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Neurons firing spontaneously in bursts in the absence of synaptic transmission have been previously recorded in different layers of cortical brain slices. It has been suggested that such neurons could contribute to the generation of alternating UP and DOWN states, a pattern of activity seen during slow-wave sleep. Here, we show that in layer 6b (L6b), known from our previous studies to contain neurons highly responsive to the wake-promoting transmitter hypocretin/orexin (hcrt/orx), there is a set of neurons, endowed with distinct intrinsic properties, which displayed a strong propensity to fire spontaneously in rhythmic bursts. In response to small depolarizing steps, they responded with a delayed firing of action potentials which, upon higher depolarizing steps, invariably inactivated and were followed by a depolarized plateau potential and a depolarizing afterpotential. These cells also displayed a strong hyperpolarization-activated rectification compatible with the presence of an I h current. Most L6b neurons with such properties were able to fire spontaneously in bursts. Their bursting activity was of intrinsic origin as it persisted not only in presence of blockers of ionotropic glutamatergic and GABAergic receptors but also in a condition of complete synaptic blockade. However, a small number of these neurons displayed a mix of intrinsic bursting and synaptically driven recurrent UP and DOWN states. Most of the bursting L6b neurons were depolarized and excited by hcrt/orx through a direct postsynaptic mechanism that led to tonic firing and eventually inactivation. Similarly, they were directly excited by noradrenaline, histamine, dopamine, and neurotensin. Finally, the intracellular injection of these cells with dye and their subsequent Neurolucida reconstruction indicated that they were spiny non-pyramidal neurons. These results lead us to suggest that the propensity for slow rhythmic bursting of this set of L6b neurons could be directly impeded by hcrt/orx and other wake-promoting transmitters.

Fast spiking (FS) GABAergic neurons are thought to be involved in the generation of high-frequency cortical rhythms during the waking state. We previously showed that cortical layer 6b (L6b) was a specific target for the wake-promoting transmitter, hypocretin/orexin (hcrt/orx). Here, we have investigated whether L6b FS cells were sensitive to hcrt/orx and other transmitters associated with cortical activation. Recordings were thus made from L6b FS cells in either wild-type mice or in transgenic mice in which GFP-positive GABAergic cells are parvalbumin positive. Whereas in a control condition hcrt/orx induced a strong increase in the frequency, but not amplitude, of spontaneous synaptic currents, in the presence of TTX, it had no effect at all on miniature synaptic currents. Hcrt/orx effect was thus presynaptic although not by an action on glutamatergic terminals but rather on neighboring cells. In contrast, noradrenaline and acetylcholine depolarized and excited these cells through a direct postsynaptic action. Neurotensin, which is colocalized in hcrt/orx neurons, also depolarized and excited these cells but the effect was indirect. Morphologically, these cells exhibited basket-like features. These results suggest that hcrt/orx, noradrenaline, acetylcholine, and neurotensin could contribute to high-frequency cortical activity through an action on L6b GABAergic FS cells.

The hypocretin-orexin (hcrt-orx) neurons are thought to maintain wakefulness because their loss results in narcolepsy. This role may be fulfilled by the excitatory action that the hcrt-orx peptide exerts on multiple brainstem and forebrain systems that, in turn, promote cortical activation. Here, we examined whether hcrt-orx may also exert a postsynaptic excitatory action at the level of the cortex, where hcrt-orx fibers project. However, we found that neurons in layers 2-5 in the primary somatosensory cortex (SSp) were unresponsive to hcrt-orx. We then found that although all neurons tested in sublayer 6a were also unresponsive to hcrt-oxr, all those tested in sublayer 6b were highly sensitive to the peptide. The sublayer selectivity of hcrt-oxr was not restricted to the somatosensory cortex, because it was also found to be present in the primary visual cortex, the motor cortex, and the cingulate cortex. In the SSp, in which the hcrt-oxr effect was investigated further, it was demonstrated to be postsynaptic, to result from an interaction with Hcrtr2-OX2receptors and to depend on the closure of a potassium conductance. Similar to the selectivity of action in the thalamus, where hcrt-oxr excites the nonspecific thalamocortical projection neurons and not the specific sensory relay neurons, here in the cortex, it excites a specific subset of cortical neurons which, through corticocortical projections, may also be involved in promoting widespread cortical activation.

