G‐PROTEIN COUPLED RECEPTOR MEDIATED METAPLASTICITY AT THE HIPPOCAMPAL CA1 SYNAPSE
BY
BIKRAMPAL SINGH SIDHU
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto ©Copyright by Bikrampal Singh Sidhu (2009)
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G‐PROTEIN COUPLED RECEPTOR MEDIATED METAPLASTICITY AT THE HIPPOCAMPAL CA1 SYNAPSE By Bikrampal Singh Sidhu Master of Science Department of Physiology University of Toronto 2009
Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.
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Acknowledgements Writing acknowledgements seems like a lose‐lose situation. On the one hand, there are tens if not hundreds of people who have helped me get to where I am today and I will undoubtedly not have space to thank them all; on the other hand, there is no way I could complete this work without thanking them. And so in advance, to everyone who thinks there name ought to appear here, and it doesn’t, rest assured that I am grateful for all of your contributions, physical, mental, emotional, intellectual or even financial. Without them, this work wouldn’t have been possible.
The first person I would specifically like to thank is John MacDonald. I appreciate the opportunities you have given me, from having faith in me the first time around in the lab and then seeing something (who knows what?) and bringing me back again and again. Thank you for fostering and perhaps even instilling a love for research and teaching that I hope I can carry through to my career.
To all the people who helped me out, Mike J, Mike B, Hongbin, Waldy: for holding my hand through various periods of cluelessness. To them and everyone else in the lab, Lidia, Ella, Cat, Rohit, Oies, Nat, Kai: for keeping me sane (because we know I’m at least slightly off my rocker) and making the lab a place I wanted to be.
And of course, my experience wouldn’t have been complete without my roommate Michelle. I strain to understand how on earth you put up with my crazies. How on earth I put up with yours is unfathomable. (Go Team!)
To my committee, Drs. Charlton, Josselyn, Wojtowicz, I have to thank you for all the positive feedback and help in facilitating this thesis.
Lastly, but never leastly, I have to thank my family for supporting my academic aims always. I appreciate your pushing me to finish what I set my mind to, and doing everything you can to help me get there.
Two degrees. Maybe only two more to go. One day I’ll finish with school and all of you will have had a tremendous part to play. Thank you.
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I. Abstract…ii II. Acknowledgements…iii III. Table of Contents…iv IV. List of Figures…vi V. Abbreviations Used…vii VI. Section One – Introduction…1 1.1 Synaptic Transmission…2 1.1.1 Excitatory Neurotransmission…4 1.1.2 Glutamate Receptors…4 1.1.3 AMPA Receptors…5 1.1.4 NMDA Receptors…5 1.1.5 NMDA Receptor Subunits…7 1.2 G‐protein Coupled Receptors…10 1.2.1 Pituitary Adenylate Cyclase Activating Polypeptide…13 1.2.2PAC1 Receptor…15 1.2.3 PAC1 Receptor Signal Transduction…16 1.2.4 Dopamine…17 1.2.5 D1 Receptor…19 1.2.6 D1 Receptor Signal Transduction…19 1.3 Hippocampus…21 1.4 Synaptic Plasticity…22 1.4.1NMDA Receptor Dependent Synaptic Plasticity…23 1.4.2 Src Family Kinases, NMDARs and Synaptic Plasticity…26 1.4.3 Metaplasticity…28 VII. Section Two – Rationale and Hypothesis…31 2.1 GPCRs Modulate NMDA Receptor Function…32 2.1.1 GPCR Mediated Effects are Subunit Specific…32 2.2 NR2 Subunit Contributions to Metaplasticity…36 2.3 Hypothesis…36 VIII. Section Three – Methods…38 3.1 Hippocampal Slice Recordings…39 3.2 Animals…41 3.3 Drugs and Peptides…41 3.4 Statistical Analysis…41 IX. Section Four – Results…43 4.1 PACAP38 has no effect on baseline synaptic transmission…44 4.2 PACAP38 alters synaptic plasticity induction…46 4.3 PACAP38 mediated reversal of LTD at 10Hz is PAC1R dependent…53 4.4 D1 agonist SKF81297 alters synaptic plasticity induction…55 4.5 SCH23390 blocks the effect of SKF81297 at 10Hz…62 4.6 Neither PACAP38, nor SKF81297 alters presynaptic release…64 4.7 PAC1 antagonist M65 attenuates LTP induction at 100Hz…67 4.8 PACAP38 preferentially increases NR2A phosphorylation…69 4.9 SKF81297 preferentially increases NR2B phosphorylation…71
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X. Section Five – Discussion…74 5.1 Summary of Key Findings…75 5.2 PAC1R and D1R Modulation of NMDARs…75 5.3 PAC1 and D1 Receptor Mediated Effects on Synaptic Plasticity…77 5.4 Functional Target Specificity…80 5.5 Physiological Specificity of PAC1 and D1 Activation…82 5.6 Extensions and Future Directions…83 5.7 Overall Conclusions…85 XI. Section Six – References…87
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List of Figures Figure 1.1 The Hippocampus, Synapse and Excitatory Neurotransmission…3
Figure 1.2 The NMDA Receptor…9
Figure 1.3 The Gq‐signaling Pathway…11
Figure 1.4 The Gs‐signaling Pathway…12
Figure 1.5 NMDA Receptor Dependent Long‐term Potentiation…25
Figure 1.6 The Bienenstock‐Cooper‐Munro Model of Metaplasticity…30
Figure 2.1 PACAP38 Mediated Modulation of NMDA Receptors…34
Figure 2.2 D1 Mediated Modulation of NMDA Receptors…35
Figure 3.1 Acutely Prepared Hippocampal Slice Recordings…42
Figure 4.1 Effect of PACAP38 on Baseline Evoked fEPSPs…45
Figure 4.2 Effects of PACAP38 on Induction of Synaptic Plasticity…47
Figure 4.3 M65 Blocks Effects of PACAP38 at 10Hz…54
Figure 4.4 Effects of SKF‐81297 on Induction of Synaptic Plasticity…56
Figure 4.5 SCH23390 Blocks Effects of SKF81297 at 10Hz…63
Figure 4.6 Effects of PACAP38, SKF81297, and SCH23390 on Presynaptic Release…66
Figure 4.7 M65 Mediated Attenuation of LTP Induction…68
Figure 4.8 PACAP38 Induces NR2A Phosphorylation…70
Figure 4.9 SKF81297 Induces NR2B Phosphorylation…72
Figure 4.10 NR2B Phosphorylation by SKF81297 is Fyn mediated…73
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Abbreviations Used
AC – adenylyl cyclase AMPA(R) – ‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazolepropionic acid (receptor) ATP – adenosine triphosphate BCM – Bienenstock‐Cooper‐Munro CAK/Pyk2 – cell adhesion kinase /proline rich tyrosine kinase 2 CaMKII – calcium/calmodulin‐dependent protein kinase II cAMP – 3’,5’‐cyclic adenosine monophosphate Csk – C‐terminal Src kinase DAG ‐ diacylglycerol DG – dentate gyrus EC – entorhinal cortex EPSP/EPSC – excitatory postsynaptic potential/current ER – endoplasmic reticulum GPCR – G‐protein coupled receptor IP3(R) – inositol triphosphate (receptor) LTD/LTP – long‐term depression/potentiation mGluR – metabotropic glutamate receptor NMDA(R) – N‐methyl‐D‐aspartate (receptor) PACAP38/PACAP27 – pituitary adenylyl cyclase activating polypeptide with 38/27 residues PIP2 – phosphatidylinositol‐4,5‐bisphosphate PKA – cAMP dependent protein kinase A PKC – protein kinase C PLC – phospholipase C PP1/PP2B – protein phosphatase 1/protein phosphatase 2B (calcineurin) PPi ‐ pyrophasphate PTP – protein tyrosine phosphatase RACK1 – receptor for activated c‐kinase 1 SCH‐23390 – R(+)‐7‐chloro‐8‐hydroxy‐3‐methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐ benzazepine SFK – Src family kinase SKF‐81297 – 6‐chloro‐2,3,4,5‐tetrahydro‐1‐phenyl‐1H‐3‐benzazepine STEP/PTPN5 – striatal enriched phosphatase VDCC – voltage dependent calcium channel VIP – vasoactive intestinal peptide
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SECTION ONE
INTRODUCTION
1
The mammalian hippocampus is a requisite structure for the formation and
consolidation but not storage of long‐term memories. The prevalent theories state that
at the cellular level this is achieved by long‐lasting changes in synaptic strength, termed
synaptic plasticity. In the CA1 region of the hippocampus, synaptic plasticity has been
determined to be dependent on the N‐methyl‐D‐aspartate (NMDA) receptor and is
modifiable by upstream signaling pathways. Altered signaling of the NMDA receptor has
therefore been studied with respect to the ability to learn and remember as well as
being implicated in pathological signaling states, particularly schizophrenia.
1.1 Synaptic Transmission
The transmission of signals between neurons is achieved at specialized locations termed
synapses. Synapses themselves are made up of a presynaptic neuron terminal, or
bouton, a postsynaptic membrane, and the intervening space called the synaptic cleft.
Transmission is begun by the firing of an action potential in the presynaptic cell, and the
propagation of this action potential to the presynaptic terminal. There, voltage‐
dependent calcium channels (VDCCs) rapidly increase intracellular calcium (Ca2+)
concentrations and cause the fusion of docked synaptic vesicles immediately inside the
presynaptic membrane (Rizo and Rosenmund, 2008). The fusion of these vesicles results
in the exocytosis and diffusion of the contained neurotransmitter across the synaptic
cleft. The transmitter binds its postsynaptic receptors, where the effects are dependent
on the receptor and cell type (Figure 1.1D; Rizo and Rosenmund, 2008).
2
Figure 1.1 The Hippocampus, Synapse, and Excitatory Neurotransmission A. Position of the hippocampus in the human brain. The hippocampus is positioned underneath the medial temporal cortex of the mammalian brain (Gray, 1918). B. A schematic of the circuitry of the hippocampus showing the CA regions, entorhinal cortex, subiculum, and dentate gyrus (Ramon y Cajal, 1952). C. A scanning electron micrograph clearly showing a synaptic regions (s1,s2). Transmitter vesicles are clearly shown in the axon termini (At1,At2) and the postsynaptic density is shown in the dendrite (Den) (Peters et al., 1991). D. A simplified schematic of basal excitatory neurotransmission. Glutamate release from the presynaptic bouton activates AMPA and NMDA receptors. The NMDA receptor is blocked by magnesium and the AMPA receptor allows the influx of sodium. A postsynaptic depolarization is shown (inset).
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1.1.1 Excitatory Neurotransmission
By far the most common type of postsynaptic effect is one in which the postsynaptic
neuron becomes more likely to fire an action potential of its own, or is excited
(Kennedy, 2000). The effect on the postsynaptic cell is most commonly cation influx
through ligand‐gated ion channels and a resultant depolarization, an excitatory
postsynaptic potential (EPSP). In the case of glutamatergic synapses, the most common
excitatory synapse, the basal EPSP is mediated by postsynaptic glutamate receptors
(Kennedy, 2000).
1.1.2 Glutamate Receptors
Glutamate as a neurotransmitter can act on target cells via a number of different
signaling pathways initiated by different receptors. The receptors can be broadly
classified in terms of their method of action and further classified based on their specific
agonists. Broadly, glutamate mediated signal transduction can be metabotropic or
ionotropic. The metabotropic glutamate receptors (mGluRs) make up a class of 8 G‐
protein coupled receptors (GPCRs, mGluR1‐mGluR8) (Nakanishi, 1994; Conn and Pin,
1997). The ionotropic glutamate receptors, on the other hand, are all ligand gated ion
channels; these can be subdivided based on their specific agonists as the ‐amino‐3‐
hydroxy‐5‐methyl‐isoxazolepropionic acid (AMPA), NMDA, and kainate receptors
(Dingledine et al., 1999). The work of this thesis centers on the AMPA and NMDA
receptors and they are discussed in more detail.
