Inhibitory synaptic plasticity in the cerebellum.

Learning and memory within the brain are thought to be based on long lasting changes in synaptic efficacy. Purkinje neurones, which focus their output on descending projection pathways and constitute the sole inhibitory output from the cerebellum, display two forms of synaptic plasticity termed ‘depolarisation-induced suppression of inhibition’ (DSI) and ‘rebound potentiation’ (RP). Purkinje neurone depolarisation induces a rapid rise in [Ca '̂ ĵi triggering both the release of a retrograde transmitter and activation of a variety of protein kinases. The phenomena of DSI underlies a transient (lasting <60s) decrease in the mean frequency of mIPSCs, occurring immediately after stimulus cessation, while RP manifests itself as a robust increase in the mean amplitude of spontaneous and miniature inhibitory postsynaptic currents (IPSCs). The present study examined the relationship between preand postsynaptic plasticity during the induction phase of DSI and rebound potentiation in cultured cerebellar Purkinje neurones. Depolarisation caused an immediate decrease in the frequency of mIPSCs (lasting -40s), followed by a transient increase in mIPSC frequency lasting approximately 5 minutes before recovering. A robust potentiation of the mean mIPSC amplitude was observed throughout all experiments and persisted for the duration of recording. The initial frequency decrease (DSI), was abolished by a specific group II mGluR antagonist, LY 341495, while the subsequent transient frequency potentiation was abolished by the specific N-methyl-D-aspartate receptor (NMDAR) antagonist, d-APV. Removal of extracellular sodium, the main current carrying ion through NMDARs, mimicked the application of d-APV by abolishing the frequency potentiation while having no effect on the induction of DSI. Immunocytochemical staining of mixed cerebellar preparations identified cerebellar basket/stellate cells as displaying immunoreactivity for NMDAR NRl subunits but not mGluR2/3 at putative presynaptic release sites. These results provide the first evidence for, 1) the involvement of presynaptic NMDARs in the transient enhancement of G ABA release during rebound potentiation and 2) the possibility that a novel group II mGluR splice variant/subtype underlies the induction of cerebellar DSI. A model is proposed to explain the relationship between DSI and rebound potentiation. INHIBITORY SYNAPTIC PLASTICITY IN THE CEREBELLUM ABSTRACT. 1. 1 LIST OF TABLES. 7 LIST OF FIGURES. 9 Chapter 1 GENERAL INTRODUCTION The cerebellum. 13 Mammalian GABAa receptors.20 Metabotropic glutamate receptors. 30 N-methyl-D-aspartate receptors. 36 AMP A, Kainate & NMD A receptors: Presynaptic regulation of transmitter release. 44 Excitatory amino acid transporters (EAATs). 47 Câ "̂ in Purkinje neurones: Sources and homeostasis.52 Long-term depression (LTD) in the cerebellum. 55 Depolarisation-induced suppression of inhibition (DSI). 57 Rebound potentiation (RP). 59 Tables 1.1 Figures 1.1-1.12 Chapter 2 MATERIALS AND METHODS 2.1. Mixed cerebellar cultures. 63 2.2. Culture solution.63 2.3. Composition of superfusing media.64 2.4. Composition of patch clamp internal solution.64 2.5. Electrophysiological techniques.65 2.6. Data recording and electrical stimulation.67 2.7. Immunocytochemical staining.69 2.8. Confocal microscopy.71 2.9. Off-line data analysis and statistical analysis.73 2.10. Drugs and their application. 75 Table 2.1-2.4 Figures 2.1-2.6 Chapter 3 MORPHOLOGICAL CHANGES DURING DEVELOPMENT OF PURKINJE NEURONES IN CULTURE Introduction.78 Results.80 Discussion. 90 Table 3.1 Figures 3.1-3.8 Chapter 4 DEPOLARISATION-INDUCED SUPPRESSION OF INHIBITION AND REBOUND POTENTIATION Introduction.94 Results 4.1. Membrane and synaptic properties of cultured PNs. 97 4.2. Depolarisation-induced suppression of inhibition (DSI). 101 4.3. Rebound potentiation (RP). 101 4.4. Timecourse of PN mlPSC amplitude and frequency modulation after stimulus cessation.106 4.5. mlPSC kinetic changes during DSI and R P . 108 Discussion 4.1. Membrane and synaptic properties of cultured PNs.I l l 4.2. Depolarisation-induced suppression of inhibition (DSI). 113 4.3. Rebound potentiation (RP). 116 4.4. mlPSC kinetic changes during DSI and R P . 118 Tables 4.1-4.3 Figures 4.1-4.9 Chapter 5 PRESYNAPTIC mGIuR-MEDIATED CONTROL OF INHIBITORY SYNAPTIC TRANSMISSION AT THE INTERNEURONE-PN SYNAPSE Introduction. 120 Results 5.1. Effects of the non-specific mGluR antagonist (S)-MCPG on DSI & RP induction. 123 5.2. Effects of the specific group II mGluR antagonist LY 341495 on DSI & RP induction.133 Discussion 5.1. Effects of the non-specific mGluR antagonist (S)-MCPG on DSI & RP induction. 146 5.2. Effects of the specific group II mGluR antagonist LY 341495 on DSI & RP induction . 147 5.3. Immunohistochemistry & pharmacology: The anomalies that exist. 149 Tables 5.1-5.8 Figures 5.1-5.14 Chapter 6 PRESYNAPTIC NMDAR-MEDIATED CONTROL OF INHIBITORY SYNAPTIC TRANSMISSION AT THE INTERNEURONE-PN SYNAPSE Introduction.152 Results 6.1. Effects of the specific NMDAR antagonist d-APV on DSI and RP induction. 154 6.2. Effects of extracellular Na"̂ removal on the induction/ maintenance of DSI & R P.163 6.3. Effects of Na"̂ removal on NMDAR-mediated currents in intemeurones. 174 6.4. Immunocytochemical identification of presynaptic NMDARs. 176 6.5. NMDA enhancement of G ABA release from cerebellar Intemeurones.183 Discussion 6.1. NMDA receptors: Involvement in DSI & R P . 188 6.2. Removal of extracellular Na"̂ : Effects on Câ '*' sequestration and EAATs.190 6.3. Removal of extracellular Na"̂ : Effects on resting membrane potential. 192 6.4. Removal of extracellular Na"̂ : Effects on presynaptic NMDARs. 195 6.5. Cerebellar intemeurone axon terminals: Putative sites fo