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Key Points: Mouse chromaffin cells in acute adrenal slices exhibit two distinct spiking patterns, a repetitive mode and a bursting mode. A sodium background conductance operates at rest as demonstrated by the membrane hyperpolarization evoked by a low Na + -containing extracellular saline. This sodium background current is insensitive to TTX, is not blocked by Cs + ions and displays a linear I-V relationship at potentials close to chromaffin cell resting potential. Its properties are reminiscent of those of the sodium leak channel NALCN. In the adrenal gland, Nalcn mRNA is selectively expressed in chromaffin cells. The study fosters our understanding of how the spiking pattern of chromaffin cells is regulated and adds a sodium background conductance to the list of players involved in the stimulus-secretion coupling of the adrenomedullary tissue., Abstract: Chromaffin cells (CCs) are the master neuroendocrine units for the secretory function of the adrenal medulla and a finely-tuned regulation of their electrical activity is required for appropriate catecholamine secretion in response to the organismal demand. Here, we aim at deciphering how the spiking pattern of mouse CCs is regulated by the ion conductances operating near the resting membrane potential (RMP). At RMP, mouse CCs display a composite firing pattern, alternating between active periods composed of action potentials spiking with a regular or a bursting mode, and silent periods. RMP is sensitive to changes in extracellular sodium concentration, and a low Na + -containing saline hyperpolarizes the membrane, regardless of the discharge pattern. This RMP drive reflects the contribution of a depolarizing conductance, which is (i) not blocked by tetrodotoxin or caesium, (ii) displays a linear I-V relationship between -110 and -40 mV, and (iii) is carried by cations with a conductance sequence g Na  > g K  > g Cs . These biophysical attributes, together with the expression of the sodium-leak channel Nalcn transcript in CCs, state credible the contribution of NALCN. This inaugural report opens new research routes in the field of CC stimulus-secretion coupling, and extends the inventory of tissues in which NALCN is expressed to neuroendocrine glands., (© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Autistic subjects frequently display sensory anomalies. Those regarding nociception and its potential outcome, pain, are of crucial interest. Indeed, because of numerous comorbidities, autistic subjects are more often exposed to painful situation. Despite being often considered as less sensitive, experimental studies evaluating this point are failing to reach consensus. Using animal model can help reduce variability and bring, regarding autism, an overview of potential alterations of the nociceptive system at the cellular and molecular level., (© 2021 médecine/sciences – Inserm.)

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, causes serious neurological disorders in animals. ETX can bind to the white matter of the brain and the oligodendrocytes, which are the cells forming the myelin sheath around neuron axons in the white matter of the central nervous system. After binding to oligodendrocytes, ETX causes demyelination in rat cerebellar slices. We further investigated the effects of ETX on cerebellar oligodendrocytes and found that ETX induced small transmembrane depolarization (by ~ +6.4 mV) in rat oligodendrocytes primary cultures. This was due to partial inhibition of the transmembrane inward rectifier potassium current (Kir). Of the two distinct types of Kir channel conductances (~25 pS and ~8.5 pS) recorded in rat oligodendrocytes, we found that ETX inhibited the large-conductance one. This inhibition did not require direct binding of ETX to a Kir channel. Most likely, the binding of ETX to its membrane receptor activates intracellular pathways that block the large conductance Kir channel activity in oligodendrocyte. Altogether, these findings and previous observations pinpoint oligodendrocytes as a major target for ETX. This supports the proposal that ETX might be a cause for Multiple Sclerosis, a disease characterized by myelin damage., Competing Interests: The authors declare no conflict of interest.

Autism is a neuro-developmental pathology affecting 1 out of 100 children worldwide. The trauma and social consequences induced by autism are a real public health issue. Clinically, autism is characterized primarily by communications and social interactions deficits associated with repetitive behaviors and restricted interests. The term of autism spectrum disorders (ASD) is used to account for the diversity of symptoms that characterize this pathology. Based on observations made in humans, a rodent (rats and mice) model of autism was obtained and validated by prenatal exposure to sodium valproate. Using this model, mechanisms that concern both the functioning of neural networks and the properties of neurons have been proposed to account for some disorders that characterize autism. This model is also widely used in pre-clinical studies to evaluate new therapies against ASD., (© 2019 médecine/sciences – Inserm.)

Neuropathological and neuroimaging studies indicate a decrease in Purkinje cell (PC) density in the cerebellum of autistic patients and rodent models of autism. Autism is far more prevalent in males than females, and sex-specific properties of PCs have been reported recently. We investigated the differential sensitivity of PCs in the valproate acid (VPA) mouse model of autism by estimating the linear density of PCs immununolabelled with calbindin in the cerebellum of males and females. Whereas prenatal VPA treatment surprisingly increased PC linear density in both sexes 13 days after birth (P13), it significantly reduced the linear density of PCs in the cerebellum of 40-day-old (P40) males, but not females. In males, PC loss was more pronounced in the posterior part of the cerebellum and was significant in the VIth, VIIth, IXth and paramedian lobules. In females, PC loss was restricted to the paramedian lobule. These results suggest that this sex-specific sensitivity of PCs to VPA may contribute towards the motor disturbances and behavioural abnormalities observed in autism.

