Inhibitory synaptic transmission mediated by GABAA receptors on cerebellar Purkinje neurones (PNs) exhibits a long-lasting ‘rebound potentiation’ (RP) subsequent to repetitive heterosynaptic climbing fibre activation (Kano et al. 1992). We report that during rebound potentiation a novel, enhancement in GABA release termed depolarization-induced potentiation of inhibition (DPI) that involves the retrograde release of a glutamate-like transmitter and subsequent activation of presynaptic N-methyl-D-aspartate (NMDA) receptors.
To induce DPI, cultured PNs (16-21 DIV) from Sprague-Dawley rats were voltage clamped at -70mV in normal ACSF containing both tetrodotoxin (TTX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to block action potentials and non-NMDA receptor-mediated currents. Cells were then subjected to a single depolarising pulse protocol that involved rapid depolarisation of the cell eight times from -70 to 0 mV (2 s intervals). RP (Llano et al. 1991; Kano et al. 1992) was associated with an increase in the mean mIPSC amplitude (146 ± 9 % of control; n = 10; P < 0.01; t test) while the mean mIPSC frequency displayed a clear biphasic profile. Initially, a rapid reduction in the mean mIPSC frequency was observed (79 ± 5% of control; lasting ~60 s), before developing into a novel, transient increase in the mean mIPSC frequency observed at 3 min post-stimulus (149 ± 8% of control; n = 10; P < 0.01). We examined whether the coincident presynaptic component to RP occurred also in immature (P6-8) slice preparations, during a stage in development when morphology and synaptic connectivity closely resembles that of cultured PNs. Induction of RP resulted in a similar increase in the mean mIPSC amplitude (154 ± 21% of control; n = 7; P < 0.02) and frequency (160 ± 30% of control; n = 7; P < 0.02) measured 5 min post-stimulus. The involvement of presynaptic NMDA receptors during the increase in GABA release was implicated by three main findings. Firstly, the antagonist, D-(2)-amino-5-phosphovalerate (D-APV; 50 µM), completely abolished the mIPSC frequency potentiation in both cultured and immature (P6-8) PNs (98 ± 11 % of control and 101 ± 13% of control, respectively). Secondly, brief (4 s), focal applications of NMDA (100 µM) induced a sustained increase in the release of GABA from basket/stellate cells persisting for > 13 min (no postsynaptic NMDA currents were observed). Finally, triple immunocolocalisation of NMDA receptor subunits (NR1, NR2A,C&D) provided further evidence for the existence of presynaptic NMDA receptors at putative basket/stellate cell axon terminals.
This presynaptic regulation of transmitter release, during the induction of cerebellar synaptic plasticity, will ensure control over PN excitability and subsequent sensorimotor co-ordination in the cerebellar cortex.
This work was supported by the MRC.