Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2010 Jan 23.
Published in final edited form as: Neuroscience. 2008 Oct 8;158(2):635–641. doi: 10.1016/j.neuroscience.2008.09.052

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

Karina Alviña 1,2, Graham Ellis-Davies 3, Kamran Khodakhah 1
PMCID: PMC2649676  NIHMSID: NIHMS94644  PMID: 18983899

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 to 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 have 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, 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.

Keywords: cerebellum, calcium channels, rebound, inhibition, rat

Introduction

Neurons of the deep cerebellar nuclei (DCN) form the major output of the computational circuitry of the cerebellum (Ito, 1984). Their rate and pattern of activity encode the information necessary for motor coordination. An interesting biophysical feature of DCN neurons is rebound depolarization (Jahnsen, 1986a; Llinas and Muhlethaler, 1988; Aizenman et al., 1998; Aizenman and Linden, 1999; Molineux et al., 2006; Pugh and Raman, 2006; Wetmore et al., 2007). When the membrane potential of a DCN neuron recovers from hyperpolarization induced by a strong inhibitory input, it transiently rises to a more depolarized level than that preceding the inhibitory input. This rebound depolarization significantly increases the firing rate of the DCN neuron after the stimulus-evoked pause. Given the inhibitory GABAergic nature of Purkinje cell synapses onto DCN neurons, rebound firing has been extensively incorporated into recent theories of cerebellar function (Medina et al., 2000; Kistler and De Zeeuw, 2003; Wetmore et al., 2007) and several functional roles, from timing to encoding information and mediating plasticity have been assigned to it (Aizenman et al., 1998; Kistler and De Zeeuw, 2003; Pugh and Raman, 2006; Wetmore et al., 2007).

However, despite the biophysical robustness of rebound firing in DCN neurons, there is a great deal of debate as to its prevalence and function in response to physiological stimuli (Telgkamp and Raman, 2002; Aksenov et al., 2004; Rowland and Jaeger, 2005; Holdefer et al., 2005; Rowland and Jaeger, 2008; Alvina et al., 2008). Full characterization of rebound firing and scrutiny of its physiological role in vivo requires pharmacological tools to manipulate it. These tools are presently lacking.

Low threshold voltage-gated calcium channels are thought to contribute to rebound firing in DCN neurons (Llinas and Muhlethaler, 1988; Aizenman and Linden, 1999; Molineux et al., 2006; Pugh and Raman, 2006). These cells express both high- and low-threshold calcium channels (Muri and Knopfel, 1994; Volsen et al., 1995; Gauck et al., 2001). These channels play an essential role in rebound depolarization in different cells because they activate and inactivate with relatively little depolarization. In fact it has recently been demonstrated that based on expression of different T-type calcium channel subtypes DCN neurons can be classified as strong or weak rebound bursters (Molineux et al., 2006). Here we report that mibefradil, a blocker of T-type calcium channels (McDonough and Bean, 1998; Martin et al., 2000), may prove to be an invaluable tool in resolving the function of rebound firing in the DCN because it effectively blocks rebound firing in these neurons without affecting their pacemaking.

Methods

Cerebellar slices

All procedures were in accordance with the policies established by the Animal Institute Committee of the Albert Einstein College of Medicine. Wistar rats (P12-20) were anesthetized with halothane and decapitated. The brain was quickly removed and placed on cold extracellular solution containing (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, and 10 glucose, pH= 7.4 when gassed with 95% O2:5% CO2. The cerebellum was dissected and mounted on a modified Oxford vibratome and 300-μm thick sagittal slices were made. The slices were kept in oxygenated extracellular solution at 34°C for one hour, and then at room temperature until use.

Electrophysiological recording

Slices were placed in a recording chamber on the stage of a Zeiss Axioskop microscope. DCN neurons were visually identified using a 40× water-immersion objective with infrared optics. The slices were superfused with the recording solution at a rate of 1.5-2 ml/min and the temperature adjusted to 35±1°C. 5 mM kynurenic acid (Spectrum Chemical MFG Corp., Gardena, CA), a broad-spectrum ionotropic glutamate receptor antagonist (Stone, 1993) was added to the recording solution to isolate inhibitory inputs.

