor-mediated Ca^"^ oscillations with KCC2 mRNA downregulation in injured facial motoneurons. J Neurophysiol 89:1353-1362.

Treiman DM (2001) GABAergic mechanisms in epilepsy. Epilepsia 42 Suppl 3:8-12.

Tsien RW, Lipscombe D, Madison D, Bley K, Fox A (1995) Reflections on Ca^^-channel diversity, 1988-1994. Trends Neurosci 18:52-54.

Tsunemi T, Saegusa H, Ishikawa K, Nagayama S, Murakoshi T, Mizusawa H, Tanabe T (2002) Novel Cay2.1 splice variants isolated from Purkinje cells do not generate P-type Ca2+ current. J Biol Chem 277:7214-7221.

Tsutsui M, Yanagihara N, Miyamoto B, Kuroiwa A, Izumi F (1994) Correlation of activation of Ca^Ycalmodulin-dependent protein kinase II with catecholamine secretion and tyrosine hydroxylase activation in cultured bovine adrenal medullary cells. Mol Pharmacol 46:1041-1047.

Turecek R, Trussell LO (2001) Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse. Nature 411:587-590.

Turecek R, Trussell LO (2002) Reciprocal developmental regulation of presynaptic ionotropic receptors. Proc Natl Acad Sci U S A 99:13884-13889.

Turner TJ, Lampe RA, Dunlap K (1995) Characterization of presynaptic calcium channels with (O- conotoxin MVIIC and co-grammotoxin SLA: role for a resistant calcium channel type in neurosecretion. Mol Pharmacol 47:348-353.

Turner TJ, Mokler DJ, Luebke JI (2004) Calcium influx through presynaptic 5 -HT3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies. Neuroscience 129:703-718.

Twyman RE, Rogers CJ, Macdonald RL (1989) Differential regulation of y-aminobutyric acid receptor channels by diazepam and phénobarbital. Ann Neurol 25:213-220.

Tyzio R, Cossait R, Khalilov I, Minlebaev M, Hubner CA, Represa A, Ben-Ari Y, Khazipov R (2006) Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery. Science 314:1788-1792.

Ubach J, Garcia J, Nittler MP, Sudhof TC, Rizo J (1999) Structure of the Janus-faced C2B domain of rabphilin. Nat Cell Biol 1:106-112.

Vacher CM, Bettler B (2003) GABAb receptors as potential therapeutic targets. Curr Drug Targets CNS Neurol Disord 2:248-259.

-253 Valtorta F, Greengard P, Fesce R, Chieregatti E, Benfenati F (1992) Effects of the neuronal phosphoprotein synapsin I on actin polymerization. I. Evidence for a phosphorylation-dependent nucleating effect. J Biol Chem 267:11281-11288. van den Pol AN, Obrietan K, Chen 0 (1996) Excitatory actions of GABA after neuronal trauma. J Neurosci 16:4283-4292.

Van Den Bosch BL, Vandenberghe W, Klaassen H, Van Houtte HE, Robberecht W (2000) Ca^^- permeable AMPA receptors and selective vulnerability of motor neurons. J Neurol Sci 180:29-34.

Van der Zee EA, Douma BR (1997) Historical review of research on protein kinase C in learning and memory. Prog Neuropsychopharmacol Biol Psychiatry 21:379-406.

Van Der Stelt SM, Di Marzo M, V (2004) Endovanilloids. Putative endogenous ligands of transient receptor potential vanilloid 1 channels. Eur J Biochem 271:1827-1834. van Rijnsoever RC, Sidler C, Fritschy JM (2005) Internalized GABA-receptor subunits are transferred to an intracellular pool associated with the postsynaptic density. Eur J Neurosci 21:327- 338. van Rijnsoever RC, Tauber M, Choulli MK, Keist R, Rudolph U, Mohler H, Fritschy JM, Crestani F (2004) Requirement of «5-GABAa receptors for the development of tolerance to the sedative action of diazepam in mice. J Neurosci 24:6785-6790.