Brain development is accompanied by a shift in gamma-aminobutyric acid (GABA) response from depolarizing-excitatory to hyperpolarizing-inhibitory, due to a reduction of intracellular chloride concentration. This sequence is delayed in Autism Spectrum Disorders (ASD). We now report a similar alteration of this shift in the cerebellum, a structure implicated in ASD. Using single GABA A receptor channel recordings in cerebellar Purkinje cells (PCs), we found two conductance levels (18 and 10 pS), the former being dominant in newborns and the latter in young-adults. This conductance shift and the depolarizing/excitatory to hyperpolarizing/inhibitory GABA shift occurred 4 days later in females than males. Our data support a sex-dependent developmental shift of GABA conductance and chloride gradient, leading to different developmental timing in males and females. Because these developmental sequences are altered in ASD, this study further stresses the importance of developmental timing in pathological neurodevelopment.

Introduction: Demyelinating disorders, characterized by a chronic or episodic destruction of the myelin sheath, are a leading cause of neurological disability in young adults in western countries. Studying the complex mechanisms involved in axon myelination, demyelination and remyelination requires an experimental model preserving the neuronal networks and neuro-glial interactions. Organotypic cerebellar slice cultures appear to be the best alternative to in vivo experiments and the most commonly used model for investigating etiology or novel therapeutic strategies in multiple sclerosis. Areas covered: This review gives an overview of slice culture techniques and focuses on the use of organotypic cerebellar slice cultures on semi-permeable membranes for studying many aspects of axon myelination and cerebellar functions. Expert opinion: Cerebellar slice cultures are probably the easiest way to faithfully reproduce all stages of axon myelination/demyelination/remyelination in a three-dimensional neuronal network. However, in the cerebellum, neurological disability in multiple sclerosis also results from channelopathies which induce changes in Purkinje cell excitability. Cerebellar cultures offer easy access to electrophysiological approaches which are largely untapped and we believe that these cultures might be of great interest when studying changes in neuronal excitability, axonal conduction or synaptic properties that likely occur during multiple sclerosis.

A growing number of receptors, often associated with the innate immune response, are being identified as targets for bacterial toxins of the beta-stranded pore-forming family. These findings raise the new question of whether the receptors are activated or merely used as docking points facilitating the formation of a pore. To elucidate whether the Staphylococcus aureus Panton-Valentine leukocidin and the leukotoxin HlgC/HlgB act through the C5a receptor (C5aR) as agonists, antagonists or differ from the C5a complement-derived peptide, their activity is explored on C5aR-expressing cells. Both leukotoxins equally bound C5aR in neutrophils and in stable transfected U937 cells and initiated mobilization of intracellular Ca(2+) . HlgC/HlgB requires the presence of robust intracellular acidic Ca(2+) stores in order to evoke a rise in free [Ca(2+) ]i , while the LukS-PV/LukF-PV directly altered reticular Ca(2+) stores. Intracellular target specificity is conferred by the F-subunit associated to the S-subunit binding the receptor. Furthermore, internalization of the two leukotoxin components (S- and F-subunits) associated to C5aR is required for the initiation of [Ca(2+) ]i mobilization. Electrophysiological recordings on living cells demonstrated that LukS-PV/LukF-PV does not alter the membrane resistance of C5aR-expressing cells. The present observations suggest that part of the pore-forming process occurs in distinct intracellular compartments rather than at the plasma membrane., (© 2015 John Wiley & Sons Ltd.)

Epsilon toxin (ET) is produced by Clostridium perfringens types B and D and causes severe neurological disorders in animals. ET has been observed binding to white matter, suggesting that it may target oligodendrocytes. In primary cultures containing oligodendrocytes and astrocytes, we found that ET (10(-9) M and 10(-7) M) binds to oligodendrocytes, but not to astrocytes. ET induces an increase in extracellular glutamate, and produces oscillations of intracellular Ca(2+) concentration in oligodendrocytes. These effects occurred without any change in the transmembrane resistance of oligodendrocytes, underlining that ET acts through a pore-independent mechanism. Pharmacological investigations revealed that the Ca(2+) oscillations are caused by the ET-induced rise in extracellular glutamate concentration. Indeed, the blockade of metabotropic glutamate receptors type 1 (mGluR1) prevented ET-induced Ca(2+) signals. Activation of the N-methyl-D-aspartate receptor (NMDA-R) is also involved, but to a lesser extent. Oligodendrocytes are responsible for myelinating neuronal axons. Using organotypic cultures of cerebellar slices, we found that ET induced the demyelination of Purkinje cell axons within 24 h. As this effect was suppressed by antagonizing mGluR1 and NMDA-R, demyelination is therefore caused by the initial ET-induced rise in extracellular glutamate concentration. This study reveals the novel possibility that ET can act on oligodendrocytes, thereby causing demyelination. Moreover, it suggests that for certain cell types such as oligodendrocytes, ET can act without forming pores, namely through the activation of an undefined receptor-mediated pathway., (© 2014 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Epsilon toxin (ET), produced by Clostridium perfringens types B and D, ranks among the four most potent poisonous substances known so far. ET-intoxication is responsible for enterotoxaemia in animals, mainly sheep and goats. This disease comprises several manifestations indicating the attack of the nervous system. This review aims to summarize the effects of ET on central nervous system. ET binds to endothelial cells of brain capillary vessels before passing through the blood-brain barrier. Therefore, it induces perivascular oedema and accumulates into brain. ET binding to different brain structures and to different component in the brain indicates regional susceptibility to the toxin. Histological examination has revealed nerve tissue and cellular lesions, which may be directly or indirectly caused by ET. The naturally occurring disease caused by ET-intoxication can be reproduced experimentally in rodents. In mice and rats, ET recognizes receptor at the surface of different neural cell types, including certain neurons (e.g. the granule cells in cerebellum) as well as oligodendrocytes, which are the glial cells responsible for the axons myelination. Moreover, ET induces release of glutamate and other transmitters, leading to firing of neural network. The precise mode of action of ET on neural cells remains to be determined., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