Extracellular recordings were obtained from single DCN neurons from all three cerebellar nuclei using a home-made differential amplifier and glass pipette electrodes filled with extracellular solution. Data was sampled at 10 kHz using an analog-to-digital converter (PCI-MIO-16XE-10; National Instruments, Austin, TX), and acquired and analyzed using custom software written in LabView (National Instruments).

Whole-cell recordings were performed using an Optopatch amplifier (Cairn Research, Kent, UK) with electrodes pulled from borosilicate glass (1–3 MΩ resistance when filled with intracellular solution). The internal solution used to measure GABA-induced currents consisted of (in mM): 70 Cs-gluconate, 10 CsF, 20 CsCl, 10 EGTA, 10 HEPES, and 3 Na2ATP, pH 7.4 (CsOH). To measure rebound firing in current-clamp mode, the internal solution used contained the following (in mM): 125 K Methylsulfate, 10 NaCl, 0.01 EGTA, 9 HEPES, 14 Creatine phosphate, 4 MgATP, and 0.3 Tris-GTP, pH 7.2 (KOH).

Mibefradil was a generous gift from Hoffmann-La Roche (Basel, Switzerland). NNC 55-0396 was purchased from Tocris (Ellisville, MO).

Photorelease of caged GABA

4-carboxy-7-nitroindolinyl-gamma-aminobutyric acid (caged GABA) was synthesized in the laboratory. Caged GABA was dissolved in the extracellular solution at a final concentration of 325 μM. 1 ms pulse of UV light from a flash lamp (Cairn Research, Kent, UK) was delivered to the epifluorescence port of the microscope using a liquid light guide and was set up as Köhler illumination to uniformly illuminate the field of view. The energy of the UV pulse was adjusted to photorelease enough GABA to produce a well-defined pause of 200-700 ms duration in the spontaneous activity of the cell. Extracellular recording was performed as described above.

Electrical stimulation

Monopolar and bipolar stimulation electrodes were used for electrical stimulation of inhibitory inputs. The monopolar electrode consisted of a glass pipette with a diameter of ≈80 μm filled with the extracellular solution. The bipolar electrode was made by twisting a pair of 50 μm nickel wires with a final tip separation of ≈200 μm. The stimulation electrode was positioned between the DCN and the cerebellar cortex to stimulate the axons of Purkinje cells and to avoid either a direct effect on the DCN neuron under study or the direct stimulation of intra-DCN axons. The electrodes were driven by constant current stimulator (Digitimer Ltd.) which was set to deliver 100-200 μs long current pulses. The stimulation paradigm consisted of either a single pulse, or a train of 10 pulses at 100 Hz, with intensities ranging from typically 50 μA to maximally 500 μA.

Pre- and post-pause firing rates were obtained by averaging 5 interspike intervals prior to, and after the stimulus-evoked pause in the spontaneous activity of the target neuron. Statistical analysis was performed by one-way ANOVA and data were considered not to be statistically different if p>0.05. All data are presented as Mean ± S.E.M.

Results

Contribution of T-type calcium channels to rebound firing in intact DCN neurons

To mimic inhibitory GABAergic inputs and avoid the complications that GABAergic inputs in slices might have been damaged by the slicing procedure, GABA was photoreleased on the soma and dendrites of DCN neurons from caged GABA pre-equilibrated with the slice. We first examined the kinetics of such GABA-evoked currents by whole cell voltage-clamping DCN neurons. As shown in Figure 1, photorelease of GABA using a 1 ms pulse of UV light resulted in a current that typically had a rise time of a few milliseconds and decayed monotonically with an average time constant of 78.7±9.0 ms (n=8 cells).

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

Figure 1

Open in a new tab

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 (Vcmd= +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.