Vawter MP, Thatcher L, Usen N, Hyde TM, Kleinman JE, Freed WJ (2002) Reduction of synapsin in the hippocampus of patients with bipolar disorder and schizophrenia. Mol Psychiatry 7:571-578.

Vazquez E, Sanchez-Prieto J (1997) Presynaptic modulation of glutamate release targets different calcium channels in rat cerebrocortical nerve terminals. Eur J Neurosci 9:2009-2018.

Verderio C, Pozzi D, Pravettoni E, Inverardi F, Schenk U, Coco S, Proux-Gillardeaux V, Galli T, Rossetto O, Frassoni C, Matteoli M (2004) SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization. Neuron 41:599-610.

Verhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, Vermeer H, Toonen RF, Hammer RE, van den Berg TK, Missler M, Geuze HJ, Sudhof TC (2000) Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287:864-869.

Verhage M, McMahon HT, Ghijsen WE, Boomsma F, Scholten G, Wiegant VM, Nicholls DG (1991) Differential release of amino acids, neuropeptides, and catecholamines from isolated nerve terminals. Neuron 6:517-524.

Vetiska SM, Ahmadian G, Ju W, Liu L, Wymann MP, Wang YT (2007) GABAa receptor- associated phosphoinositide 3-kinase is required for insulin-induced recruitment of postsynaptic GABAa receptors. Neuropharmacology 52:146-155.

Victor RG, Rusnak F, Sikkink R, Marban E, ORourke B (1997) Mechanism of Ca^^-dependent inactivation of L-type Ca2+ channels in GH3 cells: direct evidence against dephosphorylation by calcineurin. J Membr Biol 156:53-61.

Villalonga P, Lopez-Alcala C, Bosch M, Chiloeches A, Rocamora N, Gil J, Marais R, Marshall CJ, Bachs O, Agell N (2001) Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling. Mol Cell Biol 21:7345-7354.

254 - von Gerdsoff OH, Matthews G (1996) Calcium-dependent inactivation of calcium current in synaptic terminals of retinal bipolar neurons. J Neurosci 16:115-122.

Vulchanova L, Arvidsson U, Riedl M, Wang J, Buell G, Surprenant A, North RA, Elde R (1996) Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry. Proc Natl Acad Sci U S A 93:8063-8067.

Wafford KA, Bain CJ, Quirk K, McKeman RM, Wingrove PB, Whiting PJ, Kemp JA (1994) A novel allosteric modulatory site on the GABAA receptor beta subunit. Neuron 12:775-782.

Wafford KA, Thompson SA, Thomas D, Sikela J, Wilcox AS, Whiting PJ (1996) Functional characterization of human y-aminobutyric acid A receptors containing the oA subunit. Mol Pharmacol 50:670-678.

Wafford KA, Whiting P, Kemp JA (1992) Functional modulation of cloned GABAa receptors expressed in Xenopus oocytes. Adv Biochem Psychopharmacol 47:75-79.

Wagner DA, Czajkowski C (2001) Structure and dynamics of the GABA binding pocket: A narrowing cleft that constricts during activation. J Neurosci 21:67-74.

Walker SA, Lockyer PJ, Cullen PJ (2003) The Ras binary switch: an ideal processor for decoding complex Ca^^ signals? Biochem Soc Trans 31:966-969.

Wallis T, Bubb WA, McQuillan JA, Balcar VJ, Rae C (2004) For want of a nail, ramifications of a single gene deletion, dystrophin, in the brain of the mouse. Front Biosci 9:74-84.

Wallner M, Hanchar HJ, Olsen RW (2003) Ethanol enhances a4(335 and ot6(330 y-aminobutyric acid type A receptors at low concentrations known to affect humans. Proc Natl Acad Sci U S A 100:15218-15223.

