Pauline Cavelier

Control of the propagation of dendritic low-threshold Ca2+ spikes in Purkinje cells from rat cerebellar slice cultures

To investigate the ionic mechanisms controlling the dendrosomatic propagation of low‐threshold Ca2+ spikes (LTS) in Purkinje cells (PCs), somatically evoked discharges of action potentials (APs) were recorded under current‐clamp conditions. The whole‐cell configuration of the patch‐clamp method was used in PCs from rat cerebellar slice cultures. Full blockade of the P/Q‐type Ca2+ current revealed slow but transient depolarizations associated with bursts of fast Na+ APs. These can occur as a single isolated event at the onset of current injection, or repetitively (i.e. a slow complex burst). The initial transient depolarization was identified as an LTS Blockade of P/Q‐type Ca2+ channels increased the likelihood of recording Ca2+ spikes at the soma by promoting dendrosomatic propagation. Slow rhythmic depolarizations shared several properties with the LTS (kinetics, activation/inactivation, calcium dependency and dendritic origin), suggesting that they correspond to repetitively activated dendritic LTS, which reach the soma when P/Q channels are blocked. Somatic LTS and slow complex burst activity were also induced by K+ channel blockers such as TEA (2.5 × 10−4m) charybdotoxin (CTX, 10−5m), rIberiotoxin (10−7m), and 4‐aminopyridine (4‐AP, 10−3m), but not by apamin (10−4m). In the presence of 4‐AP, slow complex burst activity occurred even at hyperpolarized potentials (−80 mV). In conclusion, we suggest that the propagation of dendritic LTS is controlled directly by 4‐AP‐sensitive K+ channels, and indirectly modulated by activation of calcium‐activated K+ (BK) channels via P/Q‐mediated Ca2+ entry. The slow complex burst resembles strikingly the complex spike elicited by climbing fibre stimulation, and we therefore propose, as a hypothesis, that dendrosomatic propagation of the LTS could underlie the complex spike.

Dendro‐somatic distribution of calcium‐mediated electrogenesis in Purkinje cells from rat cerebellar slice cultures

1 The role of Ca2+ entry in determining the electrical properties of cerebellar Purkinje cell (PC) dendrites and somata was investigated in cerebellar slice cultures. Immunohistofluorescence demonstrated the presence of at least three distinct types of Ca2+ channel proteins in PCs: the α1A subunit (P/Q type Ca2+ channel), the α1G subunit (T type) and the α1E subunit (R type). 2 In PC dendrites, the response started in 66 % of cases with a slow depolarization (50 ± 15 ms) triggering one or two fast (∼1 ms) action potentials (APs). The slow depolarization was identified as a low‐threshold non‐P/Q Ca2+ AP initiated, most probably, in the dendrites. In 16 % of cases, this response propagated to the soma to elicit an initial burst of fast APs. 3 Somatic recordings revealed three modes of discharge. In mode 1, PCs display a single or a short burst of fast APs. In contrast, PCs fire repetitively in mode 2 and 3, with a sustained discharge of APs in mode 2, and bursts of APs in mode 3. Removal of external Ca2+ or bath applications of a membrane‐permeable Ca2+ chelator abolished repetitive firing. 4 Tetraethylammonium (TEA) prolonged dendritic and somatic fast APs by a depolarizing plateau sensitive to Cd2+ and to ω‐conotoxin MVII C or ω‐agatoxin TK. Therefore, the role of Ca2+ channels in determining somatic PC firing has been investigated. Cd2+ or P/Q type Ca2+ channel‐specific toxins reduced the duration of the discharge and occasionallyinduced the appearance of oscillations in the membrane potential associated with bursts of APs. 5 In summary, we demonstrate that Ca2+ entry through low‐voltage gated Ca2+ channels, not yet identified, underlies a dendritic AP rarelyeliciting a somatic burst of APs whereas Ca2+ entry through P/Q type Ca2+ channels allowed a repetitive firing mainly by inducing a Ca2+‐dependent hyperpolarization.

Dendritic low-threshold Ca2+ channels in rat cerebellar Purkinje cells: Possible physiological implications

Low-voltage activated (LVA) Ca2+ currents have been characterized in a large variety of neurons including cerebellar Purkinje cells (PCs). This review summarizes and discusses the biophysical, pharmacological properties, as well as the molecular identity of LVA Ca2+ channels described in PCs in various experimental conditions. Putative functional roles for LVA Ca2+ currents include generation of low-threshold Ca2+ spikes (LTS) that underlie burst firing, promotion of intrinsic oscillatory behaviour, Ca2+ entry close to the resting membrane potential and synaptic potentiation. Based on our recent findings on cerebellar rat PCs in slice cultures, this review presents the major evidence demonstrating that LVA Ca2+ channels produce a dendritic initiated LTS with a regulated propagation to the soma. This new role for LVA Ca2+ channels is particularly important in determining firing patterns in PCs.

Participation of low-threshold Ca2+ spike in the Purkinje cells complex spike

In Purkinje cells from cerebellar slice cultures, low-threshold Ca2+ spike (LTS) gives rise to complex bursts in the soma that resemble the complex spike induced by climbing fibers stimulation. We show that LTS is reduced by T-type and R-type Ca2+ channel blockers (SNX-482, nickel, or mibefradil). We propose that LTS is generated by openings of T-type Ca2+ channels (&agr;-1G and/or &agr;-1I subunits) and R-type Ca2+ channels (&agr;-1E subunit isoforms with a weak sensitivity to SNX-482 and to nickel). Using mibefradil we show that climbing fiber stimulation activates LTS, which contributes to the shape of the response. This Ca2+ entry may be involved in Ca2+-dependent synaptic plasticity of the parallel fiber input induced by climbing fiber activation.

Death and survival of heterozygous Lurcher Purkinje cells In vitro

The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the δ2 glutamate receptor (GluRδ2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRδ2+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRδ2+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRδ2+/+ and GluRδ2+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRδ2+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRδ2+/Lc Purkinje cell dendrites were stunted and the number of surviving GluRδ2+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1‐naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRδ2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRδ2+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival.

Cerebellar slice cultures from mice lacking the P/Q calcium channel: electroresponsiveness of Purkinje cells

To investigate the role of P/Q type Ca(2+) channels in determining the firing pattern of Purkinje cells (PCs) we compared the somatically evoked discharge of action potentials (APs) in PCs from 3 to 4 week old cerebellar slice cultures obtained with ataxic mice lacking alpha(1A)-subunit (alpha(-/-)) and with normal mice (non-ataxic alpha(+/-) or alpha(+/+)) using the whole-cell configuration of the patch-clamp recording method. Whereas evoked responses of PCs in normal mice were mainly fast APs, those of PCs from ataxic mice were mainly low-threshold Ca(2+) spikes (LTS). Furthermore, a sustained plateau potential due to the activation of cadmium sensitive Ca(2+) conductances was not observed in PCs from ataxic mice by blocking K(+) channels. These results confirm that P/Q Ca(2+) channels elicit Ca(2+)-dependent plateau potentials and control the propagation of the dendritic LTS to the soma.