T-type calcium channels mediate rebound firing in intact deep cerebellar neurons

Abstract

Neurons of the deep cerebellar nuclei (DCN) form the main output of the cerebellar circuitry and thus play an important role in cerebellar motor coordination. A prominent biophysical property observed in rat DCN neurons is rebound firing; a brief but strong hyperpolarizing input transiently increases their firing rate to much higher levels compared with that prior to the inhibitory input. Low-threshold T-type voltage-gated calcium channels have been suspected for a long time to be responsible for this phenomenon, but direct pharmacological evidence in support of this proposition is lacking. Even though a multitude of functional roles has been assigned to rebound firing in DCN neurons, their prevalence under physiological conditions is in question. Studies aimed at delineating the physiological role of rebound firing are hampered by the lack of a good pharmacological blocker. Here we show that mibefradil, a compound that blocks T-type calcium channels, potently blocks rebound firing in DCN neurons. In whole-cell experiments both mibefradil and NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]. a more selective T-type calcium channel blocker, effectively blocked rebound firing produced by direct current injection. Thus, mibefradil and other T-type channel modulators may prove to be invaluable tools for elucidating the functional importance of DCN rebound firing in cerebellar computation.

Figure 1. Photorelease of GABA induces fast inhibitory currents in DCN neurons

A, Photorelease of GABA above the soma and dendrites of voltage clamped DCN neuron (see schematic on the left) by a 1 ms pulse of UV light delivered at the time indicated by the black triangle produced a fast rising outward current (V_cmd= +30 mV). B, Average and individual (red circles) values of decay time constant of the GABA-induced outward currents measured at +30 mV in 8 cells.

Figure 2. The rebound firing post-inhibition is abolished by the T-type calcium channel blocker mibefradil in DCN neurons

The raster plots show spontaneous activity of individual DCN neurons recorded extracellularly. A, Photorelease of GABA, at the time indicated by the black triangle, induced a pause followed rebound firing in form of short high frequency burst (marked with a white triangle above the first raster). Bath application of mibefradil (added at the time marked on the left) blocked rebound firing. B, 10 electrical stimuli @ 100 Hz were delivered at the time indicated by the arrow to activate inhibitory synaptic inputs to a DCN neuron. After the stimulus-induced pause, prominent rebound firing was observed as a high-frequency burst of action potentials (white triangle). The “*”s denote traces in which a clear long lasting increase in firing rate was seen. Bath application of mibefradil (at the indicated time on the left) blocked rebound firing, including the long responses.

Figure 3. Mibefradil inhibits rebound firing without affecting baseline spontaneous activity or inhibition

A, Average time course of the effect of mibefradil on rebound firing induced by GABA photorelease and electrical stimulation (pooled data from the 10 cells examined). The inset shows the average rebound firing rate of 6 cells in the absence of mibefradil. At the times indicated on the graph, representative pre-pause and post-pause firing rates were measured by averaging the values obtained for 10 trials for each condition. B, Average and individual firing rate of the cells shown in panel A, before and after the stimulus-induced pause, in the presence and absence of mibefradil respectively. Red symbols and black symbols represent the experiments done with GABA photorelease and electrical stimulation respectively. (*) denotes p<0.001, n.s. denotes “not significant”. C, The average and individual pause durations for the 10 cells shown in panel (B) in the presence and absence of mibefradil. n.s. denotes “not significant”.

Figure 4. Blockers of T-type calcium channels abolish rebound firing evoked by direct current injection

A, Example of a whole-cell current-clamped DCN neuron showing robust rebound firing after a 500 ms long hyperpolarizing pulse to ≈ −85 mV, in control conditions (dark grey trace) and after 5 μM mibefradil was added to the bath perfusion (blue trace). B, Comparable experiment as in panel (A) but using 15 μM NNC 55-0396 (NNC, orange trace). C, Average and individual values of maximum hyperpolarization during the current injection in control and after application of T-type channel blocker (T blocker). Blue symbols represent 5 cells treated with mibefradil and orange symbols represent 4 cells treated with NNC 55-0396. “n.s.” denotes not significant. D, Average and individual values of maximum rebound firing rate in control conditions, and after the addition of T-type channel blockers (T blocker). Blue symbols represent mibefradil and orange symbols represent NNC 55-0396, (*) p<0.001. E, Average and individual values of the number of spikes after the hyperpolarizing pulse in control conditions and after adding the T-type channels blockers (T blocker). Blue symbols represent mibefradil and orange symbols represent NNC 55-0396, (*) p<0.001.