The fast rise time and decay time constant of GABA-induced currents suggests that photorelease of GABA on intact DCN neurons is likely to hyperpolarize the cells for a well-defined period of time. We thus examined the consequences of photorelease of GABA on the spontaneous activity of DCN neurons. We avoided whole-cell recordings because it is well-established that they alter the extent of contribution of calcium channels to spontaneous firing of these cells, presumably as a consequence of dialysis of the intracellular milieu (Alvina and Khodakhah, 2008). Therefore we monitored the activity of individual DCN neurons by single cell extracellular recordings in the presence of blockers of excitatory synaptic transmission (kynurenic acid). The spontaneous activity of the neuron was recorded for a minimum of 10 minutes to obtain a baseline and subsequently GABA was photoreleased onto the target cell as it was done in the whole-cell experiments described above. The energy of the pulse of photolysis UV light was adjusted such that photorelease of GABA produced a pause of 200-700 ms in the spontaneous activity (Figure 2A). In accord with our earlier observation (Alvina et al., 2008), in a small fraction of DCN neurons (8/58 cells) clear rebound firing was evident after the pause. This manifested in the form of high-frequency bursts of action potentials after the pause. The 58 DCN neurons sampled and the 8 cells that responded with rebound firing were distributed in all 3 nuclei. Recently it has been shown that the DCN are made of 3 different types of neurons, which can be partly distinguished based on their baseline firing rate (Uusisaari et al., 2007). On the basis of an analysis of the baseline firing rate of our cells, we have no evidence to believe that the cells that showed rebound firing belonged to any specific type of DCN neurons.

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

Figure 2

Open in a new tab

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 2A shows consecutive raster plots in time obtained every 45 seconds. GABA was photoreleased at the time indicated by the black triangle. The approximately 250 ms long pause produced by GABA in this cell resulted in prominent rebound firing as evidenced by the highly reproducible burst of action potentials (white triangles on the top). In all cells that showed rebound firing the bursts were robust, and were present trial after trial with repeated photorelease of GABA (Figure 2A). In 5 of these 8 cells we examined the consequences of blocking T-type calcium channels with mibefradil (5-10 μM). In all 5 cells examined, mibefradil completely abolished rebound firing (Figure 2A and 3).

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

Figure 3

Open in a new tab

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”.

We also examined whether mibefradil could equally prevent rebound firing when it was evoked synaptically, because it is possible that under these conditions the GABAergic conductance may differentially hyperpolarize electrical compartments of the cell (e.g. soma vs. dendrites). Inhibitory synaptic inputs were activated by a train of 10 constant current electrical pulses delivered at 100 Hz, or in a small number of cells with a single pulse (9 cells). Similarly to that seen when rebound firing was evoked by GABA photorelease, only 8 out of 39 cells showed rebound firing after electrical stimulation. One of these 8 cells was stimulated with a single pulse; the rest was stimulated with a train of 10 pulses. In 5 of these 8 cells (including the one cell stimulated with a single pulse), 5 μM mibefradil was applied to block T-type calcium channels. Figure 2B shows an example of one such experiment. In this cell the stimulation paradigm resulted in a pause of approximately 200 ms duration and caused clear rebound firing as evident by the robust high-frequency bursts seen in the raster plots (white triangles on the top). In some cases, again as seen with GABA photorelease, rebound firing was longer lasting than just a few initial high frequency bursts (note trials denoted by * in figure 2). Such long responses have been previously reported and may be mediated by a persistent sodium current (Llinas and Muhlethaler, 1988). As can be seen in the example shown in Figure 2B and in all 5 cells examined, application of 5 μM mibefradil effectively blocked rebound firing. Mibefradil also blocked the long rebound responses. This finding is not inconsistent with the notion that these long responses are mediated by a persistent sodium current because the T-type calcium channel mediated depolarization is likely to be required to activate the persistent sodium current.

To examine the time course of the effect of mibefradil on rebound firing, we measured the firing rate previous to the stimulus-evoked pause (pre-pause), and immediately after the pause (post-pause) in every trial for each cell examined (Figure 3A). The full effect of mibefradil on the post-pause firing rate took several minutes to complete, perhaps as a consequence of the tortuous diffusion path in the slices. The decline in the post-pause firing rate was clearly not cause by rundown because in the absence of mibefradil, the rebound firing was stable for as long as examined (≈15 minutes) (inset in Figure 3A).