4
1.1.3 AMPA Receptors
The AMPA receptor is the predominant receptor at active glutamatergic synapses. As
above, it is a ligand‐gated ion channel that opens in response to the binding of
extracellular glutamate. In baseline conditions, the AMPA receptor is non‐selective for
monovalent cations and allows the passive influx of sodium and efflux of potassium
(Dingledine et al., 1999). The measured EPSP is dominated by this AMPA receptor
mediated depolarization, but may have some contribution from the NMDA receptor.
Depending on the intracellular conditions and in particular its phosphorylation state, the
AMPA receptor can be internalized from or inserted into the postsynaptic membrane
and, furthermore, conductances through the pore can be enhanced or depressed (Man
et al., 2007; Derkach et al., 1999).
1.1.4 NMDA Receptors
The other major ionotropic glutamate receptor, the NMDA receptor, is a ubiquitous
receptor in the central nervous system with essential roles in neurotransmission.
Although originally thought to be a pentameric receptor, crystal structures have
revealed it to be heterotetrameric receptor ion channel comprised of two obligatory
NR1 subunits and two variable NR2 or NR3 subunits (Clements and Westbrook, 1991;
Laube et al., 1998; Furukawa et al., 2005). There are four possible NR2 subunits (NR2A‐
D) and two possible NR3 subunits (NR3A‐B) and therefore 18 theoretically possible
channel configurations. However, only two appear to be prevalent in the context of the
hippocampus, the NR1/NR2A/NR2A and NR1/NR2B/NR2B receptors (Paoletti and
Neyton, 2007; Groc et al., 2006; Neyton and Paoletti, 2006).
5
Functionally, the NMDA receptor pore is opened in response to the binding of
glutamate. However, the pore is quickly blocked by extracellular magnesium (Mg2+). This
block is relieved by sufficient depolarization of the postsynaptic membrane, causing an
ejection of Mg2+ (MacDonald et al., 1982; Nowak et al., 1984; Mayer et al., 1984). For
these reasons, the NMDA receptor is often called a coincidence receptor, activated only
if glutamate is present during a state of sufficient depolarization. It is possible that
sufficient depolarization is not achieved by the AMPA receptors at a single synapse and
therefore there must be spatially or temporally summated excitation in order to activate
the receptor. Also, for gating of the channel, a co‐agonist, either glycine or D‐serine (in
vivo) must bind the NR1 subunit (Schell et al., 1995; Hirai et al., 1996; Schell et al.,
1997).
Upon activation, the NMDA receptor functions as a cation channel allowing the efflux of
potassium and influx of sodium as well as, in contrast to the AMPA receptor, calcium
(Rogers and Dani, 1995) (Figure 1.2). This influx of calcium is crucial to the downstream
signaling pathways that are initiated secondary to NMDA receptor activation, such as
synaptic plasticity (Zhang et al., 1998). Kinetically, the NMDA receptors are considered
to be slow when compared to the AMPA and kainate receptors, with mean open times
of several hundred milliseconds (Jahr, 1992; Wyllie et al., 1998; Lester et al., 1999).
Relief of the Mg2+ block and “slow” calcium influx adds to the depolarization and
further relieves Mg2+ block. This positive feedback loop creates a high concentration
calcium microdomain in the postsynaptic area which can and does activate a variety of
6
calcium dependent intracellular signal pathways (Guthrie et al., 1991; Muller and
Connor, 1991; Yuste and Denk, 1995).
1.1.5 NMDA Receptor Subunits
The signaling pathways upstream and downstream of the NMDA receptor are highly
dependent on the subunit composition of the receptor (Hardingham et al., 2002; Cui et
al., 2007; Beazely et al., 2009; Xiong et al., 1998). Structurally, each subunit is comprised
of an extracellular N‐terminus, four transmembrane domains (TMI‐IV) including a re‐
entrant pore forming loop (TMII) and an intracellular C‐terminal tail. The ligand binding
site is formed by the N‐terminus in conjunction with an extracellular loop between TMIII
and TMIV (Figure 1.2) (Wood et al., 1995; Kuner et al., 1996). The NR1 binding pocket is
selective for glycine or D‐serine, whereas the NR2 subunit binds glutamate (Oh et al.,
1994; Sun YJ et al., 1998).
The NR1 subunit can exist in eight configurations by way of three distinct splice sites,
one in the N‐terminus and two in the C‐terminus. Alternately, the NR2 subunits are
distinct gene products; the NR3 subunits exist in two configurations, long and short,
based on the presence or absence of a 20 amino acid (aa) insert (Sun L et al., 1998).
The differences between the NR2A and NR2B containing receptors are both slight and
stark, structurally and functionally. Both receptors have all the functional qualities of
NMDA receptors described above; both receptors have all of the physical qualities
described above; lastly, both receptors are modifiable in the same ways. In particular,
both NR2A and NR2B subunits have long intracellular C‐termini with multiple potential
7
serine, threonine and tyrosine phosphorylation sites. However, developmentally, the
NR2A containing receptor predominates in synaptic sites at mature synapses whereas
the NR2B containing receptor predominates in synaptic sites at juvenile synapses and
extrasynaptic sites in the mature animal (Sans et al., 2000). Furthermore, activation of
the NR2A containing receptor is implicated in neuroprotection whereas the NR2B
containing receptor has been implicated in neurotoxicity (Liu et al., 2007). Lastly,
channel kinetics and calcium signaling are highly dependent on NR2 subunit
composition, with NR2A containing receptors showing higher open probabilities, higher
total charge transfer during high frequency stimulation, and lower total charge transfer
during low frequency stimulation protocols (Erreger et al., 2005) Therefore, the two
receptors appear to have different downstream signaling pathways, either via location
(Sans et al., 2000), physical coupling (Cui et al., 2007), or differences in calcium signaling
(Erreger et al., 2005; Liu et al., 2007). Both the NR2A and NR2B containing receptors
have highly modifiable intracellular C‐termini with multiple phosphorylation sites
(Leonard and Hell, 1997; Tingley et al., 1997). However, these phosphorylation sites,
particularly those affected by tyrosine kinases, are different in the two receptors. For
instance, two Src family kinases (SFKs), Src and Fyn kinases, each have three unique
phosphorylation target sites on the NR2A and NR2B C‐terminal tails respectively (Salter
and Kalia, 2004). Therefore, it appears that the two receptors also have different
upstream signaling pathways. The conjunction of these differences suggests that the
NMDA receptor has different physiological functions depending on the NR2 subunit
present.
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Figure 1.2. The NMDA Receptor A. The NMDA receptor is a membrane ion channel heterotetramer with two NR1 subunits and two NR2 subunits. Each NR1 subuit binds serine and each NR2 subunit binds glutamate. B. The NMDA receptor shown in cross‐section. The pore allows the influx of extracellular sodium and calcium, but is blocked by magnesium. The block can be relieved by sufficient depolarization. C. A schematic of NMDA receptor subunits shows the three transmembrane domains and pore forming reentrant loop (TMII). The black circles indicate splice sites for the NR1 subunit and the grey circle indicates the ligand binding domain.
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1.2 Gprotein Coupled Receptors
In contrast to the ionotropic glutamate receptors discussed above, a number of
neurotransmitters and neuromodulators can act via intracellular signaling cascades
initiated by GPCRs. Simply, a GPCR is a seven transmembrane domain containing
receptor coupled to a triheteromeric G‐protein consisting of , , and subunits. Upon
ligand binding, GTP hydrolysis by the G subunit causes a release of the G subunits and
initiation of the second messenger cascade (Neves et al., 2002). The GPCRs can be
classified by the downstream effects of the G subunits as Gi/o, Gq, or Gs. Gq and Gs type
G‐proteins are discussed further.
Gq‐type G subunits stimulate membrane docked phospholipase C (PLC), an enzyme that
cleaves the phospholipid phosphatidylinositol (PIP2) to diacyl glycerol (DAG) and
inositol‐1,4,5‐triphosphate (IP3). Membrane bound DAG activates protein kinase C (PKC),
and IP3 is stimulates its receptors (IP3Rs) on the endoplasmic reticulum (ER) releasing
intracellular calcium (Figure 1.3) (Neves et al., 2002).
Gs‐type G subunits, on the other hand, stimulate the transmembrane protein adenylyl
cyclase (AC), an enzyme that catalyzes the conversion of adenosine triphosphate (ATP)
to 3’,5’‐cyclic‐adenosine monophosphate (cAMP) and pyrophosphate (PPi). Intracellular
cAMP activates cAMP‐dependent protein kinase (PKA) (Figure 1.4) (Neves et al., 2002).
Both Gq and Gs type G proteins’ further downstream effects are dependent on the PKA,
PKC and calcium dependent pathways of the cell.
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Figure 1.3 The Gq‐signaling Pathway. The Gq alpha subunit activates PLC which cleaves PIP2, a membrane phospholipid into diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 mobilizes intracellular calcium stores from the ER and along with DAG, this activates protein kinase C (PKC).
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Figure 1.4 The Gs‐signaling Pathway. The Gs alpha subunit activates adenylyl cyclase (AC) which catalyzes
the breakdown of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and
pyrophosphate (PPi). cAMP is then able to activate protein kinase A (PKA).
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The major GPCR pathways investigated by this work are pituitary adenylyl cyclase
activating polypeptide (PACAP) and dopamine mediated and these are further
discussed.
1.2.1 Pituitary Adenylyl Cyclase Activating Polypeptide
In attempting to identify a novel hypophysiotropic hormone from hypothalamic tissue,
Miyamata et al. screened fractions for AC stimulating activity. From this approach, a
highly basic, 38 residue ‐amidated peptide that strongly stimulates cAMP production
was isolated and named pituitary adenylyl cyclase activating polypeptide (PACAP38)
(Miyata et al., 1989). Subsequently, a less basic and less active ‐amidated polypeptide
of the 27 N‐terminal residues was isolated and referred to as PACAP27. Homology
studies show 68% sequence similarity between VIP and PACAP27 suggesting that PACAP
is a member of the secretin/glucagon/VIP superfamily of peptides (Miyata et al., 1990).
Functionally, PACAP38 and PACAP27 have both been shown to be about 1000 fold more
potent AC activators than VIP and it is suggested that the physiological function of
PACAP is vital, as the sequence is highly conserved across species and murine, ovine and
human PACAP38 show sequence identity (Kimura et al., 1990).
Across species, PACAP is processed from a pre‐proprotein of 175 (rat, murine) or 176
(ovine, human) amino acids. In the pre‐proprotein, PACAP38 is preceded by a putative
signal peptide and a “pro‐region” and followed by a proteolytic processing and
amidation sequence, Gly‐Arg‐Arg. It is possible that PACAP38 is further processed into
PACAP27 via ‘re‐processing’ and amidation at a Gly‐Lys‐Arg site although it is more likely
that the pro‐protein is directly cleaved and amidated at the upstream site to create
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PACAP27. In the human, PACAP is the product of the ADCYAP1 gene with 5 exons and 4
introns. Exon 1 is untranslated and exon 5 encodes the full length PACAP38 (Hosoya et
al., 1992).
Although discovered in the hypothalamus, PACAP and its receptors have since been
localized in a wide variety of tissues with an equally wide variety of effects (Gottschall et
al., 1990; Pisegna and Wank, 1993). One source for this variety is that PACAP can act on
three distinct receptors, classified based on their differing affinities for PACAP and VIP.
PAC1 receptors demonstrate a high affinity for PACAP38 and PACAP27 and a very low
affinity for VIP, whereas the VPAC receptors, VPAC1 and VPAC2, have similar affinities
for both PACAPs and VIP. PAC1 receptors are therefore designated as PACAP specific.
