Scuola Internazionale Superiore di Studi Avanzati Trieste SISSA (Trieste) Area of Neuroscience Activity-dependent regulation of GABA release at immature mossy fibers-CA3 synapses: role of the Prion protein Thesis submitted for the degree of “Doctor Philosophiae ” 14 December 2012 Candidate: Supervisor: Maddalena Delma Caiati Prof. Enrico Cherubini 1 TABLE OF CONTENTS ABBREVIATIONS 6 ABSTRACT 10 INTRODUCTION 14 1. The hippocampus 14 2. The hippocampal mossy fibers pathway 16 2.1 Synaptic physiology 17 2.1.1 Adult MFs are glutamatergic 18 2.1.2 In particular conditions, adult MFs can co-release glutamate and GABA 19 2.1.3 Presynaptic modulation of GABA release 20 3. Synaptic plasticity 23 3.1 Short-term plasticity 23 3.2 Long-term plasticity 26 4. Functional role of MF 28 5. Development of the hippocampus 31 5.1 GABAergic signaling early in postnatal development 33 5.2 GABA is the main neurotransmitter released from immature MF terminals 36 5.3 Presynaptic modulation of GABA release by GABAA and GABA B autoreceptors 41 5.4 Activity-dependent changes of synaptic efficacy 42 2 5.4.1 Calcium transients associated with GDPs act as coincident detectors for enhancing synaptic strength 43 5.4.2 Spike-time-dependent plasticity (STDP) 45 6. Adult neurogenesis 47 7. Involvement of DG granule cells in neuropsychiatric disorders 50 AIM OF THE STUDY 55 METHODS 59 RESULTS 64 N. 1: Early in postnatal development, tonic activation of kainate receptors by ambient glutamate reduces GABA release from MF terminals. N. 2: Developmental regulation of CB1-mediated spike-time dependent depression at immature mossy fiber-CA3 synapses. N.3: Prp C controls via PKA the direction of synaptic plasticity in the immature hippocampus. CONCLUSIONS AND FUTURE PERSPECTIVES 65 BIBLIOGRAPHY 71 ANNEX ACKNOWLEDGMENTS 3 Declaration The original work presented in this thesis was carried out at the International School for Advanced Studies, Trieste, between November 2008 and November 2012. I performed and analyzed the majority of the electrophysiological experiments and contributed to papers writing. In situ hybridization experiments have been performed by Elodie Richard and Dany Verrier in Dr. Giovanni Marsicano’s laboratory in Bordeaux. N. 1: Caiati MD*, Sivakumaran S *, Cherubini E. Early in postnatal development, tonic activation of kainate receptors by ambient glutamate reduces GABA release from mossy fiber terminals. The Journal of Neuroscience 2010 Feb 3;30(5):1750-9. * equally contributed N. 2: Caiati MD , Sivakumaran S, Lanore F, Mulle C, Richard E, Verrier D, Marsicano G, Miles R, Cherubini E. Developmental regulation of CB1-mediated spike-time dependent depression at immature mossy fiber-CA3 synapses. Sci Rep. 2012;2:285. N. 3: Caiati MD , Safiulina VF, Fattorini G, Sivakumaran S, Giuseppe Legname and Enrico Cherubini Prp C controls via PKA the direction of synaptic plasticity in the immature hippocampus. Under revision in The Journal of Neuroscience 4 The following publications have been included in the present thesis in the Annex . Safiulina VF, Caiati MD , Sivakumaran S, Bisson G, Migliore M and Enrico Cherubini. Control of GABA release at single mossy fiber-CA3 connections in the developing hippocampus. Frontiers in Synaptic Neuroscience, 2010 Feb 22;2:1 doi: 10.3389/neuro.19.001. R Tyzio, C Allene, R Nardou, MA Picardo, S Yamamoto, Sivakumaran S, Caiati MD , S Rheims, M Minlebaev, P Ferré, R Khazipov, JL Romette, J Lorquin, G. Chazal, R Cossart, I Khalilov, A Nehlig, E Cherubini, Y Ben-Ari. Ketone body metabolic substrate beta-hydroxybutyrate does not alter excitatory GABA actions on neonatal cortical neurons. The Journal of Neuroscience 2011 Jan 5, 31(1):34-45. Cherubini E, Caiati MD , Sivakumaran S. In the Developing Hippocampus Kainate Receptors Control the Release of GABA from Mossy Fiber Terminals via a Metabotropic Type of Action. Adv Exp Med Biol. 2011;717:11-26. Caiati MD Is GABA co-released with glutamate from immature mossy fiber terminals? In press in The Journal of Neuroscience . 5 ABBREVIATIONS ACSF Artificial CerebroSpinal Fluid AMPA α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate ATP Adenosine 5’-triphosphate BAPTA 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid BDNF Brain-derived neurotrophic factor CA1/4 Cornu Ammonis regions 1/4 cAMP 3’-5’-cyclic Adenosine MonoPhospate CCK Cholecystokinin CNQX 6-Cyano-7-NitroQuinoXaline-2,3-dione CNS Central Nervous System D-AP5 D-2-amino-5-phosphonovalerate DCG-IV (2S,2'R,3'R)-2-(2',3'-Dicarboxycyclopropyl)glycine DG Dentate Gyrus DMSO DiMethylSulphOxide DNQX 6,7-DiNitroQuinoXaline-2,3-dione EC Entorhinal Cortex EGTA Ethylene Glycol-bis(2-aminoethylether)-N-N-N’-N’-Tetraacetic Acid EPSC Excitatory PostSynaptic Current EPSP Excitatory PostSynaptic Potential ERK Extracellular