N-methyl-D-aspartate (NMDA) receptors are associated with many forms of synaptic plasticity. Their expression level and subunit composition undergo developmental changes in several brain regions. In the mouse cerebellum, beside a developmental switch between NR2B and NR2A/C subunits in granule cells, functional postsynaptic NMDA receptors are seen in Purkinje cells of neonate and adult but not juvenile rat and mice. A presynaptic effect of NMDA on GABA release by cerebellar interneurons was identified recently. Nevertheless whereas NMDA receptor subunits are detected on parallel fiber terminals, a presynaptic effect of NMDA on spontaneous release of glutamate has not been demonstrated. Using mouse cerebellar cultures and patch-clamp recordings we show that NMDA facilitates glutamate release onto Purkinje cells in young cultures via a presynaptic mechanism, whereas NMDA activates extrasynaptic receptors in Purkinje cells recorded in old cultures. The presynaptic effect of NMDA on glutamate release is also observed in Purkinje cells recorded in acute slices prepared from juvenile but not from adult mice and requires a specific protocol of NMDA application.

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca(2+) rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.

Deletion of both alleles of the P/Q-type Ca(2+)-channel Ca(v)2.1(alpha(1A)) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q-type Ca(2+)-channels, and compared them with the effect of pharmacological inhibition of the P/Q- or N-type channels on wild-type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q-type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca(v)2.1(alpha(1A)) partially compensated. At the granule cell (GC) to PC synapses, both N- and P/Q-type Ca(2+)-channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca(v)2.1(alpha(1A)) deletion. We also found that the P/Q- but not N-type Ca(2+)-channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic gamma-aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q-type Ca(2+)-channels. Overall, our data demonstrate that both P/Q- and N-type Ca(2+)-channels play a role in glutamate release, while the P/Q-type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca(v)2.1(alpha(1A)) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q-type channels.

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.

Clostridium perfringens epsilon toxin (ET) is a potent pore-forming cytotoxin causing fatal enterotoxemia in livestock. ET accumulates in brain and kidney, particularly in the renal distal-collecting ducts. ET binds and oligomerizes in detergent-resistant membranes (DRMs) microdomains and causes cell death. However, the causal linkage between membrane permeabilization and cell death is not clear. Here, we show that ET binds and forms 220-kDa insoluble complexes in plasma membrane DRMs of renal mpkCCD(cl4) collecting duct cells. Phosphatidylinositol-specific phospholipase C did not impair binding or the formation of ET complexes, suggesting that the receptor for ET is not GPI anchored. ET induced a dose-dependent fall in the transepithelial resistance and potential in confluent cells grown on filters, transiently stimulated Na+ absorption, and induced an inward ionic current and a sustained rise in [Ca2+]i. ET also induced rapid depletion of cellular ATP, and stimulated the AMP-activated protein kinase, a metabolic-sensing Ser/Thr kinase. ET also induced mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of apoptosis-inducing factor, a potent caspase-independent cell death effector. Finally, ET induced cell necrosis characterized by a marked reduction in nucleus size without DNA fragmentation. DRM disruption by methyl-beta-cyclodextrin impaired ET oligomerization, and significantly reduced the influx of Na+ and [Ca2+]i, but did not impair ATP depletion and cell death caused by the toxin. These findings indicate that ET causes rapid necrosis of renal collecting duct cells and establish that ATP depletion-mediated cell death is not strictly correlated with the plasma membrane permeabilization and ion diffusion caused by the toxin.

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.

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.

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.

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.

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., (Copyright 2002 Elsevier Science Ireland Ltd.)

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.

Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis. Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin.

The Mandelbrot's fractal dimension (D), a measure of shape complexity, has been used to quantify the complex morphology of living cells. Previous studies on glial cells have shown that as cells increase in morphological complexity, their "D" value increases, suggesting that "D" could be used to estimate their stage of differentiation. In the present study the box-counting method was used to calculate the "D" values of rat cerebellar oligodendrocytes during their differentiation in primary culture. These values were correlated with the immunoreactivity of cells to antigenic markers commonly used for assessing their stages of differentiation: A2B5, O4 and anti-galactocerebroside (Gal-C). Our results show that changes of the fractal dimension during differentiation follow the well known pattern of markers expression by these cells. These results demonstrate that A2B5-, O4-, and Gal-C-expressing oligodendrocytes can be confidently estimated from their respective fractal dimension values. Based on this immunocytochemical calibration, the calculation of "D" allows an easy and fast determination of the developmental stage of living (unstained) oligodendrocytes before the study of their physiological characteristics. Using this method we precisely identified living oligodendrocyte progenitors and early pro-oligodendrocytes expressing voltage-activated sodium currents that is a common characteristic of these two immature developmental stages (Sontheimer et al. [1989b] Neuron 2:1135-1145)., (Copyright 2001 Wiley-Liss, Inc.)

