Dendritic glutamate release produces autocrine activation of mGluR1 in cerebellar Purkinje cells

Abstract

In recent years, it has become clear that, in addition to conventional anterograde transmission, signaling in neural circuits can occur in a retrograde manner. This suggests the additional possibility that postsynaptic release of neurotransmitter might be able to act in an autocrine fashion. Here, we show that brief depolarization of a cerebellar Purkinje cell triggers a slow inward current. This depolarization-induced slow current (DISC) is attenuated by antagonists of mGluR1 or TRP channels. DISC is eliminated by a mixture of voltage-sensitive Ca2+ channel blockers and is mimicked by a brief climbing fiber burst. DISC is attenuated by an inhibitor of vesicular glutamate transporters or of vesicular fusion. These data suggest that Ca2+-dependent postsynaptic fusion of glutamate-loaded vesicles evokes a slow inward current produced by activation of postsynaptic mGluR1, thereby constituting a useful form of feedback regulation.

Fig. 1.

Purkinje cell depolarization induces a biphasic inward current with fast and slow components. (a) A train of depolarizing command pulses from −70 to 0 mV was given, as illustrated in Inset. This train consisted of five pulses, each 10 msec long, with an interpulse interval of 100 msec. (Inset calibration bars: 2 nA, 50 msec.) This induced a biphasic inward current with fast (open circle) and slow (filled circle) components. (b) Depolarizing command pulses were given from different holding voltages, and the resultant inward currents were measured. The slow component was marked with filled circles. In these recordings, 20 μM NBQX was added, and a GABA_A receptor antagonist was omitted to allow for simultaneous recording of spontaneous IPSCs and comparison of their reversal with that of both components of the biphasic inward current. Lower traces are expanded from the squares. E_Cl was calculated to be −66.2 mV. (Calibration bars: 200 pA, 2 sec.) (c) We call the slow component of this response “depolarization induced slow current” (DISC). In this graph, DISC charge transfer was measured, and the mean ± SE was plotted as a function of time (n = 5).

Fig. 2.

DISC is Ca²⁺-triggered and involves activation of mGluR1 and TrpC1 cation channels. (a Left) When single-exemplar current traces were high-pass-filtered at 10 Hz, this revealed a significant increase in noise at the peak of DISC (red). [Calibration bars: 100 pA, 2 sec (current trace); 10 pA, 0.2 sec (filtered trace).] (Right) The standard deviation was calculated from 200-msec-long segments of the high-pass-filtered traces to yield the noise SD. Noise SD values from five sequential episodes (red circles) were scaled and superimposed on the current trace, which is an average of the same five episodes. (b–g) Both current and the high-pass-filtered traces were plotted before and after application of various receptor antagonists or channel blockers (100 μM NBQX; 100 μM CPCCOEt; 50 μM JNJ 16259685; 30 μM SKF 96365; a mixture of 0.2 μM ω-agatoxin IVA, 3 μM ω-conotoxin MVIIC, and 3 μM nifedipine; or 250 μM Cd²⁺). [Calibration bars: 200 pA, 2 sec (current traces).] The high-pass-filtered traces are displayed by using a range of −25 to 25 pA. (h) The averages of either DISC charge transfer (filled bars) or Δ noise SD (open bars) are plotted as a ratio of measurements 20 min after drug application to a preexposure baseline (con, control; N, NBQX; C, CPCCOEt; J, JNJ 16259685; S, SKF96365; a/c/n: mixture of Ca²⁺ channel blockers).

Fig. 3.

Internal application of blockers of either vacuolar H⁺-ATPase or SNARE-dependent vesicular fusion suppresses DISC. Either bafilomycin A1 (500 nM) (a) or BoTxD (100 nM) (b) was added to the internal saline. [Calibration bar: 300 pA, 2 sec (current trace).] The high-pass-filtered traces are displayed by using a range of −25 to 25 pA (a Lower). For control experiments to address side effects on mGluR1/TrpC1 signaling, an mGluR1 agonist, DHPG (150 μM), together with glutamate (10 μM), was pressure-ejected (10 psi, 50 msec) in the inner one-third of the molecular layer to induce both fast (AMPA/kainate receptor-mediated) and slow (mGluR1-mediated) inward currents. Slow currents (I_DHPG) are measured in cells perfused with bafilomycin A1. (Calibration bar: 100 pA, 2 sec.) (c) The average ratios of DISC charge transfer (filled), Δ noise SD (open), or I_DHPG amplitude (gray) are plotted. The ratio reflects the value at t = 40 min/t = 20 min.

Fig. 4.

DISC is evoked by climbing fiber burst stimulation. (a) In voltage–clamp mode with a command potential of −55 mV, a climbing fiber was stimulated five times at 10 Hz, which produced a DISC-like inward current (CF-DISC). NBQX (0.5 μM) was subsequently added. (Calibration bar: 300 pA, 2 sec.) (b) CPCCOEt was bath-applied at t = 20 min. (Calibration bar: 200 pA, 2 sec.) (c) The average ratios of CF-DISC charge transfer (filled) and Δ noise SD (open) are plotted. The ratio reflects the value at t = 40 min/t = 20 min.