Wan Q, Xiong ZG, Man HY, Ackerley CA, Braunton J, Lu WY, Becker LE, MacDonald JF, Wang YT (1997) Recruitment of functional GABAa receptors to postsynaptic domains by insulin. Nature 388:686-690.

Wang G, Dayanithi G, Newcomb R, Lemos JR (1999) An R-type Ca^^ current in neurohypophysial terminals preferentially regulates oxytocin secretion. J Neurosci 19:9235-9241.

Wang H, Bedford FK, Brandon NJ, Moss SJ, Olsen RW (1999c) GABAA-receptor-associated protein links GABAa receptors and the cytoskeleton. Nature 397:69-72.

Wang JK (1991) Presynaptic glutamate receptors modulate dopamine release from striatal synaptosomes. J Neurochem 57:819-822.

Wang JK, Andrews H, Thukral V (1992) Presynaptic glutamate receptors regulate noradrenaline release from isolated nerve terminals. J Neurochem 58:204-211.

Wang L, Kitai ST, Xiang Z (2005) Modulation of excitatory synaptic transmission by endogenous glutamate acting on presynaptic group II mGluRs in rat substantia nigra compacta. J Neurosci Res 82:778-787.

Wang M, He Y, Eisenman LN, Fields C, Zeng CM, Mathews J, Benz A, Fu T, Zorumski E, Steinbach JH, Covey DF, Zorumski CF, Mennerick S (2002) 3p-hydroxypregnane steroids are -like GABAa receptor antagonists. J Neurosci 22:3366-3375.

255 - Wang Q, Liu L, Pei L, Ju W, Ahmadian G, Lu J, Wang Y, Liu F, Wang YT (2003) Control of synaptic strength, a novel function of Akt. Neuron 38:915-928.

Wang SJ, Sihra TS (2003) Opposing facilitatory and inhibitory modulation of glutamate release elicited by cAMP production in cerebrocortical nerve terminals (synaptosomes). Neuropharmacology 44:686-697.

Wang X, Zhong P, Y an Z (2002) Dopamine D 4 receptors modulate GABAergic signaling in pyramidal neurons of prefrontal cortex. J Neurosci 22(21): 9185-9193.

Wassef A, Baker J, Kochan LD (2003) GABA and schizophrenia: a review of basic science and clinical studies. J Clin Psychopharmacol 23:601-640.

Wei W, Faria LC, Mody I (2004) Low ethanol concentrations selectively augment the tonic inhibition mediated by delta subunit-containing GABA a receptors in hippocampal neurons. J Neurosci 24:8379-8382.

Welsby PJ, Wang H, Wolfe JT, Colbran RJ, Johnson ML, Barrett PQ (2003) A mechanism for the direct regulation of T-type calcium channels by Ca^Ycalmodulin-dependent kinase II. J Neurosci 23:10116-10121.

Westenbroek RE, Ahlijanian MK, Catterall WA (1990) Clustering of L-type Ca^^ channels at the base of major dendrites in hippocampal pyramidal neurons. Nature 347:281-284.

Wheeler DB, Randall A, Tsien RW (1994) Roles of N-type and Q-type Ca^^ channels in supporting hippocampal synaptic transmission. Science 264:107-111.

Whiting PJ (1999) The GABAa receptor gene family: new targets for therapeutic intervention. Neurochem Int 34:387-390.

Whiting PJ, McAllister G, Bonnert TP, Heavens RP, Smith DW, Hewson L, ODonnell R, Rigby MR, Sirinathsinghji DJ, Marshall G, Thompson SA, Wafford KA, Vasilatis D (1997) Neuronally restricted RNA splicing regulates the expression of a novel GABA a receptor subunit conferring atypical functional properties. Journal of Neuroscience 17:5027-537.

Whiting P, McKeman RM, Iversen LL. (1990) Another mechanism for creating diversity in y- aminobutyrate type A receptors: RNA splicing directs expression of two forms of y2 phosphorylation site. Proc Natl Acad Sci USA. 87(24):9966-70.