All three receptors are class‐II G‐protein coupled receptors, having: i) large N‐terminal
extracellular domains conferring ligand specificity, ii) N‐terminal hydrophobic signal
sequence, iii) six strictly conserved cysteine residues, iv) multiple N‐glycosylation sites,
and v) a strictly conserved 83 amino acid sequence (Pisegna and Wank, 1993). The VPAC
receptors are expressed in a variety of brain regions, but where they are co‐expressed
they appear to be complementary; in general, VPAC1 and VPAC2 receptors do not
appear in the same cells. In terms of the hippocampal formation, the VPAC1 receptor
has been identified in both the hippocampus proper as well as the dentate gyrus with
little if any VPAC2 expression (Usdin et al., 1994). Although classically all three PACAP
receptor subtypes are Gs coupled, all three can also be Gq coupled and the VPAC
receptors can also be Gi coupled. Therefore, signal transduction through all three PACAP
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receptors appears to be heavily dependent upon the specific cell type’s G‐protein and
second messenger complement.
1.2.2 PAC1 Receptor
The PAC1 receptor, like PACAP and the VPAC receptors has been localized throughout
the mammalian brain and peripheral tissues. In the hippocampus, PAC1R mRNA has
been localized in the CA1‐CA4 pyramidal cells (Cauvin et al., 1991).
The PAC1 receptor is considered one of the most heavily spliced GPCRs discovered with
variations in intracellular loops, transmembrane domains and N‐terminal regions. Six
common and important splice variants arise from the presence of absence of two 28aa
cassettes (hip, hop) in the third intracellular loop. The six variants are: neither cassette
(PAC1‐s), one cassette (PAC1‐hip, PAC1‐hop1, PAC1‐hop2), or two cassettes (PAC1‐
hiphop1, PAC1‐hiphop2) (Zhou et al., 2000). Hop1 and hop2 (27aa) are thought to arise
via different splice acceptor sites in the hop cassette. All of these variants stimulate
cAMP production through a Gs signaling mechanism, but only PAC1R‐hip appears to
have no Gq related activity (Spengler et al., 1993). Another important splice variant is
PAC1R‐TM4 with a sequence modification in transmembrane region IV. PAC1R‐TM4 is
unique in that it does not appear to couple to G‐proteins at all but instead activates L‐
type calcium channels (Chatterjee et al., 1996). A third class of PAC1 variants with
alternative splicing in the N‐terminal domain appears to attenuate or abolish Gs
signaling while maintaining or enhancing PLC activity (Dautzenberg et al., 1999).
15
Pharmacological investigations of the PAC1 receptor are limited by the relative scarcity
of tools. Maxadilan, a very potent agonist of the PAC1 receptor with no sequence
homology with PACAP, and subsequently no VPAC activity, was isolated from the
leishmaniasis causing sand‐fly Lutzomia lingipalpis and modified to produce the potent
PAC1 antagonist, M65 (Moro and Lerner, 1997; Uchida et al., 1998). In addition, N‐
terminal truncated PACAP, PACAP(6‐38), has been shown to be a potent antagonist of
the PAC1 receptor (Robberecht et al., 1992).
1.2.3 PAC1 Receptor Signal Transduction
Although investigations into PACAP38’s effects on neurotransmission are relatively
young, there have been a number of promising developments in the field. As above, the
CA1 hippocampus contains both PACAP as well as the PAC1 receptor and the PAC1
receptor can activate both PKA and PKC dependent intracellular pathways. Therefore, it
follows that activation of the PAC1 receptor should directly affect the NMDA receptor
and by extension have effects on synaptic plasticity. Indeed, it has been shown that low
concentrations of PACAP can potentiate NMDA mediated responses and modulate LTP.
Furthermore, studies on PACAP and PAC1 knockout mice have demonstrated impaired
memory retention and consolidation, two hippocampus dependent processes.
Briefly, it has been hypothesized that PACAP can affect the NMDA receptor by a Fyn
kinase dependent phosphorylation of NR2B subunits. In this situation, Gs pathways
stimulate PKA dependent phosphorylation of the receptor for activated C‐kinase
(RACK1), causing a disinhibition of Fyn. A separate set of experiments has demonstrated
16
that very low concentrations of PACAP38 can induce a potentiation of CA1 field EPSPs
that occludes the induction of LTP, whereas high concentrations depress the same. In
our own investigations into the effects of PACAP on CA1 activity, we have not been able
to elicit any effects on NMDA receptors with very low concentrations, but have been
able to demonstrate the same high concentration inhibition. Moreover, although PAC1
can be both Gs and Gq coupled, we have been able to show that it is the Gq‐PKC‐Src
pathway that is responsible for the enhancement in NMDA activity.
The results of the relatively few studies into PACAP38’s effects on CA1 transmission
suggest that there is a concentration dependent effect of PACAP38 on NMDA receptors.
Although not yet thoroughly investigated, it is possible that these effects are dependent
on very different molecular mechanisms: directly at the NMDA receptor, via VPAC
receptors, and via PAC1 receptors, both Gs and Gq coupled.
1.2.4 Dopamine
In contrast to PACAP, dopamine, or 4‐(2‐aminoethyl)benzene‐1,2‐diol, has a long
history, being first synthesized in the early twentieth century. It was not, however,
studied extensively until it was recognized as a physiologically important molecule in its
discovery as a precursor in the formation of norepinephrine. Subsequently, the
discovery of high concentrations of dopamine in the striatum, with little concurrent
norepinephrine, sparked investigations into dedicated functions of dopamine (Raab and
Gigee, 1951; Vogt, 1954; Bertler and Rosengren, 1959).
17
The synthesis of dopamine is a two step process from the precursor amino acid tyrosine:
in the first step, tyrosine hydroxylase converts tyrosine into L‐3,4‐
dihydroxyphenylalanine (L‐DOPA) and in the second step, L‐DOPA is decarboxylated into
dopamine (Misu et al., 2003). The presence of dopaminergic neurons in the CNS is often
determined, therefore, by the presence of tyrosine hydroxylase activity.
The effects of dopamine in the central nervous system are mediated by its membrane
receptors, which are G‐protein coupled. Based on signal transduction mechanisms,
dopamine receptors have been divided into two broad categories: D1‐type and D2‐type
receptors. The D2‐type receptors, D2, D3, and D4, are grouped based on their negative
regulation of AC activity (Gi coupled). The D1‐type receptors, D1 and D5, are grouped
based on their positive regulation of AC activity (Gs coupled). It is suggested that the
differences in these classes is due to the differences in the third intracellular loop: D1‐
type receptors have a short loop, where D2‐type receptors have a long third
intracellular loop (Kozell et al., 1994). The D1 receptors are discussed further.
Physiologically, dopamine and its receptors have been implicated in a wide variety of
functions in a wide variety of brain areas including control of hormone secretion,
cognition, reward, and locomotion. In the hippocampus, dopamine has been implicated
in synaptic plasticity, spatial navigation, passive avoidance, reinforcement learning and
visual discrimination. Pathophysiologically, dopamine dysfunction has been implicated
strongly in a number of neurological and psychiatric disorders including Parkinson’s
disease, schizophrenia and drug abuse. Of particular relevance, pathological increases in
18
dopaminergic activity and decreases in glutamatergic activity have both been implicated
in the etiology of psychotic disorders.
1.2.5 D1 Receptor
The D1 dopamine receptor is most abundant dopamine receptor in the CNS. It has been
located in a wide variety of locations including cortical and subcortical areas, pre‐ and
post‐ synaptic membranes and in dendritic spines and axon terminals (Fremeau et al.,
1991; Huang et al., 1992). Unlike the D2 receptor, the human D1 receptor has not been
identified outside of the CNS (Dearry et al., 1990). Dopaminergic input to the
hippocampus from the mesolimbic pathway terminates on D1 receptors that display a
dentate gyrus‐CA1 gradient (Mansour et al., 1991; Amenta et al., 2001).
Unlike PACAP and the PAC1 receptor, there is a large variety of pharmacological tools
available for the study including the benzazepine derivatives 6‐chloro‐2,3,4,5‐
tetrahydro‐1‐phenyl‐1H‐3‐benzazepine (SKF‐81297) and R(+)‐7‐chloro‐8‐hydroxy‐3‐
methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine (SCH‐23390), a specific agonist
and antagonist respectively.
1.2.6 D1 Receptor Signal Transduction
When compared to the PAC1 receptor, investigations into dopaminergic effects on
neurotransmission are vast and varied. A large number of studies have shown direct
effects of D1 receptor activation on NMDA receptor function. In particular, chronic
antagonism of the D1 receptor has been shown to significantly affect NMDA receptor
function in the CA1 hippocampus (Tarazi et al., 1996), whereas activation of the same
19
enhances NMDA receptor function (Yang, 2000). Furthermore, it has repeatedly been
reported that D1 agonists and antagonists can both facilitate and inhibit the induction
and maintenance of LTP at the hippocampal and whole organismal levels (Stramiello and
Wagner, 2008; Navakkode et al., 2007; Granado et al., 2008; Otmakhova and Lisman,
1996).
These previous investigations leave a large amount of speculation as to the exact role of
D1 receptor activity on CA1 hippocampal NMDA activity and synaptic plasticity. Recent
studies into D1 activity in the hippocampus have demonstrated an enhancement of LTP
dependent on PKA, SFKs and NR2B containing receptors (Stramiello and Wagner, 2008).
A significant study has shown that D1 mediated modulation of NMDA receptors depends
on the subunit composition of the receptors, enhancing NMDAR activity in regions of
low NR2A:NR2B ratios and inhibiting NMDAR activity in regions of high NR2A:NR2B
ratios and that the ratios themselves are modifiable by D1 activity (Varela et al., 2009).
In terms of synaptic plasticity, there is a similar variance in investigations. There are
studies that have shown that D1 activation can enhance LTP (Otmakhova and Lisman,
1996), enhance LTD (Chen et al. 1996; Chen et al., 1995), and even cause a form of
chemical‐LTP not dependent on the NMDA receptor (Huang and Kandel, 1995). Still
other studies suggest D1 receptor agonism simply predisposes synapses to plasticity,
both LTP and LTD (Lemon and Manahan‐Vaughan, 2006) The unique cellular effects of
D1 receptor agonism on long‐term synaptic plasticity are therefore probably highly
dependent on cell‐type and intracellular protein complement.
20
1.3 Hippocampus
Since the beginnings of anatomy, the hippocampus has been one of the most frequently
studied mammalian structures. Its prominence in the human brain as well as its ease of
removal without damage were initially its appealing features, but it has since been an
ideal subject for study due to its demonstrable functional significance and ease of ex
vivo study with the conserved so‐called trisynaptic circuitry (Figure 1.1B).
Anatomically, the hippocampal formation is defined as the ventral elaboration of the
medial temporal cortex. Here the temporal cortex narrows into a single layer of densely
packed neurons and winds into an S‐shaped structure in the lateral ventricle (Figure
1.1A; Ramon y Cajal, 1894). Although the limbic system is no longer considered a
functionally relevant designation, the hippocampus is included as a structure.
The transverse hippocampal formation consists of two interlocking C‐shaped regions,
the dentate gyrus (DG) and the hippocampus proper. The simplified circuitry of the
hippocampus begins in the entorhinal cortex (EC), projecting onto granule cells of the
DG via the mossy fiber pathway. The perforant pathway projects from the DG into the
CA3 region, which in turn projects into the CA1 region via the Schaeffer collaterals. The
major output of the region is to the EC. This trisynaptic circuit is preserved in acutely
prepared hippocampal slices, making it an ideal candidate for study of synaptic
machinery.
Functionally, the hippocampus has been shown to be relevant for explicit memory
formation, spatial navigation and memory and is thought to be involved in numerous
21
other processes. Synaptic plasticity was first discovered in the hippocampus and is thus
thought to play a major role in the coding of memories (Bliss and Lomo, 1973).
1.4 Synaptic Plasticity
The mammalian brain is able to encode and store a remarkable variety of information
for many years and neuroscientists have repeatedly pursued the question of how this is
achieved. In many brain areas integral to memory formation, it has been shown that
there are activity dependent long‐lasting changes in synaptic efficacy and this is widely
believed to be the cellular equivalent of memory. This long‐term change can either be a
potentiation (LTP) or depression (LTD) of synaptic communication and combined they
are referred to as plasticity of the synapse. The fact that both LTP and LTD can be
reliably generated in a variety of brain regions, both in vitro and in vivo, and is activity
dependent, has been used to defend its functional relevance.