The average firing rate prior to photorelease of GABA was 15.3±1.1 spikes per second and it increased to 194.0±34.7 during the bursts associated with rebound firing (Figures 2A and 3B). Application of mibefradil reduced the high-frequency bursts such that on average the post-pause firing rate was comparable to that prior to the GABA-induced pause (15.5±1.8 spikes per second before mibefradil vs. 13.7±2.9 spikes per second after, Figures 2A and 3B, p=0.62). Similarly, the average post-pause firing rate in the electrically-induced rebound firing was 179.0±42.5 spikes per second and after application of 5 μM mibefradil it decreased to 16.8±0.8 spikes per second (Figure 2B and 3B), which was comparable to the firing rate preceding the stimulus-induced pause (13.5±1.6 spikes per second, Figure 3B, p=0.15).

In agreement with prior observations (Alvina and Khodakhah, 2008) mibefradil did not alter the rate of spontaneous activity in DCN neurons. The average pre-pause firing rate for the cells tested with GABA photolysis was 15.3±1.1 spikes per second vs. 15.5±1.8 spikes per second pre-pause after adding mibefradil (Figure 2A and 3B, p=0.71). For the cells tested with electrical stimulation in turn, the average pre-pause spontaneous firing rate was 14.8±1.5 spikes per second before mibefradil vs. 13.5±1.6 spikes per second after (Figures 2B and 3B, p=0.58). This suggests that not only T-type calcium channels do not contribute to pacemaking in these cells, but also that at the concentrations used the potential non-specific actions of mibefradil on other channels required for pacemaking in DCN neurons (such as voltage gated sodium, potassium and N-type calcium channels) is insignificant.

Application of mibefradil also did not change either the duration of the GABA-induced pauses (442.6±68.7 ms before mibefradil vs. 468.7±73.0 ms after, Figures 2A and 3C, p=0.80), or the duration of the electrically-evoked pauses (200.5±29.9 ms in control vs. 219.9±31.1 ms in mibefradil, Figure 2B and 3C, p=0.66). The fact that the synaptically-evoked pauses were not affected by mibefradil again suggests that at the concentrations used mibefradil did not affect synaptic transmission and thus any of the ions channels critical for this process (i.e. voltage-gated sodium and potassium channels and P/Q- and perhaps N- and R-type calcium channels).

Rebound firing induced by direct hyperpolarizing current injection is abolished by T-type calcium channel blockers

Using whole-cell current clamp recordings, we also examined whether blocking T-type calcium channels abolished rebound firing produced by direct current injection in DCN neurons. Figure 4A shows an example of a cell that from a membrane potential of ≈ −60 mV was hyperpolarized to ≈ −85 mV for 500 ms. The hyperpolarization produced a strong rebound firing in the form of a high-frequency burst of 2-3 action potentials (Figure 4A). Application of 5 μM mibefradil completely inhibited rebound firing in 4 out 5 cells. In the 5th cell, 5 μM only reduced the firing rate and 10 μM mibefradil was needed to completely abolish it. On average, rebound firing consisted of 2.77 ± 0.35 spikes with a average firing rate of 112.9 ± 23.9 spikes per second (Figures 4D and E). The number of spikes composing the rebound response decreased to 1.07 ± 0.32 spikes in the presence of mibefradil (Figure 4E, p= 0.009). It was not possible to quantify the rebound firing rate in the presence of mibefradil because it was either absent or consisted of a single spike. It is noteworthy that the level of maximum hyperpolarization produced by the current injection was not affected by mibefradil (−80.6 ± 3.19 mV in control vs. −83.45 ± 3.67 mV in mibefradil, Figure 4C, p=0.56).

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

Figure 4

Open in a new tab

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.

