Presynaptic NMDA receptors act as local high-gain glutamate detector in developing cerebellar molecular layer interneurons.

In the classical view, NMDA receptors (NMDARs) are located postsynaptically and play a pivotal role in excitatory transmission and synaptic plasticity. In developing cerebellar molecular layer interneurons (MLIs) however, NMDARs are known to be solely extra- or presynaptic and somewhat poorly expressed. Somatodendritic NMDARs are exclusively activated by glutamate spillover from adjacent synapses, but the mode of activation of axonal NMDARs remains unclear. Our data suggest that a volume transmission is likely to stimulate presynaptic NMDARs (preNMDARs) since NMDA puffs directed to the axon led to inward currents and Ca²âº transients restricted to axonal varicosities. Using local glutamate photoliberation, we show that pre- and post-synaptic NMDARs share the same voltage dependence indicating their containing NR2A/B subunits. Ca²âº transients elicited by NMDA puffs are eventually followed by delayed events reminding of the spontaneous Ca²âº transients (ScaTs) described at the basket cell/Purkinje cell terminals. Moreover, the presence of Ca²âº transients at varicosities located more than 5 µm away from the uncaging site indicates that the activation of preNMDARs sensitizes the Ca²âº stores in adjacent varicosities, a process that is abolished in the presence of a high concentration of ryanodine. Altogether, the data demonstrate that preNMDARs act as high-gain glutamate detectors.

Current and calcium responses to local activation of axonal NMDA receptors in developing cerebellar molecular layer interneurons.

In developing cerebellar molecular layer interneurons (MLIs), NMDA increases spontaneous GABA release. This effect had been attributed to either direct activation of presynaptic NMDA receptors (preNMDARs) or an indirect pathway involving activation of somato-dendritic NMDARs followed by passive spread of somatic depolarization along the axon and activation of axonal voltage dependent Ca(2+) channels (VDCCs). Using Ca(2+) imaging and electrophysiology, we searched for preNMDARs by uncaging NMDAR agonists either broadly throughout the whole field or locally at specific axonal locations. Releasing either NMDA or glutamate in the presence of NBQX using short laser pulses elicited current transients that were highly sensitive to the location of the spot and restricted to a small number of varicosities. The signal was abolished in the presence of high Mg(2+) or by the addition of APV. Similar paradigms yielded restricted Ca(2+) transients in interneurons loaded with a Ca(2+) indicator. We found that the synaptic effects of NMDA were not inhibited by blocking VDCCs but were impaired in the presence of the ryanodine receptor antagonist dantrolene. Furthermore, in voltage clamped cells, bath applied NMDA triggers Ca(2+) elevations and induces neurotransmitter release in the axonal compartment. Our results suggest the existence of preNMDARs in developing MLIs and propose their involvement in the NMDA-evoked increase in GABA release by triggering a Ca(2+)-induced Ca(2+) release process mediated by presynaptic Ca(2+) stores. Such a mechanism is likely to exert a crucial role in various forms of Ca(2+)-mediated synaptic plasticity.

Calcium-permeable presynaptic AMPA receptors in cerebellar molecular layer interneurones.

Axons of cerebellar molecular layer interneurones (MLIs) bear ionotropic glutamate receptors. Here, we show that these receptors elicit cytosolic [Ca2+] transients in axonal varicosities following glutamate spillover induced by stimulation of parallel fibres (PFs). A spatial profile analysis indicates that these transients occur at the same locations when induced by PF stimulation or trains of action potentials. They are not affected by the NMDAR antagonist AP-V, but are abolished by the AMPAR inhibitor GYKI-53655. Mimicking glutamate spillover by a puff of AMPA triggers axonal [Ca2+]i transients even in the presence of TTX. Addition of specific voltage-dependent Ca2+ channel (VDCC) blockers such as omega-AGAIVA and omega-conotoxin GVIA or broad range inhibitors such as Cd2+ did not significantly inhibit the signal indicating the involvement of Ca2+-permeable AMPARs. This hypothesis is further supported by the finding that the subunit specific AMPAR antagonist IEM-1460 blocks 75% of the signal. Bath application of AMPA increases the frequency and mean peak amplitude of GABAergic mIPSCs, an effect that is blocked by philanthotoxin-433 (PhTx) and reinforced by facilitating concentrations of ryanodine. By contrast, a high concentration of ryanodine or dantrolene reduced the effects of AMPA on mIPSCs. Single-cell RT-PCR experiments show that all GluR1-4 subunits are potentially expressed in MLI. Taken together, the results suggest that Ca2+-permeable AMPARs are colocalized with VDCCs in axonal varicosities and can be activated by glutamate spillover through PF stimulation. The AMPAR-mediated Ca2+ signal is amplified by Ca2+-induced Ca2+ release from intracellular stores, leading to GABA release by MLIs.