signal-Regulated Kinases 6 GABA γ-aminobutyric acid GAD Glutamic Acid Decarboxylase GAT γ-aminobutyric acid transporter GDP Giant Depolarizing Potential GYKI 52466 4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3] benzodiazepin-5-yl)-benzenamine Hydrochloride HEPES 4-(2-hydroxyethyl)-1-piperazine-1-ethanesulfonic acid KAR Kainate Receptor KCC2 K+/Cl- co-transporter isoform 2 L-AP4 L(+)-2-amino-4-phosphonobutyric acid LTD Long-Term Depression LTP Long-Term Potentiation MAPK Mitogen-activated protein (MAP) kinases MF Mossy Fiber mGluR metabotropic glutamate receptor nAChR nicotinic acetylcholine receptor NKCC1 Na-K-Cl cotransporter isoform 1 NMDA N-Methyl-D-Aspartate O-LM Oriens–Lacunosum Moleculare O-OR Oriens–Oriens and Radiatum P-LM Pyramidale–Lacunosum Moleculare PCR Polymerase Chain Reaction PKA Protein Kinase A 7 PPR Paired-Pulse Ratio PTX Picrotoxin R–LM Radiatum–Lacunosum Moleculare STD-LTD spike-timing-dependent-long-term depression STD-LTP spike-timing-dependent-long-term potentiation TTX Tetrodotoxin VGAT Vesicular GABA Transporter 8 “Study the science of art. Study the art of science. Develop your senses- especially learn how to see. Realize that everything connects to everything else.” Leonardo Da Vinci 9 ABSTRACT In adulthood, mossy fibers (MFs), the axons of granule cells of the dentate gyrus (DG), release glutamate onto CA3 principal cells and interneurons. In contrast, during the first week of postnatal life MFs release γ-aminobutyric acid (GABA), which, at this early developmental stage exerts a depolarizing and excitatory action on targeted cells. The depolarizing action of GABA opens voltage-dependent calcium channels and NMDA receptors leading to calcium entry and activation of intracellular signaling pathways involved in several developmental processes, thus contributing to the refinement of neuronal connections and to the establishment of adult neuronal circuits. The release of GABA has been shown to be down regulated by several neurotransmitter receptors which would limit the enhanced excitability caused by the excitatory action of GABA. It is worth noting that the immature hippocampus exhibits spontaneous correlated activity, the so called giant depolarizing potentials or GDPs that act as coincident detector signals for enhancing synaptic activity, thus contributing to several developmental processes including synaptogenesis. GDPs render the immature hippocampus more prone to seizures. Here, I explored the molecular mechanisms underlying synaptic transmission and activity-dependent synaptic plasticity processes at immature GABAergic MF-CA3 synapses in wild-type rodents and in mice lacking the prion protein ( Prnp 0/0 mice). In the first paper, I studied the functional role of kainate receptors (KARs) in regulating GABA release from MF terminals. Presynaptic KARs regulate synaptic transmission in several brain areas and play a central role in modulating glutamate release at adult MF-CA3 synapses. I found that functional presynaptic GluK1 receptors are present on MF terminals where they down regulate GABA release. Thus, application of DNQX or UBP 302, a selective antagonist for GluK1 receptors, strongly increased the amplitude of MF-GABA A-mediated postsynaptic currents (GPSCs). This effect was associated with a decrease in failure rate and increase in PPR, indicating a presynaptic type of action. 10 GluK1 receptors were found to be tonically activated by glutamate present in the extracellular space, since decreasing the extracellular concentration of glutamate with a glutamate scavenger system prevented their activation and mimicked the effects of KAR antagonists. The depressant effect of GluK1 on GABA release was dependent on pertussis toxin (PTx)-sensitive G protein-coupled kainate receptors since it was prevented when hippocampal slices were incubated in the presence of a solution containing PTx. This effect was presynaptic since application of UBP 302 to cells patched with an intracellular solution containing GDP βS still potentiated synaptic responses. In addition, the depressant effect of GluK1 on GABA release was prevented by U73122, which selectively inhibits phospholipase C, downstream to G protein activation. Interestingly, U73122, enhanced the probability of GABA release, thus unveiling the ionotropic type of action of kainate receptors. In line with this, we found that GluK1 receptors enhanced MF excitability by directly depolarizing MF terminals via calcium-permeable cation channels. We also explored the possible involvement of GluK1 in spike time-dependent (STD) plasticity and we found that GluK1 dynamically regulate the direction of STD-plasticity, since the pharmacological block of this receptor shifted spike-time dependent potentiation into depression. The mechanisms underlying STD-LTD