We examined the efficacy of NNC 55-0396, a modified and much more specific mibefradil analogue (Huang et al., 2004; Li et al., 2005) in blocking rebound firing in DCN neurons. We performed the same experiments in whole-cell configuration, and induced rebound firing by injecting hyperpolarizing currents as described. An example of such experiment is shown in Figure 4B. The rebound high-frequency burst of action potentials induced under control conditions was completely abolished after application of 15 μM NNC 055-0396 (a very low concentration at which NNC 055-0396 is exquisitely selective for T-type calcium channels). On average, in control conditions the maximum firing rate after the hyperpolarizing pulse was 157.07 ± 35.70 spikes per second (n=4 cells), after adding NNC 55-0396 there was on average less than 1 spike and therefore the firing rate was considered to be zero (Figure 4D, p< 0.001 vs. control). Thus, NNC 55-0396 reduced the number of spikes from 2.68 ± 0.32 to 0.59 ± 0.06 (Figure 4E, p<0.001 vs. control), without altering the maximum level of hyperpolarization (−86.03 ± 3.29 mV in control vs. −89.84 ± 1.28 mV, Figure 4C p=0.33). In summary, these results support the idea that low-threshold T-type calcium channels mediate DCN neurons rebound firing.

Discussion

A stereotypic biophysical characteristic of DCN neurons is a pronounced rebound depolarization after a strong hyperpolarization (Jahnsen, 1986a; Llinas and Muhlethaler, 1988; Aizenman and Linden, 1999; Molineux et al., 2006). This rebound depolarization, which can result in rebound firing, depends on several factors such as the membrane potential prior to the hyperpolarizing pulse, and the amplitude and duration of the inhibitory stimulus (Jahnsen, 1986a; Aizenman and Linden, 1999). The voltage ranges required to induce rebound firing in DCN neurons are incompatible with high-threshold voltage gated calcium channels and low-threshold calcium channels were implicated early on (Jahnsen, 1986b; Llinas and Muhlethaler, 1988; Aizenman and Linden, 1999). Whole-cell recordings have shown that rebound firing is obtained at membrane potentials consistent with activation and inactivation of low-threshold T-type calcium channels (Aizenman and Linden, 1999). Moreover, the expression of different T-type channel isoforms has been correlated with particular rebound behaviors in different DCN neurons (Molineux et al., 2006). However, it has been reported that blockade of T-type calcium channels with mibefradil does not abolish rebound firing (Aizenman and Linden, 1999).

In an attempt to characterize the function of voltage-gated calcium channels in the regulation of rebound firing in DCN neurons and to find a tool to manipulate it, we revisited this issue under more physiological conditions. We monitored the activity of DCN neurons extracellularly and produced a prominent pause in their spontaneous firing by photoreleasing GABA onto their soma and dendrites. In a small fraction of DCN neurons (16 out of 97 cells) this procedure was effective in producing robust rebound firing. We found that in all cells examined mibefradil, a well established T-type channel blocker (McDonough and Bean, 1998; Martin et al., 2000), completely abolished the increase in the firing rate observed after photoreleasing GABA. We similarly found that mibefradil blocked rebound firing in all cells in which inhibitory synaptic inputs were used as the stimuli to induce rebound firing. Our finding that mibefradil blocks rebound firing is in contrast to an earlier report (Aizenman and Linden, 1999). The difference is likely to be the consequence of the fact that in the earlier study a lower concentration of mibefradil was used and that we found that a relatively long time (several minutes) was required for mibefradil to equilibrate with the slice. Supporting our findings, a more recent publication reported that mibefradil decreases the maximum rebound firing rate after a very strong and prolonged hyperpolarization (Molineux et al., 2008). While in this study mibefradil did not completely block rebound firing it is noteworthy that the hyperpolarizations used were of much larger amplitude and greater duration compared to ours and may have recruited additional conductances. Given that under physiological conditions it is unlikely that DCN neurons are hyperpolarized to such a great extent, we believe that blocking T-type calcium channels is an effective approach to block rebound firing.