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 alpha1A subunit (P/Q type Ca2+ channel), the alpha1G subunit (T type) and the alpha1E subunit (R type). In PC dendrites, the response started in 66 % of cases with a slow depolarization (50 +/- 15 ms) triggering one or two fast (approximately 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. 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. Tetraethylammonium (TEA) prolonged dendritic and somatic fast APs by a depolarizing plateau sensitive to Cd2+ and to omega-conotoxin MVII C or omega-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. 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.

1. Desensitization of a metabotropic response was investigated in CA3 pyramidal neurons in hippocampal slice cultures using the patch-clamp technique. 2. 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD), an agonist at metabotropic glutamate receptors (mGluRs), and metacholine (MCh), an agonist at muscarinic receptors, induced a cationic current that appears to be activated through a G-protein-independent transduction process, as previously shown. Prolonged or repetitive bath application of agonists led to rapid desensitization of the cationic current with a time constant of approximately 20 s. 3. Complete recovery from desensitization was observed within 6 min. 4. These responses mediated by mGluRs and muscarinic receptors cross-desensitized. 5. Preventing the activation of G-proteins by loading cells with GDP beta S strongly reduced or suppressed desensitization, and resulted in a sustained inward cationic current. When cells were filled with GTP gamma S to irreversibly activate G-proteins, the desensitization process was enhanced such that a first application of agonist caused a markedly reduced response. 6. These results show that a cationic current induced by metabotropic agonists in hippocampal pyramidal cells undergoes apparent desensitization and suggests that this process occurs through a G-protein-mediated inhibition of the underlying membrane conductance.

Voltage-dependent Ca2+ conductances were investigated in Purkinje cells in rat cerebellar slice cultures using the whole-cell and cell-attached configurations of the patch-clamp technique. In the presence of 0.5 mM Ca2+ in the extracellular solution, the inward current activated with a threshold of -55 +/- 1.5 mV and reached a maximal amplitude of 2.3 +/- 0.4 nA at -31 +/- 2 mV. Decay kinetics revealed three distinct components: a fast (24.6 +/- 2 msec time constant), a slow (304 +/- 46 msec time constant), and a nondecaying component. Rundown of the slow and sustained components of the current, or application of antagonists for the P/Q-type Ca2+ channels, allowed isolation of the fast-inactivating Ca2+ current, which had a threshold for activation of -60 mV and reached a maximal amplitude of 0.7 nA at a membrane potential of -33 mV. Both activation and steady-state inactivation of this fast-inactivating Ca2+ current were described with Boltzmann equations, with half-activation and inactivation at -51 mV and -86 mV, respectively. This Ca2+ current was nifedipine-insensitive, but its amplitude was reduced reversibly by bath-application of NiCl2 and amiloride, thus allowing its identification as a T-type Ca2+ current. Channels with a conductance of 7 pS giving rise to a fast T-type ensemble current (insensitive to omega-Aga-IVA) were localized with a high density on the dendritic membrane. Channel activity responsible for the ensemble current sensitive to omega-Aga-IVA was detected with 10 mM Ba2+ as the charge carrier. These channels were distributed with a high density on dendritic membranes and in rare cases were also seen in somatic membrane patches.

1. The dominant voltage-gated K+ currents in the somatic membrane of CA3 pyramidal cells from hippocampal slice cultures were characterized using the cell-attached configuration of the patch-clamp recording method. The kinetics, the voltage dependence of activation and inactivation, and the pharmacological properties of the current were determined from ensemble averages of large numbers of episodes from multichannel patches. 2. Steady-state analysis revealed that this current was half-inactivated at the resting membrane potential (Vr), and fully inactivated when patches were held 40 mV positive to Vr. Inactivation was removed when patches were hyperpolarized by 50 mV from Vr. Inactivation was well described by the Boltzmann equation with a slope factor of 12.6 mV. Removal of inactivation of the peak outward current could be described by a time-dependent monoexponential function with a time constant of the order of 100 ms. In contrast, the time course of inactivation was very slow: a +40 mV depolarization relative to Vr of several seconds was required for complete inactivation of the total outward current. 3. When steady-state inactivation was removed by hyperpolarization, the outward current activated with a threshold 10 mV positive to Vr and was half-activated at a potential 57 mV positive to Vr. The conductance can be described in terms of a single Boltzmann equation with a slope factor of 13.5 mV. Activation and inactivation properties of the somatic conductance produce a small window current between +10 and +20 mV relative to Vr. 4. The outward current activated in a voltage-dependent manner in less than 10 ms with 500 ms depolarizing steps. A kinetic analysis of its decay revealed at least three components, with the following time constants: a fast (17 ms), a slowly (approximately 150 ms), and a very slowly inactivating component (in the range of seconds). 5. External application of 4-aminopyridine (4-AP) induced a dose-dependent block of the peak outward current with an IC50 of 28 microM. The inhibitory effect of 4-AP saturated at a concentration of 200 microM which blocked 80% of the total current. The slowly and very slowly inactivating components of the current were not observed with 20 mM tetraethylammonium (TEA) in the pipette solution. A fast transient ensemble current (mean decay time constant, 24 ms) persisted in the presence of extracellular TEA in 29% of the patches. 6. In summary, at least two distinct voltage-gated K+ currents were present at the somatic level of hippocampal pyramidal cells. The dominant one, which we named IK(AT), is sensitive to micromolar concentrations of 4-AP and millimolar concentrations of TEA, and contributes three kinetic components to the total outward current. The second is TEA insensitive, and contributes only a fast transient component of outward current probably corresponding to the classic A-type K+ current. Intracellular recordings in CA3 pyramidal cells showed that IK(AT) plays an important role in regulating the duration of the action potential.

