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

In Purkinje cells from cerebellar slice cultures, low-threshold Ca 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 Ca channel blockers (SNX-482, nickel, or mibefradil). We propose that LTS is generated by openings of T-type Ca channels (alpha-1G and/or alpha-1I subunits) and R-type Ca channels (alpha-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 Ca entry may be involved in Ca-dependent synaptic plasticity of the parallel fiber input induced by climbing fiber activation.

Synaptic organization of the mouse cerebellar cortex in organotypic slice cultures.

The cellular and synaptic organization of new born mouse cerebellum maintained in organotypic slice cultures was investigated using immunohistochemical and patch-clamp recording approaches. The histological organization of the cultures shared many features with that observed in situ. Purkinje cells were generally arranged in a monolayer surrounded by a molecular-like neuropil made of Purkinje cell dendritic arborizations. Purkinje cell axons ran between clusters of small round cells identified as granule cells by Kv3.1b potassium channel immunolabelling. The terminal varicosities of the Purkinje cells axons enwrapped presumptive neurons of the cerebellar nuclei whereas their recurrent collaterals were in contact with Purkinje cells and other neurons. Granule cell axons established contacts with Purkinje cell somata and dendrites. Parvalbumin and glutamine acid decarboxylase (GAD) immunohistochemistry revealed the presence of presumptive interneurons throughout the culture. The endings of granule cell axons were observed to be in contact with these interneurons. Similarly, interneurons endings were seen close to Purkinje cells and granule cells. Whole cell recordings from Purkinje cell somata showed AMPA receptor-mediated spontaneous excitatory post-synaptic currents (sEPSCs) and GABAA receptor-mediated spontaneous inhibitory post-synaptic currents (sIPSCs). Similar events were recorded from granule cell somata except that in this neuronal type EPSPs have both a NMDA component and an AMPA component. In addition, pharmacological experiments demonstrated a GABAergic control of granule cell activity and a glutamatergic control of GABAergic neurons by granule cells. This study shows that a functional neuronal network is established in such organotypic cultures even in the absence of the two normal excitatory afferents, the mossy fibers and the climbing fibers.

The mouse cerebellar cortex in organotypic slice cultures: an in vitro model to analyze the consequences of mutations and pathologies on neuronal survival, development, and function.

Thin acute slices and dissociated cell cultures taken from different parts of the brain have been widely used to examine the function of the nervous system, neuron-specific interactions, and neuronal development (specifically, neurobiology, neuropharmacology, and neurotoxicology studies). Here, we focus on an alternative in vitro model: brain-slice cultures in roller tubes, initially introduced by Beat Gähwiler for studies with rats, that we have recently adapted for studies of mouse cerebellum. Cultured cerebellar slices afford many of the advantages of dissociated cultures of neurons and thin acute slices. Organotypic slice cultures were established from newborn or 10-15-day-old mice. After 3-4 weeks in culture, the slices flattened to form a cell monolayer. The main types of cerebellar neurons could be identified with immunostaining techniques, while their electrophysiological properties could be easily characterized with the patch-clamp recording technique. When slices were taken from newborn mice and cultured for 3 weeks, aspects of the cerebellar development were displayed. A functional neuronal network was established despite the absence of mossy and climbing fibers, which are the two excitatory afferent projections to the cerebellum. When slices were made from 10-15-day-old mice, which are at a developmental stage when cerebellum organization is almost established, the structure and neuronal pathways were intact after 3-4 weeks in culture. These unique characteristics make organotypic slice cultures of mouse cerebellar cortex a valuable model for analyzing the consequences of gene mutations that profoundly alter neuronal function and compromise postnatal survival.

K+ channel activation and low-threshold Ca2+ spike of rat cerebellar Purkinje cells in vitro.

Using the whole-cell configuration of the patch-clamp recording method, we analyzed the role of K+ conductances in determining the characteristics of the dendritically-initiated low-threshold Ca+ spike (LTS) recorded at the somatic level of rat cerebellar Purkinje cells (PCs) in slice cultures. Blockade of tetra-ethyl-ammonium-(TEA)- and 4-aminopyridine-(4-AP)-sensitive K+ channels increased the amplitude of the LTS. This effect was prominent with 4-AP, which promotes the fast-decaying component of the LTS. Surprisingly, a shortening of the LTS was induced by the blockade of K+ channel activity instead of a broadening of spikes as generally observed. We propose that, when propagating to the soma, TEA- and 4-AP-sensitive K+ channel activity affects the electrical properties of dendrites such that the LTS is attenuated and slowed down.

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.

Control of the propagation of dendritic low-threshold Ca(2+) spikes in Purkinje cells from rat cerebellar slice cultures.

To investigate the ionic mechanisms controlling the dendrosomatic propagation of low-threshold Ca(2+) 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 Ca(2+) 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 Ca(2+) channels increased the likelihood of recording Ca(2+) 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 x 10(-4) M) charybdotoxin (CTX, 10(-5) M), rIberiotoxin (10(-7) M), and 4-aminopyridine (4-AP, 10(-3) M), but not by apamin (10(-4) M). 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 Ca(2+) 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.