An obvious concern would be non-specific actions of mibefradil on other calcium channels. In fact, mibefradil blocks some R-type calcium channels in addition to T-type calcium channels (Randall and Tsien, 1997; Viana et al., 1997; Jimenez et al., 2000). However, given that R-type calcium channels are high-threshold (Randall and Tsien, 1997; Tottene et al., 2000); our data suggests that low-threshold T -type calcium channels mediate rebound firing in DCN neurons. Moreover, the effect of the more specific T-type channel blocker NNC 55-0396 used at low concentration here (Huang et al., 2004), corroborates the fact the blocking T-type channels (as done so when using mibefradil) abolishes rebound firing. Depending on the subunit composition and expression of auxiliary subunits, mibefradil can also block other voltage-gated channels (Jimenez et al., 2000; Perchenet and Clement-Chomienne, 2000; Chouabe et al., 2000). However, our finding that at the concentrations used (mainly 5 μM and in some earlier experiments 10 μM) mibefradil neither affected spontaneous pacemaking or synaptic transmission suggests that it is extremely unlikely that its efficacy in blocking rebound firing is the consequence of its non-specific actions on other channels. It is important to note that despite the very remote possibility that mibefradil may be blocking rebound firing in DCN neurons by a non-specific action on other channels, given its lack of an effect on intrinsic conductances and synaptic transmission, mibefradil remains an effective tool for examining the physiological function of rebound firing in the cerebellum. Taking into account the effect of NNC 55-0396, it is clear than T-type channels modulators can be effectively used to study the potential role of rebound firing in the cerebellum.

A multitude of functional roles has been assigned to DCN rebound firing (Aizenman et al., 1998; Pugh and Raman, 2006; Wetmore et al., 2007). There is, however, some debate as to how prevalent rebound firing is under physiological conditions (Telgkamp and Raman, 2002; Aksenov et al., 2004; Rowland and Jaeger, 2005; Holdefer et al., 2005; Rowland and Jaeger, 2008; Alvina et al., 2008). After sensory stimulation, for example, the response of DCN neurons in vivo can consist of three components: a short-latency increase in spiking, an intermediate inhibition and a late increase in firing rate (Armstrong et al., 1975a; Armstrong et al., 1975b; Rowland and Jaeger, 2005). The late increase, which could perhaps be the consequence of rebound depolarization, was the least frequent response component in all nuclei (Rowland and Jaeger, 2005). Moreover, these late increases are more likely to be the consequence of direct excitation of DCN neurons by mossy and olivary fibers rather than rebound firing because they are often not preceded by a pause in activity (Rowland and Jaeger, 2008). It has also recently been shown that direct and strong activation of Purkinje cell inputs of a DCN neuron in vivo does not result in rebound firing (Alvina et al., 2008).

It is plausible, but remains to be demonstrated, that even if rebound firing is uncommon it may be physiologically relevant and may play an important functional role in cerebellar function, perhaps in specialized motor tasks. It is hoped that the establishment of mibefradil (and other T-type calcium channels blockers) as an effective blocker of this phenomenon will aid future studies aimed at elucidating the functional relevance of DCN rebound firing in cerebellar computation.