1. We have used the cell-attached configuration of the patch-clamp recording method to characterize the biophysical properties of the voltage-gated K+ channel underlying a 4-aminopyridine (4-AP)- and tetraethylammonium (TEA)-sensitive K+ current (IK(AT)) in pyramidal cells of hippocampal slice cultures. 2. The unitary conductance of channels carrying IK(AT) current (KAT channels) was 19.1 +/- 5.1 pS with a physiological K+ gradient (2.7 mM external K+) and 39.0 +/- 3.6 pS with high external K+ (140 mM). The reversal potential changed with the external K+ concentration as expected for a channel with a dominant K+ selectivity. Channel activity was blocked under both conditions by either external application of 4-AP at 100 microM or by including 20 mM TEA in the pipette solution. 3. An analysis of kinetic behaviour showed that open times were distributed as a single exponential. The mean open time (+/- S.D.) was 4.4 +/- 1.4 ms at a voltage 30 mV positive to resting potential and increased with further depolarization to reach a value of 16.2 +/- 7.4 ms at 70 mV positive to the resting potential. At this depolarized potential, we observed bursts of channel openings with a mean burst duration around 100 ms. 4. With repeated depolarizing pulses, response failures of the KAT channel occurred in a non-random manner and were grouped (referred to as mode 0). This mode was associated with a voltage-dependent inactivation process of the channel and was favoured when the opening probability of the channel was reduced by increasing steady-state inactivation or by bath application of 4-AP. This is consistent with the localization of the binding site for 4-AP at or near the inactivation gate of the channel. 5. When KAT channel openings were elicited by 500 ms depolarizing steps, activity was either transient or it persisted throughout the duration of the pulse. These two modes of activity alternated in a random manner or occurred in groups giving rise to transient (time constant, 20-100 ms) or sustained ensemble currents. In the presence of low concentrations of 4-AP (20-40 microM), the transient pattern of activity was more frequently observed. 6. In addition to mode 0, we propose the existence of at least two further gating modes for KAT channels: mode T (transient current) and mode S (sustained current) that underlie the three decaying components of the IK(AT) ensemble current. These gating modes are probably under the control of intracellular factors that remain to be identified.

The electrical properties of the precursor cells of the external germinal layer of rat cerebellum were assessed during their differentiation in control medium (Dulbecco's modified Eagle's medium) supplemented or not with either basic fibroblast growth factor (bFGF) or 25 mM potassium chloride (KCl). Resting potential was shown to be -10 mV in all three conditions 3 hours after plating [days in vitro (DIV)0]. By DIV 5, it reached -63 mV for cells cultured in 25 mM KCl but only -28 mV in control and bFGF media. The main voltage-sensitive ionic current measured at DIV 0 under all conditions was a composite IK consisting in a sustained K+ current blocked by tetraethylammonium (IK(TEA)), plus a rapidly activating and inactivating TEA-insensitive IK(A). Both currents increased with time in all conditions, but after 5 days IK(A) became dominant in terms of density. IK(TEA) is likely an IK(Ca), since it was blocked by 67% in 1 mM TEA. On DIV 0, INa and ICa were absent or small in amplitude. By DIV 3, 80% of the cells had currents able to generate a spike. Interestingly, ICa mean amplitude and current density measured at -10 mV in control condition on DIV 1 was significantly larger than those recorded in bFGF and 25 mM KCl. The order of appearance of the ionic currents, IK, ICa, and INa, leads directly to fast spike activity allowing for poor calcium entry. Firing rate likely depends on IK(A), which increased during the first 6 days of development but could be differentially regulated by bFGF.

In isolated chromaffin cells, the high-voltage-activated Ca2+ current, recorded using 5 mM Ca2+ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5'-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5'-triphosphate (ITP) could not replace ATP, whereas guanosine 5'-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl2 and the liberation of a phosphate group since the ATP analogue 5'-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5'-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg2+ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5'-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca2+ concentration (pCa 6 instead of pCa 7.7, where pCa = -log10[Ca2+]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca2+ channels when going to the inside-out patch recording configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