Briefly, synaptic plasticity was postulated as Hebbian theory in the late 1940s, and even
before that by the anatomist Ramon y Cajal, as the idea that if one neuron repeatedly
and persistently activates another, the synapses between them will become
strengthened and more stable (Ramon y Cajal, 1894; Hebb, 1949). The work done by the
Andersen lab in the 1960s and 1970s established that synaptic plasticity, specifically LTP,
can be induced in both anesthetized and awake animals’ hippocampi (Bliss and Lomo,
1973). The large field of research into synaptic plasticity has since demonstrated both
LTP and LTD at a variety of mammalian synapses and research into the mechanisms and
22
modes of induction have resulted in anti‐Hebbian, non‐Hebbian, NMDA receptor
dependent and independent forms of synaptic plasticity. In the hippocampus, for
instance, LTP of the mossy fiber pathway is NMDA receptor independent whereas
Schaeffer collateral – CA1 LTP is NMDA receptor dependent. Because this thesis focuses
on the CA1 synapse, NMDA receptor dependent plasticity is discussed further.
1.4.1 NMDAreceptor Dependent Synaptic Plasticity
The CA1 synapse is one of the most heavily studied regions for synaptic plasticity. It has
been repeatedly demonstrated by antagonists and calcium buffering that the NMDA
receptor, and more specifically, calcium influx through the NMDA receptor is required
for the long‐lasting changes at this synapse (Harris et al., 1984; Mulkey and Malenka,
1992). Although highly controversial, it has been reported that the NR2A‐containing
receptor mediated signaling leads to LTP whereas the NR2B‐containing receptor
mediated signaling leads to LTD (Liu et al., 2004; Morishita et al., 2007). A more recent
hypothesis suggests that it may be the ratio of contribution between the two NR2
subunits that determines the direction of plasticity (Cho et al., 2009). Whichever the
case, it is certain that calcium influx via the NMDA receptor is prerequisite for synaptic
plasticity at the CA1 synapse.
The mechanisms by which the calcium signal causes either LTP or LTD have been
extensively studied and many of the major components of the pathway have been
elucidated. In the case LTP, it has been shown that calcium‐calmodulin dependent
23
kinase II (CaMKII) activation mediates a phosphorylation and postsynaptic insertion of
AMPA receptors (Figure 1.5). More recently, LTD has been shown to be dependent on
calcium mediated activation of calcineurin (protein phosphatase 2B, PP2B). PP2B
activation dephosphorylates and disinhibits protein phosphatase 1 (PP1), which can
subsequently dephosphorylate AMPA receptors with a resultant internalization
(Kameyama et al., 1998; Lee et al., 1998; Lee et al., 2000). Whether changes in the
phosphorylation state serve only to stabilize or destabilize AMPA receptors in the
postsynaptic membrane or also change their channel properties is not definitively
known, although some studies report no change in single channel AMPA conductance following
plasticity inducing protocols (Shi et al., 1999; Oh and Derkach, 2005).
24
Figure 1.5 NMDA Receptor Dependent Long‐term Potentiation. The induction of LTP begins with sufficient depolarization caused by influx of sodium through the AMPA receptor (1) as to cause a relief of extracellular magnesium block of the NMDA receptor pore (2). Subsequent influx of calcium through the NMDA receptor activates CaMKII and PKC (3) which in turn phosphorylate existing AMPA receptors (4) and cause a rapid insertion of new AMPA receptors (5). This stabilizes them in the membrane, increases single channel conductance and overall causes increased future activity.
25
1.4.2 Src Family Kinases (SFKs), NMDA Receptors, and Synaptic Plasticity
Although the Src family of tyrosine kinases has classically been associated with the
etiology and pathogenesis of cancers, they also play an integral functional role in a
number of other cellular processes. In terms of the CA1 hippocampus, there is much
evidence to suggest that the Src family kinases (SFKs), and Src kinase in particular, are
integral to long‐lasting synaptic plasticity (Grant et al., 1992; Lu et al, 1998; Kojima et al.,
1997).
Briefly, the SFKs are a family of intracellular, modular, non‐receptor tyrosine kinases.
Thus far, nine members of the family have been described in mammals, with numerous
homologs in a wide variety of species, suggesting an integral role in cellular function
(Tatosyan and Mizenina, 2000). At the hippocampal CA1 synapse, two of the SFKs, Src
and Fyn, have been shown to be important regulators of NMDA receptor function as
well as synaptic plasticity (Le et al., 2006; MacDonald et al., 2006; Yaka et al., 2003).
Initial investigations into the role of non‐receptor tyrosine kinases in LTP showed that
non‐functional mutations in Fyn, but not Src, causes an impairment in LTP and
associated spatial learning tasks (Grant et al., 1992). However, subsequent studies have
shown that Src as well as Fyn are important for these processes (Salter and Kalia, 2004).
In particular, cultured and acutely isolated CA1 hippocampal neurons show an
enhancement of NMDA receptor mediated currents and occlusion of LTP via an activator
of endogenous Src (Lu et al., 1998; Huang et al., 2001); furthermore, inhibition of Src
kinase by the synthetic peptide Src(40‐58), can impair induction of LTP (Lu et al., 1998).
26
In concert, these results strongly suggest a pivotal role for Src in NMDA receptor
dependent LTP at the CA1 synapse.
Specific investigations into the molecular actions of Src kinase in LTP have shown that
Src kinase activity is increased by PKC or Pyk2 activation (Huang et al., 2001; McCulloch
et al., 2002; Seabold et al., 2003), and can be enhanced directly by sodium influx (Yu and
Salter, 1998). Furthermore investigations it has been shown that dephosphorylation of
tyrosine Y416 by the phosphatase STEP inhibits Src mediated enhancement of NMDARs
(Salter and Kalia, 2004), and inhibition of dephosphorylation of Y524 by a separate
phosphatase, PTPa, blocks the induction of LTP (Lei et al., 2002). A still further
complicating piece of the Src dependent pathway is the demonstration that NMDA
receptors and AMPA mediated currents are not subject to Src dependent modifications
in the basal state due to a continuous inhibition by C‐terminal Src kinase (Csk) (Xu et al.,
2008). The interplay between these and other phosphatases and kinases is therefore
strongly implicated in Src dependent NMDA receptor function during induction of
synaptic plasticity.
Along with this evidence suggesting a complex role for Src in regulating NMDA
receptors, there is also a growing body of evidence suggesting the same for Fyn kinase.
In particular, it has been demonstrated that LTP induction is correlated with
phosphorylation of the NR2B tail at tyrosine residue Y1472 and that this site has a
decreased phosphorylation with functional knockout of Fyn (Nakazawa et al., 2001).
Furthermore, it has since been demonstrated that a scaffolding protein, RACK1, binds
27
both Fyn and the NR2B subunit, and PKA‐dependent phosphorylation of RACK1 results
in phosphorylation of the NR2B subunit by Fyn and enhancement of NMDA‐mediated
currents (Yaka et al., 2003; Yaka et al., 2002).
These complex interactions of Src, Fyn and the NMDA receptor are dependent therefore
on a large number of scaffolding proteins, kinases, and phosphatases working in
concert. The evidence suggests that the SFKs are each part of multipart complexes with
the NMDA receptors, and that their interactions may therefore be receptor subtype
specific.
1.4.3 Metaplasticity
Because synaptic plasticity is an integrative and highly complex process with both pre‐
and post‐ synaptic contributors, it was naturally thought that any modulation of
intermediaries in synaptic plasticity can alter the nature of the plasticity induced. As one
example, partial agonists of the NMDA receptor glycine binding site can enhance LTP
and attenuate LTD (Zhang et al., 2008). These changes in synaptic plasticity,
enhancements or attenuations, are together defined as metaplasticity, or the “plasticity
of plasticity” (Abraham and Bear, 1996).
Originally, the idea of metaplasticity was postulated and modeled mathematically on
visual cortical neurons’ responses to binocular and monocular inputs (Bienenstock et al.,
1982). This Bienenstock‐Cooper‐Munro (BCM) theory of metaplasticity suggests that
present postsynaptic activity is a function of previous postsynaptic activity and that this
28
function is itself modifiable (Figure 1.6). The predictions of the BCM model have since
been confirmed in visual cortical neurons as well as in the hippocampus.
The BCM model can be simplified and made less mathematical by constraining it to
NMDA receptor dependent plasticity in the CA1 region. Briefly, a range of presynaptic
stimulations can elicit plasticity to varying degrees at the CA1 synapse. The degree to
which any given presynaptic stimulation will affect the synaptic activity is dependent not
only on the chosen stimulation, but also on other proximal factors, such as the state of
the NMDA receptor. In particular, the total relationship between presynaptic
stimulation and postsynaptic response can be described simply by the modification
threshold, , the point at which response switches between depression and
potentiation. This modifiable threshold is predicted to be dependent on any factors that
the induction of plasticity is dependent upon; in the case of CA1 synaptic plasticity, it is
expected that the state of the NMDA receptors is a particularly important determinant
of .
29
Figure 1.6 The Bienenstock‐Cooper‐Munro Model of Metaplasticity. The BCM model predicts that with varied levels of presynaptic activity, varied levels of future postsynaptic activity can be elicited. Theta represents the setpoint activity at which LTD inducing events are changed to LTP inducing events and this setpoint can be shifted leftward, enhancing LTP, or rightward, enhancing LTD. The BCM model is modified with presynaptic stimulation frequency as a proxy measure for presynaptic activity.
30
SECTION TWO
RATIONALE AND HYPOTHESIS
31
2.1 GPCRs Modulate NMDA Receptor Function
Modification of NMDA receptor function has been studied extensively by way of a
variety of experimental approaches. Our group has been able to demonstrate the
modulation of NMDA receptor function by way of stimulation of different upstream
pathways. Of particular importance, we have been able to show that GPCRs have the
ability to enhance NMDA mediated currents by way of different intracellular pathways.
Through the use of genetic manipulations, biochemical techniques and
electrophysiological recordings, we have been able to elucidate the pathways by which
these upstream GPCRs can modulate the NMDA receptor. Of particular relevance,
activation of the PAC1 receptor leads to a long‐lasting enhancement of NMDA mediated
EPSCs by way of activation of a Gq/PKC mediated signaling pathway. In contrast,
activation of the D1 receptor leads to a similar enhancement by way of activation of a
Gs/PKA pathway.
2.1.1 GPCR Mediated Effects are Subunit Specific
Although PKC and PKA phosphorylation sites do exist on the NR1 subunit and these
could contribute to the observed enhancements, we have also demonstrated differing
pathways for the enhancement (Leonard and Hell, 1997; Tingley et al., 1997). Activation
of the PAC1 receptor and subsequent activation of PKC causes activation of a different
signaling cascade that results in NR2A subunit phosphorylation. Alternately, activation
of the D1 receptor and subsequent activation of PKA causes activation of a signaling
cascade terminating in NR2B phosphorylation. The NR2 subunit specificity of these
cascades, coupled with the stark differences in physiological role of these subunits,
32
suggests that the balance of competing GPCR signaling could play a role in maintaining
or changing the function of a given neuron.
33
Figure 2.1 PACAP38 Mediated Modulation of NMDA Receptors. Briefly, the PAC1 receptor is Gq coupled and mobilizes ER calcium stores as well as liberates DAG via PLC activation, causing PKC to be activated. PKC phosphorylates Pyk2, which in turn phophorylates RACK1 and releases Src in the vicinity of the NMDA receptor. Src phosphorylates the intracellular NR2A tail, enhancing the activity of NR2A containing receptors. (Modified from MacDonald et al., 2005)
34
Figure 2.2 D1 Mediated Modulation of NMDA Receptors. Briefly, the D1 receptor is Gs coupled and causes formation of cAMP via AC activation, causing PKA to be activated. PKA directly phosphorylates RACK1 releasing Fyn in the vicinity of the NMDA receptor. Fyn phosphorylates the intracellular NR2B tail, enhancing the activity of NR2B containing receptors.
