Jean-Louis Bossu

Patch-clamp study of the tetrodotoxin-resistant sodium current in group C sensory neurones

Conditions were devised to isolate in cranial sensory neurones transfer of Na ions: K and Ca were omitted from the extracellular medium, and simultaneously cells were intracellularly loaded with 120 mM caesium and 20 mM TEA at [Ca]i = 10(-8) M. A tetrodotoxin (TTX)-resistant current was shown to be elicited by step depolarization from -25 MV upwards. This current successively activates and inactivates at increasing rates on further depolarization and at 0 mV (where peak amplitude is reached) its time course is of 20-50 ms. Absence of TTX-sensitivity (up to 15 microM), slow time course and an activation curve shifted by 15 mV towards the depolarized potentials differentiate this current from the more classical fast Na current which can be elicited on the same cells. Inactivation was provoked by a prepulse of varying amplitude and duration: with a prepulse command to -20 mV, inactivation was of 50% within a delay of 300 ms and almost 100% in about 1 min. After complete inactivation by command to 0 mV for 300 ms, recovery by holding the potential at -80 mV was of 50% in 205 ms, and of 100% after 1-4 s. It is concluded that a charge transfer of Na accounts for most of the hump which prolongs the action potential of these sensory neurones, and thus it can be proposed that spike duration as modulated by neurotransmitters may also involve Na in addition to Ca.

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements.

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1–4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10−8 M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I>Br>Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23–31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR 42641, 1 μM) blocked activity evoked by GABA (1–10 μM), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 μM, and were practically abolished at Cai 1 μM. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Cai reached 1 μM.

Voltage-gated Ca entry in isolated bovine capillary endothelial cells: evidence of a new type of BAY K 8644-sensitive channel

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.

Two types of calcium channels are expressed in adult bovine chromaffin cells

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.

Effect of internal calcium concentration on calcium currents in rat sensory neurones

Using the patch-clamp technique in whole-cell configuration we have investigated the effect of increasing the internal calcium concentration (Cai) from below 10−8 M to 10−6 M on the three calcium currents: ICa, T (T for transient), ICa, S (S for sustained), ICa, N (N for neither), recently described in rat sensory neurones> Increasing Cai led to a dose-dependent reduction of the amplitude of ICa, S and, as Cai reached 5×10−7M ICa, S was nearly abolished. ICa, N is well evidenced from 5×10−10 M to 10−7 M where its is a large current. Preliminary observations indicate an increase of its inactivation rate following, as expected for a possible Cai dependent-inactivation, the increase of Cai from 5×10−10 M to 10−7 M. With Ca=5×10−7 M, all the cells displayed ICa, T and half of the cells in addition ICa, N, but it was of small amplitude. At Cai=10−6 M, most of the recorded cells only exhibited ICa, T.

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.

Inactivation characteristics reveal two calcium currents in adult bovine 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.

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

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 Ca2+ 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.

Identification of living oligodendrocyte developmental stages by fractal analysis of cell morphology

The Mandelbrots 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). J. Neurosci. Res. 65:439–445, 2001.