Supplementary Material

01

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Aizenman CD, Linden DJ. Regulation of the rebound depolarization and spontaneous firing patterns of deep nuclear neurons in slices of rat cerebellum. J Neurophysiol. 1999;82:1697–1709. doi: 10.1152/jn.1999.82.4.1697. [DOI] [PubMed] [Google Scholar]
  2. Aizenman CD, Manis PB, Linden DJ. Polarity of long-term synaptic gain change is related to postsynaptic spike firing at a cerebellar inhibitory synapse. Neuron. 1998;21:827–835. doi: 10.1016/s0896-6273(00)80598-x. [see comments] [DOI] [PubMed] [Google Scholar]
  3. Aksenov D, Serdyukova N, Irwin K, Bracha V. GABA neurotransmission in the cerebellar interposed nuclei: involvement in classically conditioned eyeblinks and neuronal activity. J Neurophysiol. 2004;91:719–727. doi: 10.1152/jn.00859.2003. [DOI] [PubMed] [Google Scholar]
  4. Alvina K, Khodakhah K. Selective regulation of spontaneous activity of neurons of the deep cerebellar nuclei by N-type calcium channels in juvenile rats. J Physiol. 2008;586:2523–2538. doi: 10.1113/jphysiol.2007.148197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Alvina K, Walter JT, Kohn A, Ellis-Davies GC, Khodakhah K. Questioning the role of rebound firing in the cerebellum. Nat Neurosci. 2008 doi: 10.1038/nn.2195. In press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Armstrong DM, Cogdell B, Harvey R. Effects of afferent volleys from the limbs on the discharge patterns of interpositus neurones in cats anaesthetized with alpha-chloralose. J Physiol. 1975a;248:489–517. doi: 10.1113/jphysiol.1975.sp010985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Armstrong DM, Cogdell B, Harvey RJ. Responses of interpositus neurones to stimulation of forelimb cutaneous afferents in chloralose-anaesthetized cats. J Physiol. 1975b;245:28P–29P. [PubMed] [Google Scholar]
  8. Chouabe C, Drici MD, Romey G, Barhanin J. Effects of calcium channel blockers on cloned cardiac K+ channels IKr and IKs. Therapie. 2000;55:195–202. [PubMed] [Google Scholar]
  9. Gauck V, Thomann M, Jaeger D, Borst A. Spatial distribution of low- and high-voltage-activated calcium currents in neurons of the deep cerebellar nuclei. J Neurosci. 2001;21:RC158. doi: 10.1523/JNEUROSCI.21-15-j0004.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Holdefer RN, Houk JC, Miller LE. Movement-related discharge in the cerebellar nuclei persists after local injections of GABA(A) antagonists. J Neurophysiol. 2005;93:35–43. doi: 10.1152/jn.00603.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huang L, Keyser BM, Tagmose TM, Hansen JB, Taylor JT, Zhuang H, Zhang M, Ragsdale DS, Li M. NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluo ro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]: a new selective inhibitor of T-type calcium channels. J Pharmacol Exp Ther. 2004;309:193–199. doi: 10.1124/jpet.103.060814. [DOI] [PubMed] [Google Scholar]
  12. Ito M. The cerebellum and neural control. Raven Press; New York: 1984. [Google Scholar]
  13. Jahnsen H. Electrophysiological characteristics of neurones in the guinea-pig deep cerebellar nuclei in vitro. J Physiol. 1986a;372:129–147. doi: 10.1113/jphysiol.1986.sp016001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jahnsen H. Extracellular activation and membrane conductances of neurones in the guinea-pig deep cerebellar nuclei in vitro. J Physiol. 1986b;372:149–168. doi: 10.1113/jphysiol.1986.sp016002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jimenez C, Bourinet E, Leuranguer V, Richard S, Snutch TP, Nargeot J. Determinants of voltage-dependent inactivation affect Mibefradil block of calcium channels. Neuropharmacology. 2000;39:1–10. doi: 10.1016/s0028-3908(99)00153-7. [DOI] [PubMed] [Google Scholar]
  16. Kistler WM, De Zeeuw CI. Time windows and reverberating loops: a reverse-engineering approach to cerebellar function. Cerebellum. 2003;2:44–54. doi: 10.1080/14734220309426. [DOI] [PubMed] [Google Scholar]
  17. Li M, Hansen JB, Huang L, Keyser BM, Taylor JT. Towards selective antagonists of T-type calcium channels: design, characterization and potential applications of NNC 55-0396. Cardiovasc Drug Rev. 2005;23:173–196. doi: 10.1111/j.1527-3466.2005.tb00164.x. [DOI] [PubMed] [Google Scholar]