35
2.2 NR2 Subunit Contributions to Metaplasticity
The prevalent hypotheses today concerning the induction of plasticity at the CA1
synapse are centered on the NR2 subunit. There is currently a large body of
electrophysiological evidence suggesting that the NR2B subunit is required for LTP
whereas the NR2A subunit is required for LTD, although this theory is not uncontested
(Morishita et al., 2007; but see Liu et al., 2004). More recently, in vivo experiments have
uncovered roles for both NR2 subtypes in both LTP and LTD induction (Fox et al., 2006).
The compromise that is materializing amongst all possible theories is that a ratio of
activity of the NR2 subtypes determines the degree and direction of synaptic plasticity in
this area (Cho et al., 2009). Still, whether an increase in the NR2A:NR2B activity ratio
preferentially results in LTP or LTD is not by any means a decided point.
2.3 Hypothesis
The convergence of the NR2 subunit hypotheses of metaplasticity and our own work
determining subunit specific enhancement of NMDA currents suggests a physiological
mechanism governing synaptic plasticity in the CA1 subfield of the hippocampus. Since
both Gs and Gq receptors generally, and the D1 and PAC1 receptor specifically, do exist
in CA1 postsynaptic neurons and because the signaling cascades terminating at the
NMDA receptor are NR2 subunit specific, we suggest that synaptic plasticity may be
altered by the balance amongst upstream neuromodulators.
More specifically, because the initial investigations of Yu Tian Wang’s group suggested a
close association between LTD and the NR2B subunit, we hypothesize that the D1
36
receptor signaling pathway, terminating in the NR2B subunit, will enhance LTD over LTP,
shifting the BCM relationship rightward; also, PAC1 receptor signaling, terminating in
the NR2A subunit, will enhance LTP over LTD, shifting the BCM relationship leftward.
However, considering that initial investigations were based on imperfect pharmacology,
and a separate hypothesis put forth by Mark Bear’s group, suggesting increases in the
NR2A:NR2B results in increased LTD, we also considered as an alternative hypothesis
that decreasing the NR2A:NR2B by D1 receptor activation will enhance LTP over LTD,
with the reverse situation for PAC1 receptor activation.
37
SECTION THREE
METHODS
38
3.1 Hippocampal Slice Recordings
3‐4 week old Wistar rats were anesthetized using isoflurane and immediately
decapitated. The brains were rapidly removed and submerged in chilled (4oC)
oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (aCSF) composed of (in mM):
NaCl (124), KCl (3), CaCl2 (2.6), MgCl2 (1.3), NaHCO3 (26), NaH2PO4 (1.25) and D‐
glucose (10) with osmolarity adjusted to 300‐310 mOsM and pH to 7.4. Both hippocampi
were acutely isolated and mounted on a block of 3% agar. Transverse hippocampal
slices of a thickness of 350 um were cut using a vibrotome (VT1000E; Leica). Slices were
allowed to recover in a submerged holding chamber for at least 90 minutes under
continuous oxygenation until needed. Slices were transferred to a holding chamber,
mechanically fixed using a thin platinum wire grid and continuously perfused with warm
(30oC) oxygenated aCSF. Slices were allowed 10 minutes to recover before any
recording. Field excitatory postsynaptic potentials (fEPSPs) were evoked at a frequency
of 0.05Hz by electrical stimulation (100us duration) delivered to the Schaffer‐collateral
pathway using a concentric bipolar stimulating electrode (25um exposed tip, David Kopf
Instruments), and recorded using glass microelectrodes (3‐5M filled with aCSF)
positioned in the stratum radiatum layer of the CA1 subfield at a distance of
approximately 50um from the cell layer. Electrode depth was varied until a maximal
response was elicited (approximately 175um from surface). The input‐output
relationship was first determined in each slice by varying stimulus intensity (10‐1000 uA)
and recording the corresponding fEPSP. Using a stimulus intensity that evoked 30‐40%
of maximal fEPSP, paired‐pulse responses were measured every 20s by delivering two
39
stimuli in rapid succession with intervals (interstimulus interval, ISI) varying from 10‐
1000 ms. Following this protocol, fEPSPs were evoked and measured for twenty minutes
at 0.05Hz using the same stimulus intensity to test for stability of the response. At this
time, plasticity was induced by 1, 10, 20, 50 or 100Hz stimulation with train pulse
number constant at 600. Any treatments were added to aCSF and perfused the slice for
the ten minutes immediately prior to induction of plasticity. Determination of degree of
synaptic plasticity was done for the period 50‐60 minutes post‐induction.
Fields were recorded using an Axopatch‐1D amplifier (Axon Instruments), filtered at
2kHz, digitized using a Digidata 1320 (Axon Instruments) and acquired using Clampex
(Axon Instruments). Data were analyzed using Clampex and Graphpad Prism software.
Animals were chosen to ensure maximum randomness in experiments. Briefly, to ensure
there were no specific animal dependent effects, no same day consecutive slices were
chosen to be part of the same sample set and no sample set contained more than two
slices from the same animal. All animals used for a treatment sample were also used for
control experiments. In order to ensure the health of each slice, input‐output
relationships and paired‐pulse ratios were measured prior to each recording, but after
recovery periods. Any slices that showed a variance of greater than two standard
deviations from the mean input/output slope or paired‐pulse facilitation ratio with
interstimulus interval of 40ms were discarded.
40
3.2 Animals
All animal experimentation was conducted in accordance with the Policies on the Use of
Animals at the University of Toronto and the University of Western Ontario.
3.3 Drugs and Peptides
The sources of drugs used for this study are as follows: PACAP38 (Calbiochem);
SKF81297, SCH23390 (Tocris Bioscience). M65 and Maxadilan were generously provided
by Dr. EA Lerner.
3.4 Statistical Analysis
All population data are expressed as means ± standard error of the mean (SEM). Paired
t‐test was used to compare within groups, student’s t‐test was used to compare
between two groups, and analysis of variance (ANOVA) was used to analyze multiple
groups. P values were constant at 0.05.
41
Figure 3.1 Acutely Prepared Hippocampal Slice Recordings. The conserved trisynaptic circuitry is shown. Stimulating electrodes are placed between the CA3 and CA1 regions to activate Schaffer collaterals (SC) and recordings are obtained from the CA1 region (GC: granule cell; MF: mossy fibre; PP: perforant pathway; drawn by Salter MW)
42
SECTION FOUR
RESULTS
43
4.1 PACAP38 has no effect on baseline synaptic transmission
In order to assess the effects of the PAC1 signaling pathway on the induction of synaptic
plasticity, it was first undertook to investigate any action PACAP38 may have on baseline
fEPSPs. As above, we have previously described a maximal enhancement of the NMDA
receptor through Gq‐type signaling at a concentration of 1nM and so this concentration
was chosen for all experiments (MacDonald et al., 2005). Therefore, fEPSPs were
recorded for 10 minutes at 0.05Hz in standard aCSF followed by 10 mins at 0.05Hz with
addition of 1nM PACAP38 to the bath. fEPSPs were then recorded for 60 mins without
any plasticity inducing protocols in order to demonstrate long term effects of 1nM
PACAP38 on synaptic transmission. 1nM PACAP38, applied in this way, did not affect
fEPSP slope (1.04±0.05, n=4 at t=60 mins, vs. control 1.06±0.02, n=3) and did not induce
any plasticity (Figure 4.1).
44
Figure 4.1
Effect of PACAP38 (1nM) on Baseline (0.05Hz) Evoked fEPSP 1.6 +PACAP38 (1nM) (n=4) 1.5 Control (n=3) 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5
Normalized fEPSP Slope 0.4 0.3 0.2 0.1 0.0
Baseline t=25-35 mins t=50-60 mins During ControlApplication 0.3mV 20ms
+PACAP38
0.3mV 20ms
Figure 4.1 PACAP38 has no effect on baseline fEPSPs when applied for ten minutes. PACAP38 was applied for ten minutes after ten minutes of baseline recording. fEPSP slope was measured for the subsequent sixty minutes. At t=60 mins post application, PACAP38 had no effect on fEPSP slope (control: 1.06±0.02, n=3; PACAP38: 1.04±0.05, n=4). Sample traces in the presence and absence of PACAP38 at t=‐20 (baseline), t=0 (during application), t=35 mins post application, and t=60 mins post application are shown below.
45
4.2 PACAP38 alters synaptic plasticity induction
Having determined that 1nM PACAP38 applied exogenously does not affect baseline
synaptic transmission, we undertook to determine its effects on synaptic plasticity
induction. Briefly, fEPSPs were evoked for ten minutes at 0.05Hz prior to a ten minute
period of PACAP38 application. Bath application of PACAP38 (1nM) for 10 minutes
caused no immediate changes to baseline fEPSPs. After 10 minutes of drug application,
LTP or LTD was induced using a 600 pulse varying frequency protocol. This protocol was
chosen as a compromise between 900 pulse LTD inducing stimulations and 200 pulse
LTP inducing stimulations. The pulses were kept constant in order to control for total
charge transfer to the slice. Pre‐tetanus application of 1nM PACAP38 caused no change
in LTD induction at 1Hz (0.63±0.04, n=5, vs. control 0.70±0.11, n=5) or LTP induction at
50Hz (1.63±0.24, n=6, vs. control 1.40±0.12, n=4) or 100Hz (1.50±0.20, n=4, vs. control
1.25±0.13, n=4) (Figure 4.2A,D,E). The effects of bath application of 1nM PACAP38
caused a reversal of LTD induction with 10Hz stimulation (1.40±0.15, n=7, vs. control
0.65±0.08, n=6; p=0.0015) and a similar reversal at 20Hz (1.19±0.09, n=5, vs. control
0.87±0.11, n=5; p<0.05 (Mann‐Whitney U)) (Figure 4.2B,C). Together, the results show
an alteration in the expression of synaptic plasticity and therefore the BCM relationship
in the presence of PACAP38 (Figure 4.2F).
46
Figure 4.2A
1 Hz Stimulation 1.2 1 2
1.0
0.8
0.6 Normalized Normalized fEPSP Slope
0.4 Control +PACAP38 (1nM)
0.2 -20 -10 0 10 20 30 40 50 60 Time (min)
Control +PACAP38
2 2 1 0.25mV 1 0.25mV 10ms 10ms
Figure 4.2A PACAP38 causes no change in LTD induction at 1Hz. Application of 1 nM PACAP38 for ten minutes (t=‐ 10 to t=0 mins) did not change LTD induction relative to control slices (control: 0.63±0.04, n=5; PACAP38: 0.70±0.11, n=5). Sample traces with and without PACAP38 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
47
Figure 4.2B
10 Hz Stimulation 1.5
1.3
1.1
0.9 +PACAP38 (1nM) (n=7) Control (n=6) Normalized fEPSP Slope fEPSP Normalized
0.7
2 0.5 1 -20 -10 0 10 20 30 40 50 60 Time (min)
Control +PACAP38
2 1 0.25mV 1 10ms 0.25mV 10ms 2
Figure 4.2B PACAP38 reversed LTD with 10Hz induction. Application of 1 nM PACAP38 for ten minutes (t=‐10 to t=0 mins) reversed LTD induction relative to control slices (control: 0.65±0.08, n=6; PACAP38: 1.40±0.15, n=7; p=0.0015). Sample traces with and without PACAP38 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
48
Figure 4.2C
20 Hz Stimulation 1.6
+PACAP38 (1nM) (n=6) 1.4 Control (n=5)
1.2
1.0 Normalized fEPSP Slope
0.8
1 2 0.6 -20 -10 0 10 20 30 40 50 60 Time (min)
Control +PACAP38
1 2 0.2mV 0.2mV 10ms 10ms 2 1
Figure 4.2C PACAP38 reversed LTD with 20Hz induction. Application of 1 nM PACAP38 for ten minutes (t=‐10 to t=0 mins) reversed LTD induction relative to control slices (control: 0.87±0.11, n=5; PACAP38: 1.19±0.09, n=5; p<0.05). Sample traces with and without PACAP38 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
49
Figure 4.2D
50 Hz Stimulation 1.8
1.6
1.4
1.2 1 Normalized fEPSP Slope fEPSP Normalized 2 1.0
Control (n=4) +PACAP38 (1nM) (n=6)
0.8 -20 -10 0 10 20 30 40 50 60 Time (min)
Control +PACAP38
1
1 0.2mV
0.2mV 10ms 10ms 2 2
Figure 4.2D PACAP38 did not change LTP induction at 50Hz. Application of 1 nM PACAP38 for ten minutes (t=‐10 to t=0 mins) did not change LTP induction relative to control slices (control: 1.40±0.12, n=4; PACAP38: 1.63±0.24, n=6). Sample traces with and without PACAP38 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
50
Figure 4.2E
100 Hz Stimulation 2.2
2.0
1.8
1.6
1.4
Normalized fEPSP Slope 1.2
1.0 Control (n=4) 2 1 +PACAP38 (1nM) (n=4) 0.8 -20 -10 0 10 20 30 40 50 60 Time (min)
Control +PACAP38
1 0.2mV 0.2mV 10ms 2 10ms
Figure 4.2E PACAP38 did not change LTP induction at 100Hz. Application of 1 nM PACAP38 for ten minutes (t=‐10 to t=0 mins) did not affect LTP induction relative to control slices (control: 1.25±0.13, n=4; PACAP38: 1.50±0.20, n=4). Sample traces with and without PACAP38 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
51
Figure 4.2F. BCM Relationship for PACAP38 (1nM)
1.50 * * 1.25
1.00
Normalized fEPSP Amplitude fEPSP Normalized PACAP38, 1nM 0.75 Control
0.50 1 10 20 50 100 Stimulus Frequency (Hz)
Figure 4.2F Bath applied PACAP38 for ten minutes prior to induction of plasticity causes a leftward shift in the plasticity modification threshold and BCM relationship. *p<0.05.