The Ca2+ current recorded by the whole-cell technique in chromaffin cells shows, before the often described rundown, a transient facilitation or runup. Initial current amplitude was 570 +/- 165 pA and then it increased by 49 +/- 23% (n = 19, SD) over 2 +/- 1 min in the absence of adenosine 5'-triphosphate (ATP). In the presence of ATP, this process occurred with the same magnitude but it was slowed in a dose-dependent manner, lasting 17 +/- 2 min with 2 mM ATP (n = 8). Since adenosine 5'-diphosphate (ADP) does not reproduce this ATP effect, a complex series of phosphorylations is likely to intervene and we show that, at least, a cAMP-dependent i.e., cyclic adenosine monophosphate) phosphorylation occurs. Pertussis toxin (PTX) pretreatment yielded an already maximal Ca2+ current (around 1000 pA) at the time of the patch rupture, which only slightly increased thereafter (10%, n = 11). Also, guanosine 5'-diphosphate (GDP) and guanosine 5'-O-(2-thiodiphosphate) (GDP[ beta s]), induced a fast runup, which was absent in the presence of GTP. Furthermore, we show that facilitation does not occur in the presence of dihydrophyridine (DHP) antagonists. Globally, our data suggest that an ATP-dependent phosphorylation stabilizes the inhibitory control exerted by a PTX-sensitive G protein and, as a result, slows down the facilitation of L-type Ca2+ channels. The recruitment of L-type channels can also be facilitated by the application of a DHP agonist or a depolarizing prepulse protocol.l We show that these processes are only effective over a period which parallels the runup and are not additive to it.(ABSTRACT TRUNCATED AT 250 WORDS)

Mature rat cerebellar oligodendrocytes were isolated in culture using a serum free medium and identified using anti-galactocerebroside (GalC) and OL-1 antibodies. The morphology of such oligodendrocytes changes with time in culture from a multipolar shape at about 4 days in vitro (DIV) to a monopolar shape at about 8 DIV, a transition that has been previously described only in situ. Voltage-gated ionic currents were characterized at both oligodendrocyte developmental stages using the whole cell configuration of the patch-clamp technique. No differences between these two stages were detected, both types expressed a large K+ inward rectifying current similar to that described for oligodendrocytes from other vertebrate neuronal structures. This current could play an important role in the control of oligodendrocyte resting membrane potential. Our culture system provides a valuable model, close to the situation encountered in situ, not only to study the process of oligodendrocyte maturation, but also to identify the possible interactions between oligodendrocytes and cerebellar neurons such as granular and Purkinje cells during development.

In cultured cerebellar granule cells, we examined the effects of dihydropyridines (DHPs) on K+ currents, using the whole-cell recording configuration of the patch-clamp technique and on Ca(2+)-activated K+ channels ("maxi K+ channels") using outside-out patches. We found that micromolar concentrations of nicardipine, nifedipine, (+) and (-) BAY K 8644, nitrendipine, nisoldipine and (-) nimodipine block 10-60% of macroscopic K+ currents. The most potent of these DHPs was nicardipine and the least potent, (-) BAY K 8644. (+) Nimodipine had no effect on this current. The inhibitory effects of nifedipine and nicardipine were not additive with those of 1 mM tetraethylammonium (TEA). Outside-out recordings of "maxi K+ channels" showed a main conductance of 200 pS (in 77% of the patches) and two subconductance states (in 23% of the patches). Neither nifedipine nor nicardipine affected the main conductance, but decreased the values of the subconductance levels. In 10% of these patches, nicardipine induced a flickering activity of the channel. These findings show that both Ca2+ and K+ channels have DHP-sensitive sites, suggesting similarity in electrostatic binding properties of these channels. Furthermore, cerebellar granule cells may express different subtypes of "maxi K+ channels" having different sensitivities to DHPs. These drugs may provide new tools for the molecular study of K+ channels.

We have examined the internal Ca(2+)-dependence of the long-term evolution of whole cell high voltage activated Ca current in chromaffin cells. The evolution of the peak Ca current was characterized by 2 distinct phases: after an initial facilitation, there followed a rundown, which represented a reduction by 70% within some 10 min. The rundown process was shown not to depend on Ca2+ entry nor on membrane depolarization. It resulted from cell dialysis with a saline solution and, once initiated, it proceeded at a rate of 0.28 min-1 at 4 different Ca2+ concentrations (pCa 5-9). The facilitation is also initiated by cell dialysis but this process developed faster at higher internal Ca2+ concentrations. Thus, globally, high-voltage activated Ca2+ current runs down faster when using a recording pipette solution with a higher internal Ca2+ concentration (pCa 5 or 6). Some leupeptin-sensitive proteases may be involved in the initiation of facilitation and rundown processes.

The properties and characteristics of calcium channel openings in cerebellar granule cells were analysed by the cell-attached patch-clamp technique. At depolarized potentials, with 110 mM Ba2+ as the divalent charge carrier, 36% of the patches displayed activity that consisted of elementary events whose amplitude ranged from -0.3 to -1.75 pA at 0 mV, giving rise to a high threshold current. In this population of events at least four different types of channel openings were identified by their distinct biophysical and pharmacological properties. Two types of channel openings, with conductances around 24 and 7 pS, had similar characteristics in that both opened following two modes of gating characterized by brief (approximately 2 ms) and longer openings (approximately 8 ms) and both were sensitive to dihydropyridines. A further type of channel opening, with a conductance around 11 pS gated mainly with brief openings (approximately 1 ms), was shown to be insensitive to dihydropyridines but was undetectable in recordings from the cells that had been treated with omega-conotoxin. The last type of event was revealed after treatment of the cell with nicardipine or nifedipine and omega-conotoxin. The corresponding channel had a conductance of 19 pS and opened in one dominant mode characterized by brief openings (approximately 1 ms). The data obtained on single-channel activity of cerebellar granule cells are compared with the properties of the total current recorded in whole-cell conditions.