Wieland HA, Luddens H, Seeburg PH (1992) A single histidine in GABAa receptors is essential for benzodiazepine agonist binding. J Biol Chem 267:1426-1429.

Williams JR, Sharp JW, Kumari VG, Wilson M, Payne JA (1999) The neuron-specific K-Cl cotransporter, KCC2. Antibody development and initial characterization of the protein. J Biol Chem 274:12656-12664.

Williams ME, Feldman DH, McCue AF, Brenner R, Velicelebi G, Ellis SB, Harpold MM (1992) Stmcture and functional expression of a l, a2, and (3 subunits of a novel human neuronal calcium channel subtype. Neuron 8:71-84.

Williams RW, Herrup K (1988) The control of neuron number. Annu Rev Neurosci 11:423-453.

- 256 - Witcher DR, De WM, Sakamoto J, Franzini-Armstrong C, Pragneli M, Kahl SD, Campbell KP (1993) Subunit identification and reconstitution of the N-type Ca^^ channel complex purified from brain. Science 261:486-489.

Wohlfarth KM, Bianchi MT, Macdonald RL (2002) Enhanced neurosteroid potentiation of ternary GABAa receptors containing the Ô subunit. J Neurosci 22:1541-1549.

Wolff JR, Joo F, Dames W (1978) Plasticity in dendrites shown by continuous GABA administration in superior cervical ganglion of adult rat. Nature 274:72-74.

Wonnacott S (1997) Presynaptic nicotinic ACh receptors. Trends Neurosci 20:92-98.

Woodhall G, Evans DI, Cunningham MO, Jones RS (2001) NR2B-containing NMDA autoreceptors at synapses on entorhinal cortical neurons. J Neurophysiol 86:1644-1651.

Wright SC, Schellenberger U, Ji L, Wang H, Larrick JW (1997) Calmodulin-dependent protein kinase II mediates signal transduction in apoptosis. FASEB J 11:843-849.

Wu LG, Borst JG, Sakmann B (1998b) R-type Ca^^ currents evoke transmitter release at a rat central synapse. Proc Natl Acad Sci U S A 95:4720-4725.

Wu LG, Saggau P (1997) Presynaptic inhibition of elicited neurotransmitter release. Trends Neurosci 20:204-212.

Xiao C, Zhou C, Li K, Ye JH (2007) Presynaptic GABAa receptors facilitate GABAergic transmission to dopaminergic neurons in the ventral tegmental area of young rats. J Physiol 580:731-743.

Xu JC, Lytle C, Zhu TT, Payne JA, Benz E Jr, Forbush B, III (1994) Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter. Proc Natl Acad Sci U SA 91:2201-2205.

Xu W, Lipscombe D (2001) Neuronal CavL3al L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. J Neurosci 21:5944-5951.

Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, Fukuda A (2004) Cl- uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCCl. J Physiol 557:829-841.

Yanagihori S, Terunuma M, Koyano K, Kanematsu T, Ho RS, Hirata M (2006) Protein phosphatase regulation by PRIP, a PLC-related catalytically inactive protein-implications in the phospho- modulation of the GABAa receptor. Adv Enzyme Regul 46:203-222.

Yang E, Schulman H (1999) Structural examination of autoregulation of multifunctional calcium/calmodulin-dependent protein kinase II. J Biol Chem 274:26199-26208.

Yang J, Woodhall GL, Jones RS (2006) Tonic facilitation of glutamate release by presynaptic NR2B-containing NMDA receptors is increased in the entorhinal cortex of chronically epileptic rats. J Neurosci 26:406-410.

Yang SH, Armson PF, Cha J, Phillips WD (1997) Clustering of GABAa receptors by rapsyn/43kD protein in vitro. Mol Cell Neurosci 8:430-438.

- 257 - Yang Z, Webb TI, Lynch JW (2007) Closed-state cross-linking of adjacent betal subunits in alpha 1 beta 1 GABAa receptors via introduced 6' cysteines. J Biol Chem 282:16803-16810.