52
4.3 PACAP38 mediated reversal of LTD at 10Hz is PAC1R dependent
It has been well studied that the effects of endogenous and exogenous PACAP38 can be
mediated by any of three receptors, the PAC1, VPAC1 and VPAC2 receptors. The varying
affinities for PACAP38 and VIP along with our previous work in acutely isolated
hippocampal neurons suggested that the effect of 1nM PACAP38 could only be
mediated by the PAC1 receptor (MacDonald et al., 2005). However, to confirm this
result, we used the selective PAC1 antagonist M65 (1uM) in conjunction with PACAP38
(1 nM) at a plasticity induction frequency of 10Hz. Bath co‐application of M65 and
PACAP38 caused no change in induction of LTD with a stimulation protocol of 10Hz for 1
minute. From above, PACAP38 alone caused a dramatic reversal of LTD to LTP under the
same protocol.
53
Figure 4.3
2 *
1 60 mins post 60 tetanus
Normalized Normalized fEPSP Slope; 0
+M65 (n=3) Control (n=6) +PACAP38 (n=7)
+PACAP38 M65 (n=4)
Figure 4.3 The effect of PACAP38 on LTD induction at 10Hz was PAC1 receptor mediated. The effect of 1nM PACAP38 was abolished by coapplication of M65, a specific PAC1 antagonist (0.86±0.09, n=4; PACAP38 alone: 1.40±0.15, n=7; p=0.032). M65 application alone had no effect relative to control (0.82±0.01, n=3; control: 0.65±0.08, n=6).
54
4.4 D1 agonist SKF81297 alters synaptic plasticity induction
Having demonstrated a metaplastic effect for PACAP38, it was undertaken to determine
whether our observed enhancements in NMDA EPSCs in acutely isolated hippocampal
neurons via Gs signaling pathways also elicited a metaplastic effect. To that end,
hippocampal slices were prepared and recordings were made as in 4.2 excepting the
replacement of PACAP38 (1nM) with SKF81297 (10uM), a specific D1 agonist. Bath
application of SKF81297 had no immediate effect on baseline fEPSPs. SKF81297 did not
elicit an observed change in LTP induction at 50Hz (1.00±0.05, n=4, vs. control
1.18±0.10, n=5) nor in LTD induction at 1Hz (0.82±0.10, n=6, vs. control 0.65±0.13, n=6)
(Figure 4.4D,A). SKF81297 attenuated LTP at 60 min induced by 100Hz stimulation
(1.07±0.09, n=5, vs. control 1.32±0.09, n=5; p=0.04) and enhanced LTD induction using
10Hz (0.54±0.11, n=6, vs. control 0.81±0.17, n=5) and 20Hz (0.67±0.07, n=5, vs. control
0.86±0.09, n=5; p<0.05) induction protocols (Figure 4.4E,B,C). Together, it was
illustrated that the D1 agonist SKF81297 can alter the expression of synaptic plasticity
and therefore the BCM relationship at the CA1 synapse in slice (Figure 4.4F).
55
Figure 4.4A
1 Hz Stimulation 1.4
1.2
Control (n=6) +SKF81297 (10uM) (n=6)
1.0
0.8 1 Normalized fEPSP Normalized Slope
0.6
2 0.4 -20 -10 0 10 20 30 40 50 60 Time (min)
Control SKF81297
2 0.2mV 0.2mV 10ms 2 10ms 1 1
Figure 4.4A SKF81297 did not change LTD induction at 1Hz. Application of 10uM SKF81297 for ten minutes (t=‐10 to t=0 mins) did not affect LTD induction relative to control slices (control: 0.65±0.13, n=6; SKF81297: 0.82±0.10, n=6). Sample traces with and without SKF81297 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
56
Figure 4.4B
10Hz Stimulation 1.3 Control, (n=5) +SKF81297 (10uM) (n=6)
1.1 2
0.9
0.7 Normalized Normalized fEPSP Slope
0.5
1 0.3 -20 -10 0 10 20 30 40 50 60 Time (min)
SKF81297 Control
2
2 0.2mV 1 0.2mV 1 10ms 10ms
Figure 4.4B SKF81297 enhanced LTD induction at 10Hz. Application of 10uM SKF81297 for ten minutes (t=‐10 to t=0 mins) increased LTD induction relative to control slices (control: 0.81±0.17, n=5; SKF81297: 0.54±0.11, n=6). Sample traces with and without SKF81297 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
57
Figure 4.4C
20Hz Stimulation 1.4
1.2 Control (n=5) 1 +SKF81297 (10uM) (n=5)
2 1.0
0.8 Normalized fEPSP Slope Normalized
0.6
0.4 -20 -10 0 10 20 30 40 50 60 Time (min)
SKF81297 Control 0.2mV 0.2mV 10ms 2 10ms 2 1 1
Figure 4.4C SKF81297 enhanced LTD induction at 20Hz stimulation protocol. Application of 10uM SKF81297 for ten minutes (t=‐10 to t=0 mins) increased LTD relative to control slices (control: 0.86±0.08, n=5; SKF81297: 0.67±0.06, n=5; p<0.05 by Mann‐Whitney U test). Sample traces with and without SKF81297 application at time points 1 (t=‐ 20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
58
Figure 4.4D
50 Hz Stimulation 1.6
1.4
1.2
1.0 Normalized fEPSP Slope
0.8 2 Control (n=5) +SKF81297 (10uM) (n=5) 1 0.6 -20 -10 0 10 20 30 40 50 60 Time (min)
Control SKF81297
1 1 0.2mV 2 0.2mV 10ms 10ms 2
Figure 4.4D SKF81297 did not alter LTP induction at 50Hz. Application of 10uM SKF81297 for ten minutes (t=‐10 to t=0 mins) did not affect LTP induction relative to control slices (control: 1.18±0.10, n=5; SKF81297: 1.00±0.05, n=4). Sample traces with and without SKF81297 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
59
Figure 4.4E
100Hz Stimulation 1.7
1.5
1.3
1.1 Normalized Normalized fEPSP Slope
0.9 Control (n=5) +SKF81297 (10uM) (n=5)
1 2 0.7 -20 -10 0 10 20 30 40 50 60 Time (min)
Control SKF81297
1 0.2mV 10ms 1 0.2mV 10ms 2 2
Figure 4.4E SKF81297 attenuated LTP induced by 100Hz stimulation. Application of 10uM SKF81297 for ten minutes (t=‐10 to t=0 mins) prior to stimulation attenuated LTP induction relative to control slices (control: 1.32±0.09, n=5; SKF81297: 1.07±0.09, n=5; p=0.04). Sample traces with and without SKF81297 application at time points 1 (t=‐20 ‐ ‐18 mins) and 2 (t=58‐60 mins) are shown below.
60
Figure 4.4F. BCM Relationship for SKF81297 (10uM)
1.25
1.00 *
0.75 *
Normalized fEPSP Amplitude fEPSP Normalized 0.50 SKF81297, 10uM Legend *
0.25
1 10 20 50 100 Stimulus Frequency (Hz)
Figure 4.4F Bath applied SKF81297 for ten minutes prior to induction of plasticity causes a rightward shift in the plasticity modification threshold and BCM relationship. *p<0.05.
61
4.5 SCH23390 blocks the effect of SKF81297 at 10Hz
To further demonstrate that the effects of bath application of SKF81297 were indeed
due to D1 activation and not to non‐specific effects or agonism at a different receptor,
the specific D1 antagonist SCH23390’s effects were investigated using a 10Hz induction
protocol. Using the same paradigm as above, SCH23390, in the presence and absence of
the agonist SKF81297, was bath applied immediately prior to 10Hz stimulation.
SCH23390 was able to completely abolish the enhancement of LTD in the presence of
the agonist (0.85±0.13, n=5, vs. SKF81297 alone 0.54±0.11, n=6; p=0.049), but had no
independent effect (0.88±0.12, n=5, vs. control 0.88±0.08, n=5) (Figure 4.5). This
suggests strongly that the effects of SKF81297 are in fact mediated by stimulation of the
Gs coupled D1 receptor, and not attributable to nonspecific activity.
62
Figure 4.5
1.25
1.00
0.75
0.50
0.25 60 mins tetanus post 60
Normalized Normalized fEPSP Slope; 0.00
Control (n=5) +SKF81297 (n=6)+SCH23390 (n=5)
+SKF81297, SCH23390 (n=5)
Figure 4.5 The effect of SKF81297 on LTD induction at 10Hz was D1 receptor mediated. The effect of 10uM SKF81297 was abolished by coapplication of SCH23390, a specific D1 antagonist (0.85±0.13, n=5; SKF81297 alone: 0.54±0.11, n=5; p=0.049). SCH23390 application alone had no effect relative to control (0.88±0.12, n=5; control: 0.88±0.08, n=5).
63
4.6 Neither PACAP38, nor SKF81297 alter presynaptic transmitter release
The effects of PACAP38 and SKF81297 on synaptic plasticity have been shown above to
be dependent on the D1 and PAC1 receptors. However, whether these receptors are
acting at a presynaptic or postsynaptic site had not been investigated. Therefore, to
determine whether PACAP38 or SKF81297 caused an alteration in presynaptic
transmitter release, a comparison of paired‐pulse ratios was undertaken. Briefly, CA1
synapses are synapses of medium release probability (P=0.3‐0.6; Thomson and
Bannister 1999; Markram et al 1998) and therefore demonstrate facilitation when
stimuli are paired. The release probability is determined by the fraction of release sites
that are fully activated by a presynaptic stimulus; the remainder are partially activated
and therefore primed for release by a second stimulus (Thomson, 2000). Any change in
presynaptic release probability will affect both the magnitude of the measured response
to a single stimulus, but also the facilitation induced by paired stimuli. Explicitly, a higher
release probability would impair facilitation.
Briefly, as above, after initial determination of input‐output relationship and paired‐
pulse ratio, fEPSPs were evoked at 0.05Hz for ten minutes in control aCSF followed by
ten minutes in the presence or absence of treatments. Subsequently, the paired‐pulse
ratios were again measured. Compared with time matched controls (1.59±0.02, n=5),
none of PACAP38 (1.42±0.07, n=5), SKF81297 (1.42±0.07, n=5), and SCH23390
(1.54±0.03, n=5) affected the paired‐pulse ratio with an interstimulus interval of 40ms.