Confluent bovine capillary endothelial cells display, when examined for voltage-dependent calcium entries using cell-attached channel recordings, two types of Ca2+ channels (4 and 23.5 pS in 110 mM Ba2+) both sensitive to the dihydropyridine Ca agonist BAY K 8644. In contrast to isolated cells, confluent cells display no T-type, low threshold activity, and Ca currents were typically only elicited at very depolarized potentials. In these cells, voltage-dependent calcium entries will only be made operative by substances able to shift their activation towards the resting potential.

Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl2 above -40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl2) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above -40 mV which generates the low threshold sustained current.

1. Calcium channel activity was recorded in chromaffin cells in the cell-attached condition, using 110 mM-Ba2+ as the permeant ion. 2. One type of calcium channel had a conductance of 16 pS, was completely inactivated at a holding potential of -20 mV and was insensitive to dihydropyridine agonists and antagonists. These characteristics correspond to a calcium channel of the N-type. 3. A second type of calcium channel was active at holding potentials of -30 mV and above, had a channel conductance of 31 pS, and was sensitive to the dihydropyridine agonist, Bay K 8644. The channel opened along two dominant modes with characteristic time constants of 0.5 and 5 ms. The main effect of Bay K 8644 was to increase the probability of both short and long openings with no change in their relative proportions (6 to 1 respectively). These characteristics correspond to a calcium channel of the L-type. 4. omega-Conotoxin affected the activity of both N- and L-type channels. It drastically reduced the number of N-type channel openings and produced complex changes in L-type channel activity. Long openings were less frequent and the conductance during short openings was slightly smaller than that measured in the presence of Bay K 8644. 5. The discussion focuses on modulation of L-type channel activity. Openings of L-type channels are rarely recorded in the cell-attached configuration under control conditions. Addition of Bay K 8644 is needed to reveal the presence of L-type channels. By contrast, L-type currents recorded in the whole-cell configuration are always observed and are insensitive to Bay K 8644. These results indicate that L-type channels are normally inoperable in chromaffin cells.

1. Two calcium currents were identified by differences in their inactivation characteristics in adult chromaffin cells maintained in short-term primary culture (3-5 days). Calcium currents were recorded by means of the whole-cell configuration using an intracellular medium highly buffered for pH and pCa. 2. Calcium current evoked from a holding potential of -90 mV inactivated along two components: an initial transient with a time constant of 250 ms followed by a plateau. 3. Steady-state inactivation followed two processes which developed at two distinct membrane potentials. One process was half-inactivated at low voltages around -55 mV and affected mainly the initial transient component. The other process, which affected mainly the sustained component of the calcium current, was half-inactivated at voltages around -10 mV. The proportions of these two processes varied greatly from cell to cell. 4. The dihydropyridine antagonists (nicardipine and nifedipine applied at 10(-5) M) and the phenylalkylamine D600 (5 x 10(-6) M) shifted the half-inactivation value towards -55 mV, indicating the suppression of the sustained component. The snail toxin, omega-conotoxin, had the opposite effect; it shifted the half-activation value towards -10 mV. 5. The calcium channel agonist Bay K 8644 (10(-5) M) either had no effect or induced only a slight increase of the response, as did its (-)-enantiomer (10(-6) M). To interpret the present results, we suggest that the L-component was maximally activated in our recording conditions. 6. In chromaffin cells, the calcium current recorded in whole-cell conditions is composed of two components with properties close to those of N- and L-type currents described in sympathetic neurons.

Trans-1-amino-cyclopentyl-1,3-dicarboxylic acid (trans-ACPD), a specific agonist of the glutamate phosphoinositide-coupled receptor (Qp receptor), increased the amplitude of the outward K+ current recorded in the whole-cell configuration of the patch-clamp technique in mouse cultured cerebellar granule cells. This effect was abolished by buffering internal Ca2+ with BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. Activation of a large-conductance K+ channel was observed when trans-ACPD or quisqualic acid (QA), another Qp receptor agonist, was applied outside the cell-attached patch pipettes. No activation was observed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a specific agonist of ionotropic non-N-methyl-d-aspartate (non-NMDA) receptors. The effects of trans-ACPD or QA were potentiated in the presence of external Ca2+. The channel was also directly activated by both micromolar concentrations of internal Ca2+ and membrane depolarization. Its unitary conductance was 100 - 115 pS under asymmetrical K+ and 195 - 235 pS under high symmetrical K+ conditions. In the absence of agonist, the channel was blocked by 1 mM external tetraethylammonium. This is the first description of a large conductance Ca2+-activated K+ channel in cultured cerebellar granule cells. It possesses properties similar to those of the so-called 'big K+ channel' described in other preparations. Our cell-attached experiments demonstrated an indirect coupling between Qp receptors and this channel. The most likely hypothesis is that the second messenger system inositol 1,4,5-triphosphate (IP3)-Ca2+ was involved in the coupling process. This hypothesis was further strengthened by our whole-cell experiments. On the basis of the voltage- and Ca2+-sensitivities of the studied channel, we estimated an increase of 350 to 570 nM in internal Ca2+ concentration when Qp receptors were stimulated by 100 microM trans-ACPD. Under physiological conditions, stimulation of Qp receptors by the endogenous neurotransmitter should lead to similar K+ channel activation and therefore would tend to reduce the efficacy of ionotropic glutamate synaptic receptor stimulation responsible for cell excitation.