  18. Llinas R, Muhlethaler M. Electrophysiology of guinea-pig cerebellar nuclear cells in the in vitro brain stem-cerebellar preparation. J Physiol. 1988;404:241–258. doi: 10.1113/jphysiol.1988.sp017288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martin RL, Lee JH, Cribbs LL, Perez-Reyes E, Hanck DA. Mibefradil block of cloned T-type calcium channels. J Pharmacol Exp Ther. 2000;295:302–308. [PubMed] [Google Scholar]
  20. McDonough SI, Bean BP. Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons. Mol Pharmacol. 1998;54:1080–1087. doi: 10.1124/mol.54.6.1080. [DOI] [PubMed] [Google Scholar]
  21. Medina JF, Nores WL, Ohyama T, Mauk MD. Mechanisms of cerebellar learning suggested by eyelid conditioning. Curr Opin Neurobiol. 2000;10:717–724. doi: 10.1016/s0959-4388(00)00154-9. [DOI] [PubMed] [Google Scholar]
  22. Molineux ML, McRory JE, McKay BE, Hamid J, Mehaffey WH, Rehak R, Snutch TP, Zamponi GW, Turner RW. Specific T-type calcium channel isoforms are associated with distinct burst phenotypes in deep cerebellar nuclear neurons. Proc Natl Acad Sci U S A. 2006;103:5555–5560. doi: 10.1073/pnas.0601261103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Molineux ML, Mehaffey WH, Tadayonnejad R, Anderson DM, Tennent AF, Turner RW. Ionic factors governing rebound burst phenotype in rat deep cerebellar neurons. J Neurophysiol. 2008 doi: 10.1152/jn.90427.2008. [DOI] [PubMed] [Google Scholar]
  24. Muri R, Knopfel T. Activity induced elevations of intracellular calcium concentration in neurons of the deep cerebellar nuclei. J Neurophysiol. 1994;71:420–428. doi: 10.1152/jn.1994.71.1.420. [DOI] [PubMed] [Google Scholar]
  25. Perchenet L, Clement-Chomienne O. Characterization of mibefradil block of the human heart delayed rectifier hKv1.5. J Pharmacol Exp Ther. 2000;295:771–778. [PubMed] [Google Scholar]
  26. Pugh JR, Raman IM. Potentiation of mossy fiber EPSCs in the cerebellar nuclei by NMDA receptor activation followed by postinhibitory rebound current. Neuron. 2006;51:113–123. doi: 10.1016/j.neuron.2006.05.021. [DOI] [PubMed] [Google Scholar]
  27. Randall AD, Tsien RW. Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels. Neuropharmacology. 1997;36:879–893. doi: 10.1016/s0028-3908(97)00086-5. [DOI] [PubMed] [Google Scholar]
  28. Rowland NC, Jaeger D. Coding of tactile response properties in the rat deep cerebellar nuclei. J Neurophysiol. 2005;94:1236–1251. doi: 10.1152/jn.00285.2005. [DOI] [PubMed] [Google Scholar]
  29. Rowland NC, Jaeger D. Responses to tactile stimulation in deep cerebellar nucleus neurons result from recurrent activation in multiple pathways. J Neurophysiol. 2008;99:704–717. doi: 10.1152/jn.01100.2007. [DOI] [PubMed] [Google Scholar]
  30. Stone TW. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev. 1993;45:309–379. [PubMed] [Google Scholar]
  31. Telgkamp P, Raman IM. Depression of inhibitory synaptic transmission between Purkinje cells and neurons of the cerebellar nuclei. J Neurosci. 2002;22:8447–8457. doi: 10.1523/JNEUROSCI.22-19-08447.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tottene A, Volsen S, Pietrobon D. alpha(1E) subunits form the pore of three cerebellar R-type calcium channels with different pharmacological and permeation properties. J Neurosci. 2000;20:171–178. doi: 10.1523/JNEUROSCI.20-01-00171.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Uusisaari M, Obata K, Knopfel T. Morphological and electrophysiological properties of GABAergic and non-GABAergic cells in the deep cerebellar nuclei. J Neurophysiol. 2007;97:901–911. doi: 10.1152/jn.00974.2006. [DOI] [PubMed] [Google Scholar]
  34. Viana F, Van Den BL, Missiaen L, Vandenberghe W, Droogmans G, Nilius B, Robberecht W. Mibefradil (Ro 40-5967) blocks multiple types of voltage-gated calcium channels in cultured rat spinal motoneurones. Cell Calcium. 1997;22:299–311. doi: 10.1016/s0143-4160(97)90068-3. [DOI] [PubMed] [Google Scholar]
  35. Volsen SG, Day NC, McCormack AL, Smith W, Craig PJ, Beattie R, Ince PG, Shaw PJ, Ellis SB, Gillespie A. The expression of neuronal voltage-dependent calcium channels in human cerebellum. Brain Res Mol Brain Res. 1995;34:271–282. doi: 10.1016/0169-328x(95)00234-j. [DOI] [PubMed] [Google Scholar]
  36. Wetmore DZ, Mukamel EA, Schnitzer MJ. Lock-and-key mechanisms of cerebellar memory recall based on rebound currents. J Neurophysiol. 2007 doi: 10.1152/jn.00344.2007. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS94644-supplement-01.pdf (1.4MB, pdf)

RESOURCES