Furthermore, no group differed significantly from pretreatment controls (1.49±0.03,
n=5), reaffirming the health of the slice (Figure 4.6). Paired pulse ratios at 10, 20, 80,
64
150, 300, 500, and 1000 ms were also measured and no effects were elicited (Data not
shown).
65
Figure 4.6
2.0
1.5
1.0
0.5 Paired Pulse Paired RatioPulse
0.0
Pretreatment PACAP38 (1nM) SKF81297 (10uM)SCH23390 (0.5uM) Time Matched Control
Treatment (10min)
Baseline Stimulation (0.05Hz,20min)
Figure 4.6 PACAP38, SKF81297 and SCH23390 did not alter presynaptic machinery. The paired pulse ratio was measured using a 40ms interstimulus interval at t=0 and t=20 minutes with or without drug application (t=10‐20 minutes). SKF81297 (1.42±0.07, n=5), SCH23390 (1.54±0.03, n=5) and PACAP38 (1.42±0.07, n=5) did not affect paired pulse ratio relative to negative control, at t=0, (1.49±0.03, n=5) or time‐matched control, at t=20, (1.59±0.02, n=5).
66
4.7 PAC1 antagonist M65 attenuates LTP induction by 100Hz Stimulation
As in 4.3, we undertook to confirm that the effects of PACAP38 on LTP induction were in
fact mediated by the PAC1 receptor. Therefore, the specific PAC1 antagonist M65 was
used with a 100Hz stimulation protocol. In the absence of exogenous PACAP38, M65
was able to attenuate LTP induction at 100Hz (1.16±0.04, n=4, vs. control 1.48±0.03,
n=5; p=0.0003) (Figure 4.7). Because this suggests that PAC1 antagonism can itself be
metaplastic, any results using M65 could be confounding. The attenuation of LTP
induction by M65 suggests that high frequency stimulation somehow causes the PAC1
receptor to be activated, or else M65 has some other non‐specific effect. Given that
exogenous PACAP38 did not have an effect on LTP induction at 100Hz, we hypothesize
that PACAP38’s effects were occluded by the stimulation protocol. The most
parsimonious and attractive explanation is that 100Hz stimulation itself caused a release
of endogenous PACAP38 and thus M65 could attenuate this effect and the effects of
exogenous PACAP38 were occluded.
67
Figure 4.7
100Hz Stimulation 2.8
2.6
2.4
2.2
2.0 Control (n=5) 1.8 M65 (1 uM) (n=4)
1.6
Normalized fEPSP Normalized Slope fEPSP 1.4
1.2
1.0
0.8 -20 -10 0 10 20 30 40 50 60 Time (min)
Control +M65
1 0.2mV
0.2mV 1 10ms 10ms
2 2
Figure 4.7 M65 attenuates LTP induction. Application of 1 uM M65 for ten minutes (t=‐10 to t=0 mins) attenuated LTP induction (control: 1.48±0.03, n=5; M65: 1.16±0.04, n=4; p=0.0003). Sample traces with and without M65 application are shown below.
68
4.8 PACAP38 preferentially increases NR2A phosphorylation
The pathway we have proposed for the effects of PACAP38 on hippocampal neurons
suggests the effects are mediated by the NR2A subunit of the NMDA receptor. In order
to demonstrate that PACAP38 does have a specific effect on the NR2A subunit, we
undertook biochemical experiments to investigate Src kinase and NR2 phosphorylation
in the presence of PACAP38. Briefly, hippocampal slices were treated for 10 minutes at
37oC with 1nM PACAP38 and then homogenized for experiments. Either NR2A or NR2B
was immunoprecipitated followed by Western blot analysis. NR2A, but not NR2B,
tyrosine phosphorylation increased in the PACAP38 treated slices when compared to
controls, as measured by the non‐specific phosphotyrosine antibody 4G10(Figure
4.8A,B,C). Furthermore, Src tyrosine residue 416 showed an increased phosphorylation
in the PACAP38 treated group when compared to controls (Figure 4.8D). Because it has
been shown that Src phosphorylates the NR2A intracellular tail, the results suggest that
PACAP38 mediated effects on the NMDA receptor, and thereby on CA1 plasticity, is
dependent on preferential phosphorylation of the NR2A subunit by Src kinase.
69
Figure 4.8 Low concentrations of PACAP38 induce NR2A but not NR2B tyrosine phosphorylation. Rat pup (PN17) hippocampi were cut to slices and treated with 1nM PACAP38 at 37oC prior to homogenization for experiments. Immunoprecipitations were performed using anti‐NR2A antibody (A) or NR2B antibody (B). A. The blot was first probed with anti‐phosphotyrosine antibody (4G10), then probed with anti‐NR2A after membrane stripping. B. Quantification of results from A. Tyrosine phosphorylation is increased by PACAP38 treatment. C. The blot was probed with anti‐phosphotyrosine antibody, then probed with anti‐NR2B after membrane stripping. PACAP38 application did not increase NR2B phosphorylation. D. Western blot analysis was performed using anti‐pSrcY416 and total Src after membrane stripping. PACAP38 treatment increased phosphorylation of the Src activating residue Y416. (*p<0.05, n=3; Performed by Gang Lei)
70
4.9 SKF81297 preferentially increases NR2B phosphorylation
To complement the above, we also undertook to investigate the effects of D1 agonism
on NR2 subunit phosphorylation. The pathway we propose for D1 receptor mediated
effects is dependent on NR2B and the Src family kinase Fyn. With a similar procedure as
above, we demonstrated that SKF81297 increases NR2B and not NR2A phosphorylation
and that the increase is blocked completely by the D1 antagonist SCH23390 (Figure 4.9).
Furthermore, we demonstrated an increase in Fyn Y420, but not Src Y416,
phosphorylation with SKF81297 treatment, and the abolition of this phosphorylation by
the D1 antagonist SCH23390 (Figure 4.10A,B). The results taken together suggest that
the effects of D1 activation on the NMDA receptor are caused by a Fyn, but not Src,
phosphorylation of the NR2B tail. Confirmatory blots for NR2B pY1472, a substrate for
Fyn, showed an increase in the SKF81297 treated group (Figure 4.10C) and the global
increase in NR2B tyrosine phosphorylation in the treated group could be abolished by
an inhibitor peptide for Fyn (Figure 4.10D).
71
Figure 4.9 SKF81297 increases NR2B but not NR2A tyrosine phosphorylation. Hippocampi from rat pups (PN17) were cut to slices and treated with 10uM of SKF81297 with or without SCH23390 (10uM) at 37oC for 15 minutes. A. Tissue lysate containing 400ug protein was incubated with anti‐NR2B antibody overnight and precipitated with protein A/G plus agarose beads. The precipitates were subject to Western blot analysis, probing sequentially with 4G10 and anti‐NR2B antibodies respectively after membrane stripping. B. Quantification of results of A. C. The same procedure as A was performed excepting precipitation with anti‐NR2A antibody. (*p<0.05, one‐way ANOVA, n=4; Performed by Gang Lei).
72
Figure 4.10 NR2B tyrosine phosphorylation by SKF81297 is mediated through Fyn kinase activation. A. SKF81297 (10uM) has no observed effect on tyrosine phosphorylation of Src kinase, but PACAP38 (100nM) does. B. SKF81297 facilitates tyrosine phosphorylation of Fyn kinase and this effect can be blocked completely by the D1 receptor antagonist SCH23390 (10uM) or an inhibitor peptide of Fyn (10uM). C. D1 receptor activation increases phosphorylation of pNR2BY1472, a Fyn kinase substrate. D. An inhibitor peptide of Fyn kinase fully blocks the tyrosine phosphorylation of NR2B by SKF81297. (Performed by Gang Lei).
73
SECTION FIVE
DISCUSSION
74
5.1 Summary of Key Findings
The major aim of this thesis was to provide a possible physiological mechanism for
metaplasticity at the hippocampal CA1 synapse. We propose that metaplasticity at the
synapse is governed by the interplay between a variety of upstream signaling pathways
terminating at the NR2 subunit of the NMDA receptor. Furthermore, we have
demonstrated that at least two neuromodulators that are present and active in the CA1
hippocampus can have these metaplastic effects. The major conclusions are outlined
here:
1. PACAP, acting through PAC1 receptors, enhances the propensity for LTP at the CA1
synapse.
2. SKF81297, acting through D1‐type dopamine receptors, enhances the propensity for
LTD at the CA1 synapse.
3. PACAP38 modulation of NMDA receptors is Src‐dependent and NR2A subunit
specific; SKF81297 modulation of NMDA receptors is fyn‐dependent and NR2B
subunit specific.
5.2 PAC1R and D1R dependent modulation of NMDARs
Previous work on PACAP mediated effects on CA1 neurotransmission has shown an
ability for PACAP to alter baseline synaptic transmission (Costa et al., 2009; Roberto et
al., 2001; Roberto and Brunelli, 2000; Kondo et al., 1997) and, in fact, induce a form of
chemical plasticity (Roberto et al., 2001). We have previously shown that the effects of
75
PACAP38 on NMDA activity are concentration dependent and the range of effects is
probably dependent on different modes of action (MacDonald et al., 2005). Briefly, we
have shown that concentrations below 0.1nM do not elicit an enhancement in NMDA
peak currents, whereas concentrations above 100nM directly inhibit the NMDA
receptor. To ensure that there was also no direct inhibition or enhancement of mixed
field EPSPs, it was demonstrated that 1nM PACAP38 does not affect baseline CA1
neurotransmission. As the PAC1 receptor can be both Gq and Gs coupled (Ohtaki et al.,
1993; McCulloch et al., 2002; Spengler et al., 1993), and as VPAC receptors respond to
higher concentrations of PACAP38 (Gottschall et al., 1990), we hypothesize that study
paradigms that use higher concentrations of PACAP do not elicit their effects through
the PAC1‐Gq pathway alone, but instead through a mixed PACAP‐mediated response.
Similarly, with sufficiently lower concentrations of PACAP38, we have not been able to
observe any effect on NMDAR function and therefore suggest that any effects here must
also be attributed to a separate PACAP‐mediated response. Our investigations,
therefore, suggest that the results presented here are indicative of actions of the PAC1‐
Gq receptor pathway and not some mixed effect.
Similar to our work with PACAP38, we have been able to show a direct effect of the D1‐
specific agonist SKF81297 on NMDAR mediated EPSCs. In particular, we have been able
to demonstrate an enhancement in peak current that is insensitive to inhibitors of Src
kinase or NR2A knockout, enhanced by NVP‐AAM077, and is sensitive to Ro25‐6981,
inhibitors of Fyn kinase, and the specific D1 antagonist SCH23390 (Trepanier,
unpublished). Our demonstrations of NR2B‐dependent enhancement, and potential
76
NR2A‐dependent depression by SKF81297 are strengthened by other investigations and
other preparations (Hallett et al., 2006; Gao and Wolf, 2008; Schilstrom et al., 2006;
Varela et al., 2009). Moreover, the absence of effect on presynaptic release probability
is corroborated by other investigations determining dopamine mediated presynaptic
effects to by D2‐type receptor dependent (Hsu, 1996). The strength of our evidence
along with the demonstrations of other groups suggests that the effects elicited by the
D1‐agonist SKF81297 are elicited by a postsynaptic Gs‐NR2B pathway.
5.3 PAC1 and D1 Receptor Mediated Effects on Synaptic Plasticity
The major results of this thesis suggest a physiological role for receptors upstream of the
NMDA receptor in terms of synaptic plasticity. The model put forth by Bienenstock,
Cooper and Munro suggests that the degree and direction of synaptic plasticity is
dependent on prior postsynaptic activity and a modification threshold, . This
modification threshold is itself modifiable in a process termed metaplasticity. PAC1 and
D1 receptor activation both appear to modify this threshold and therefore show direct
metaplastic effects.