Postmitotic cerebellar granule cells, maintained for 5 - 6 days in Dulbecco's modified essential medium supplemented with 25 mM KCl, have been studied in whole-cell recording conditions to characterize calcium currents. With 10 mM Ba2+ as the divalent charge carrier, and using a pipette solution highly buffered for Ca2+ (30 mM EGTA, 100 mM HEPES - Tris, pH 7.2), only a high-threshold voltage-activated barium current was recorded from a holding potential of -90 mV. The addition of 1 mM ATP to the pipette medium allowed stable recording for an average duration of 10 min, compatible with pharmacological studies of the barium current. Ninety-six per cent of the current was half-inactivated at low negative holding potential (-76 mV). A total block of current was obtained with 1 microM Cd2+. Sixty-three per cent of the mean current was abolished by 3 microM omega-conotoxin (omega-CgTx; Ki=10 nM for a 15 min application), but individual cells showed either full sensitivity to this toxin or incomplete sensitivity. Seventy-eight per cent of the mean current was also abolished by 10 microM nicardipine but with a higher Ki of 0.5 microM. After exposure to omega-CgTx, BAY K 8644 had no effect on the remaining current, though it was suppressed by nicardipine. No sensitivity to diltiazem, desmethoxyverapamil or flunarizine could be detected. Our major conclusion is that at least half of the channels have a mixed pharmacology, showing sensitivity to both omega-CgTx and dihydropyridine antagonists.

Cell-attached patch recordings were used to study calcium channels on the dendritic membrane of rat cerebellar Purkinje cells maintained in culture. Experiments were performed with isotonic BaCl2 (110 mM) in the pipette and isotonic potassium gluconate in the bath to zero the cell membrane potential. Two distinct types of voltage-activated calcium channels were identified. The first one had a small conductance (9 pS), was activated at a low threshold (congruent to -50 mV) and could be inactivated by holding the membrane potential at -30 mV. This channel had the same characteristics as the T channel described in other neuronal preparations. The second type of Ca channel activated at a high threshold (-30 or +10 mV depending on whether BAY K 8644 was added or not to the pipette solution) and was still activatable even when the membrane was held at -40 mV. In the presence of BAY K 8644 this channel had a conductance of 21 pS with long openings. All these characteristics are similar to those of the S (L) Ca channel described in many preparations. The present study is in agreement with our previous experiments on Purkinje dendrites, where we identified low and high threshold Ca currents using the whole-cell configuration. Up to now, no channel corresponding to the N current has been observed but we cannot exclude its presence.

The effect of 0.5-1.0 microM taxol, a potent promoter of microtubule polymerization in vitro, was studied on the secretory activity of chromaffin cells of the adrenal medulla. Taxol was found to have a dual effect: the long-term effect (after a 1-h incubation) of taxol was to induce almost complete inhibition of catecholamine release, whereas after a short incubation (10 min) a massive, nicotine-independent release of catecholamine was produced. From results obtained using the patch-clamp technique to study the Ca++-dependent K+ channels (Ic channels), it was possible to conclude that taxol probably provokes an augmentation of free [Ca++]i in the cytoplasm, values increasing from 10(-8) M at rest to several 10(-7) M. The increased spontaneous release of stored neurohormones and the increased frequency of opening of Ic channels occur simultaneously and could both originate from a rise of [Ca++]i upon taxol addition. Immunofluorescence and ultrastructural studies showed that 13-h taxol treatment of chromaffin cells led to a different distribution of secretory organelles, and also to microtubule reorganization. In treated cells, microtubules were found to form bundles beneath the cell membrane and, at the ultrastructural level, to be packed along the cell axis. It is concluded that in addition to its action on microtubules, the antitumor drug taxol has side effects on the cell secretory activity, one of them being to modify free [Ca++]i.

The calcium currents of rat sensory neurones (of the IX and X cranial nerves) grown in culture were studied using whole cell recordings. In cells loaded with CsCl, and bathed in a solution where Na was replaced by choline or Tris, a step depolarization from -80 mV to 0 mV elicited the well-documented sustained Ca current (iCa,s). In contrast, depolarization from -80 mV to -60 mV and up to -20 mV evoked a distinct transient inward current (iCa,t) which could be isolated by imposing an internal pCa 7. It relaxed in about 100 ms and could possibly occur independently of the former current. The transient current was only affected by manipulation of the Ca concentration in the external medium and therefore was considered to be also a transfer of Ca. Ba was shown to act as a substitute with a lower affinity than Ca. The maximal amplitude of this current was in the order of a few hundred pA in Ca 5 mM and Mg 2 mM. Both activation and inactivation occurred in the same voltage span. The underlying event was studied using noise analysis and compared to the Ca transfer occurring during the sustained current as measured in chromaffin cells by Fenwick et al. (1982). We found them to be of similar amplitude.