Yao J, Davies LA, Howard JD, Adney SK, Welsby PJ, Howell N, Carey RM, Colbran RJ, Barrett PQ (2006) Molecular basis for the modulation of native T-type Ca^^ channels in vivo by Ca^Ycalmodulin-dependent protein kinase II. J Clin Invest 116:2403-2412.

Yarwood SJ, Steele MR, Scotland G, Houslay MD, Bolger GB (1999) The RACKl signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem 274:14909-14917.

Yasuda R, Sabatini BL, Svoboda K (2003) Plasticity of calcium channels in dendritic spines. Nat Neurosci 6:948-955.

Yatani A, Brown AM (1985) The calcium channel blocker nitrendipine blocks sodium channels in neonatal rat cardiac myocytes. Circ Res 56:868-875.

Yawo H, Chuhma N (1993a) Preferential inhibition of co-conotoxin-sensitive presynaptic Ca^^ channels by adenosine autoreceptors. Nature 365:256-258.

Ye J (2000) Physiology and pharmacology of native glycine receptors in developing rat ventral tegmental area neurons. Brain Res 862:74-82.

Ye JH, Wang F, Kmjevic K, Wang W, Xiong ZG, Zhang J (2004) Presynaptic glycine receptors on GABAergic terminals facilitate discharge of dopaminergic neurons in ventral tegmental area. J Neurosci 24:8961-8974.

Yuste R, Katz LC (1991) Control of postsynaptic Ca^^ influx in developing neocortex by excitatory and inhibitory neurotransmitters. Neuron 6:333-344.

Zazpe A, Artaiz I, Del RJ (1994) Role of 5 -HT3 receptors in basal and K^-evoked dopamine release from rat olfactory tubercle and striatal slices. Br J Pharmacol 113:968-972.

Zeilhofer HU, Blank NM, Neuhuber WL, Swandulla D (2000) Calcium-dependent inactivation of neuronal calcium channel currents is independent of calcineurin. Neuroscience 95:235-241.

Zeller A, Arras M, Jurd R, Rudolph U (2007) Mapping the contribution of (33-containing GABAa receptors to volatile and intravenous general anesthetic actions. BMC Pharmacol 7:2.

Zhang JF, Randall AD, Ellinor PT, Horne WA, Sather WA, Tanabe T, Schwarz TL, Tsien RW (1993) Distinctive pharmacology and kinetics of cloned neuronal Ca^^ channels and their possible counterparts in mammalian CNS neurons. Neuropharmacology 32:1075-1088.

Zhang JZ, Davletov BA, Sudhof TC, Anderson RG (1994) Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78:751-760.

Zhang SJ, Jackson MB (1995) GABAa receptor activation and the excitability of nerve terminals in the rat posterior pituitary. J Physiol 483 (3):583-595.

Zhang SJ, Jackson MB (1993) GABA-activated chloride channels in secretory nerve endings. Science 259:531-534.

-258 - Zhang W, Basile AS, Gomeza J, Volpicelli LA, Levey AI, Wess J (2002a) Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock­ out mice. J Neurosci 22:1709-1717.

Zhang W, Yamada M, Gomeza J, Basile AS, Wess J.(2002b) Multiple muscarinic acetylcholine receptor subtypes modulate striatal dopamine release, as studied with M 1-M5 muscarinic receptor knock-out mice. J Neurosci. 22(15):6347-52.

Zucker RS, Fogelson AL (1986) Relationship between transmitter release and presynaptic calcium influx when calcium enters through discrete channels. Proc Natl Acad Sci U SA 83:3032-3036.

Zucker RS, Regehr WG (2002) Short-term synaptic plasticity. Annu Rev Physiol 64:355-405.

Zuhlke RD, Pitt GS, Deisseroth K, Tsien RW, Reuter H (1999) Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399:159-162.

-259