Investigations into synaptic plasticity at the CA1 synapse have predominantly focused on
the molecular mechanisms responsible for the expression of LTP and LTD. A significant
number of necessary components as well as modulators have thus been discovered and
arguably the NMDA receptor is foremost amongst them. Application of the BCM model
77
to plasticity must therefore be concerned with how is modified by the state of the
NMDA receptor.
Because the role of the NMDA receptor is in part conferred by the subunit composition
of the receptor, and because the NMDA receptor is inextricably linked to synaptic
plasticity, the NR2 subunit has been repeatedly studied in terms of its role in the same.
In the visual cortex it has been demonstrated that a modification in the functional
contribution of the NR2A and NR2B subunits directly affects the induction of synaptic
plasticity (Cho et al., 2009; Philpot et al., 2007). In particular, it has been shown that a
decrease in the functional NR2A:NR2B ratio facilitates potentiation (Cho et al., 2009).
In the context of the CA1 hippocampus, the same general hypotheses have guided
study. Initially, it was shown that inhibition of NR2B preferentially inhibited LTD,
whereas inhibition of NR2A preferentially inhibited LTP (Liu et al., 2004; Massey et al.,
2004). This interpretation would suggest that the LTP inducing stimuli activate NR2A
containing receptors and LTD inducing stimuli activate NR2B containing receptors.
Further evidence that NR2B receptors are selectively implicated in the internalization of
AMPARs and LTD of NMDARs during low frequency stimuli strengthen the suggestion
that NR2B receptors are integral to LTD (Sobczyk and Svoboda, 2007; Tigaret et al.,
2006). However, since this demonstration, a number of groups have collaborated to
show that pharmacological blockers of NR2B containing receptors do not prevent LTD
and similarly that NR2A receptors are not required for induction of LTP (Morishita et al.,
2007; Berberich et al., 2005; Weitlauf et al., 2005). The extension of these experiments
78
to the in vivo situation has reinforced the contribution of NR2A in LTP and NR2B in LTD
(Fox et al., 2006).
Reconciliation of these mutually exclusive hypotheses has suggested a NR2 ratio based
determination of synaptic plasticity where both subunits can potentially cause both
types of plasticity, but where the differential contribution of each is the ultimate
determinant (Philpot et al., 2001; Chen and Bear, 2007; Morishita et al., 2007). Indeed,
this is the very same hypothesis that has been demonstrated in ocular dominance
plasticity in the visual cortex and is suggested in varying other areas of the brain.
By extending the discussion of metaplasticity outside of the realm of pharmacological
blockers and genetic knockout animals, we have been able to demonstrate a
physiological mechanism for metaplasticity at the CA1 synapse. In particular, it is shown
that PACAP38 activation, through the NR2A subunit, is able to decrease the threshold
for LTP, and D1 activation, through the NR2B subunit, is able to increase the threshold
for LTP. Although superficially it appears that this would simply strengthen the original
hypothesis regarding subunit contribution, we suggest that within this subunit‐ratio‐
based hypothesis it is more physiologically relevant to discuss endogenous upstream
regulators of the NMDA receptor. Indeed, the specific manner in which the NMDA
receptor subunits are modulated may be pivotal in this determination of plasticity and
that may be leading to a controversy in results. The work of the Bear group on ocular
dominance plasticity determining an NR2A:NR2B ratio based model is rooted in genetic
manipulation of the NR2A subunit (Cho et al., 2009) versus an acute effect on the NMDA
79
receptor, either via pharmacological blockers (Liu et al., 2004) or upstream signaling.
Because of this difference in approach, between acute effects and systemic effects,
there are reasons to believe the conclusion that the specific manner in which the NMDA
receptor is affected is critical to plasticity. Our paradigm of acute effects and specificity
is discussed further.
5.4 Functional Target Specificity
Although many groups have been able to show that through manipulation of the NMDA
receptor we can observe metaplastic effects, very few have investigated how these
metaplastic effects may occur in vivo. We suggest that the balance between a variety of
upstream signaling pathways terminating at the NMDA receptor and specifically the NR2
subunits provide the basis for metaplasticity of NMDAR dependent synaptic plasticity
and further show that the SFKs play a particularly important role in distinguishing NR2A‐
from NR2B‐ containing receptors.
We as well as other groups have previously investigated the subunit specificity of
actions of the SFKs Src and Fyn. Investigations into Src kinase dependent enhancement
of NMDA EPSCs has shown that the effect can be pharmacologically blocked by NVP‐
AAM077 and Zn2+, but not Ro25‐6981, and is not present in NR2A knockout mice (Yang,
unpublished). Alternatively, we have been able to show that Fyn kinase dependent
enhancement of NMDA EPSCs can be blocked by Ro25‐6981, but not Zn2+ or NVP‐
AAM077, and is not absent in NR2A knockout mice. Other groups have similarly been
able to show an NR2B effect via Fyn activity (Abe et al., 2005; Nakazawa et al., 2001) and
80
an NR2A effect via Src activity (Yang and Leonard, 2001). The physical interactions of the
same have also been investigated, including a RACK1 binding of both Fyn and NR2B
(Yaka et al., 2002), and PSD‐95 association of NR2A and Src (Kalia and Salter, 2003). This
mounting evidence suggests that Src is able to preferentially target NR2A and Fyn is able
to preferentially target NR2B and therefore that each of these NMDAR‐complexes is
functionally separate in the postsynaptic neuron.
Because of this target‐specificity of the SFKs, upstream activators of the same should be
able to produce similarly target‐specific effects. Indeed, we have shown that activation
of the PAC1 receptor Gq signaling cascade is able to increase the phosphorylation of Src
at tyrosine Y416, a residue required for catalytic activity (Smart et al., 1981; Xu et al.,
1999). Furthermore, PAC1 receptor activation is able to preferentially increase NR2A
phosphorylation, suggesting that the target specificity of Src is translated upstream to
the PAC1 receptor. Similarly, we have been able to show that activation of the D1 Gs
signaling cascades are able to preferentially phosphorylate Fyn at the activating residue
Y420 (Sun G et al., 1998; Superti‐Furga et al., 1993). Moreover, D1 activity is able to
preferentially increase NR2B phosphorylation. The target‐specificity previously
demonstrated by SFKs, therefore, is translatable to the upstream GPCRs, whether Gs or
Gq coupled.
This demonstration of target‐specificity previously and above displays that specific
signaling cascades can have direct differential effects on the NR2 subunit of the NMDA
receptor. This linear relationship between affector and effector could be particularly
81
useful in eliciting predictable effects on synaptic plasticity by alterations in
neuromodulator release by upstream neurons and glia.
5.5 Physiological Specificity of PAC1 and D1 Activation
The target‐specificity of Src and Fyn kinases, and by extension the PAC1 and D1
receptors, is augmented by the functional specificity of those same receptors. We have
been able to show that not only does activity at these receptors directly affect the
NMDA receptor, but also that the effects change the induction of synaptic plasticity at
the CA1 synapse.
It has been reported that activity of the NMDA receptor directly determines the
direction and degree of synaptic plasticity at any given synapse. By showing that
upstream GPCRs can directly target the NMDA receptor, and that this targeting is
subunit specific, we have hypothesized that upstream GPCRs can directly modify the
induction of synaptic plasticity.
In the course of this thesis, it has been shown that activation of the PAC1 receptor at
middle frequencies can directly affect the induction of synaptic plasticity. In particular,
at these middle frequencies, CA1 synapses have an increased propensity towards LTP
versus LTD when the PAC1 receptor is activated prior to induction. From our previous
results and the observations of others, we suspect that PAC1 activation causes Src
mediated enhancement of NR2A containing receptors and that this enhancement during
induction causes this shift in plasticity. In this model, NR2A is phosphorylated prior to
82
induction of plasticity by Src kinase whereby effectually increasing the functional
contribution of NR2A containing receptors when they are activated during induction.
Similarly, activation of the D1 receptor has been shown to directly affect the induction
of synaptic plasticity by increasing the propensity towards LTD. Given the results that
suggest that activation of the D1 receptor increases NR2B Y1472 phosphorylation
mediated by Fyn kinase, and that D1 receptor activation increases NMDA activity, we
suggest that D1 activity enhances NR2B containing receptors leading to this alteration in
plasticity. Similar to the PAC1 situation, D1 activation causes NR2B to be phosphorylated
prior to induction at Y1472 by Fyn kinase which effectively increases the contribution of
NR2B containing receptors and promotes depression.
The results presented above, both in terms of Fyn and Src mediated phosphorylation,
and PAC1 and D1 mediated shifts in plasticity, suggest a physiological specificity of the
PAC1 and D1 signaling cascades. In particular, not only do PAC1 and D1 receptors target
NR2A and NR2B in a physically and functionally specific way, they also target synaptic
plasticity in a physiologically specific manner.
5.6 Extensions and Future Directions
The demonstrations here of metaplastic effects initiated by Gq and Gs type GPCRs
suggests that there may be a great number of different signal transduction pathways
that affect synaptic plasticity in a physiological way. The intracellular messengers that
we have shown play intermediary roles between GPCR and NMDA receptor are
83
activated by a truly enormous variety of other pathways. As an extension therefore, it is
probable that all Gq and Gs pathways have metaplastic effects to varying degrees.
Potential avenues for advancement in the areas of hippocampal synaptic plasticity and
metaplasticity are numerous. In terms of the specific effects demonstrated in this work,
a small number of future directions are discussed.
The causal linkage between the GPCR mediated changes in the NMDA receptor and
metaplasticity, although suggested strongly, has not been definitively shown. The
evidence that is presented here would be strengthened therefore by a demonstration
that Src(40‐58), an inhibitor peptide of Src kinase (Yu et al., 1997), can block the effects
elicited by PACAP38. Similarly, blocking the D1 mediated effect via an inhibitor peptide
of Fyn kinase would strengthen the causal linkage. Blockade of this last step in the
intracellular pathway while applying plasticity inducing protocols would be one possible
way to solidify the GPCR‐SFK‐NR2 metaplasticity pathways.
Outside of this Gq/Gs paradigm, there are other signal transduction systems that elicit
metaplastic effects with regards to NMDA receptor dependent plasticity. As an example,
hormones released by the global stress response alter the induction of LTP in the
hippocampus (Hirata et al., 2009). This stress‐induced metaplasticity is dependent on
disruptions in calcium homeostasis (Kim and Yoon, 1998). With this evidence in hand,
we can predict that other GPCR systems, such as Gi‐coupled serotonergic and
dopaminergic systems, or calcium homeostasis modifiers may play a role in determining
the direction and degree of synaptic plasticity.
84
More broadly, investigations into metaplasticity are a very new and interesting field of
neuroscience and as such there is still a wealth of directions in which we can investigate.
We have and are demonstrating that through a variety of upstream GPCR pathways, the
NR2 subunit can be regulated affecting metaplasticity. In this vein in particular, there are
a number of different receptors including the cholinergic, adrenergic and metabotropic
glutamatergic that can and do affect the NMDA receptor and each of these probably
plays a differing role in the regulation of synaptic function . Furthermore, a number of
non‐GPCR mediated affectors of the NMDA receptor exist and all of these lend
themselves to further investigation.
In a more global vein, metaplasticity has been investigated most thoroughly in the visual
cortex and the hippocampus, but should be investigated elsewhere as well. A better
understanding of the whole CNS mechanisms of in vitro and ex vivo metaplasticity
should lead to better investigations into the functional physiological role of
metaplasticity in vivo.
5.7 Overall Conclusions
The work presented here has centered on demonstrating one possible physiological
mechanism for metaplasticity in the CA1 hippocampus. In summary, it has been shown
that the Gq signaling pathway initiated by PACAP38 activation of the PAC1 receptor
causes an increase in Src and NR2A phosphorylation and shifts the modification
threshold leftward, increasing the propensity for LTP. Conversely, the Gs signaling
pathway initiated by SKF81297 activation of the D1 receptor causes an increase in Fyn
85
and NR2B phosphorylation and shifts the modification threshold rightward, increasing
the propensity for LTD. Upstream modulators of the NMDA receptor are therefore
implicated in a physiological mediation of long term synaptic plasticity in the CA1
hippocampus.
86
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