As is evident from the pathological consequences of its absence in narcolepsy, orexin (hypocretin) appears to be critical for the maintenance of wakefulness. Via diffuse projections through the brain, orexin-containing neurons in the hypothalamus may act on a number of wake-promoting systems. Among these are the intralaminar and midline thalamic nuclei, which project in turn in a widespread manner to the cerebral cortex within the nonspecific thalamocortical projection system. Testing the effect of orexin in rat brain slices, in two nuclei of this system, centromedial (CM) nuclei and rhomboid nuclei, we found that it depolarized and excited all neurons tested through a direct postsynaptic action. An additional analysis of this effect in CM neurons indicates that it results from the decrease of a potassium conductance. By a detailed comparison of the effects of orexin A and B, we established that orexin B was more potent than orexin A, indicating the probable mediation by orexin type 2 receptors. In contrast to its effect on the nonspecific thalamocortical projection neurons, orexin had no effect on the specific sensory relay neurons of the somatic, ventral posterolateral, and visual dorsal lateral geniculate nuclei. Orexin differs in this regard from norepinephrine and acetylcholine, to which neurons in the specific and nonspecific systems are sensitive. Orexin may thus act in the thalamus to promote wakefulness by exciting neurons of the nonspecific thalamocortical projection system, which, through widespread projections to the cerebral cortex, stimulate and maintain cortical activation.

The orexins (orexin A and B, also known as hypocretin 1 and 2) are two recently identified neuropeptides (de Lecea et al., 1998; Sakurai et al., 1998) which are importantly implicated in the control of wakefulness (for reviews see Hungs and Mignot, 2001; van den Pol, 2000; Willie et al., 2001 ). Indeed, alteration in these peptides' precursor, their receptors or the hypothalamic neurones that produce them leads to the sleep disorder narcolepsy (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000; Thannickal et al., 2000). The mechanisms by which the orexins modulate wakefulness, however, are still unclear. Their presence in fibres coursing from the hypothalamus (Peyron et al., 1998) up to the preoptic area (POA) and basal forebrain (BF) suggests that they might influence the important sleep and waking neural systems situated there (Jones, 2000). The present study, performed in rat brain slices, demonstrates, however, that the orexins have no effect on the GABA sleep-promoting neurones of the POA, whereas they have a strong and direct excitatory effect on the cholinergic neurones of the contiguous BF. In addition, by comparing the effects of orexin A and B we demonstrate here that orexins' action depends upon orexin type 2 receptors (OX(2)), which are those lacking in narcoleptic dogs (Lin et al., 1999). These results suggest that the orexins excite cholinergic neurones that release acetylcholine in the cerebral cortex and thereby contribute to the cortical activation associated with wakefulness.

Wakefulness has recently been shown to depend upon the newly identified orexin (or hypocretin) neuropeptides by the findings that alteration in their precursor protein, their receptors or the neurons that produce them leads to the sleep disorder narcolepsy in both animals and humans. The questions of how and where these brain peptides act to maintain wakefulness remain unresolved. The purpose of the present study was to determine whether the orexins could directly affect hypothalamic histaminergic neurons, which are known to contribute to the state of wakefulness by their diffuse projections through the brain. Using brain slices, we recorded in the ventral tuberomammillary nuclei from neurons identified as histaminergic on the basis of their previously described morphological and electrophysiological characteristics and found that they were depolarized and excited by the orexins through a direct postsynaptic action. We then compared the depolarizing effect of orexin A and B and found that they were equally effective upon these cells. This latter finding suggests that the effect of orexins is mediated by orexin type 2 receptors, which are those lacking in narcoleptic dogs. Our results therefore show that the histaminergic neurons of the tuberomammillary nuclei represent an important target for the orexin system in the maintenance of wakefulness.

Vestibular compensation for the postural and oculomotor deficits following unilateral labyrinthectomy is a model of functional plasticity in the brain of adult vertebrates. The mechanisms involved in this recovery are still controversial. The post-lesional lack of vestibular input might be compensated by changes in the efficacy of the remaining sensory inputs involved in gaze and posture stabilization. However, the compensation process could also rapidly become independent of these external cues, and thus be detectable in vitro in preparations obtained from lesioned animals. In agreement with this hypothesis, we have shown recently that prominent traces of the compensation process appeared three days after the lesion on in vitro isolated brains taken from labyrinthectomized guinea-pigs, where the connectivity of the central vestibular-related networks is preserved. We report here that, one week after the lesion, a slight increase in the intrinsic, spontaneous activity of the deafferented, central vestibular neurons was found in brainstem slices. This increase became stronger in slices taken after one month of compensation, and was associated at this stage with a significant decrease in the intrinsic activity of the vestibular neurons on the contralesional side. Vestibular compensation could thus follow a "top-down" strategy: it would first rely on the external cues given by the intact sensory systems, then on an internal reorganization of the vestibular-related networks, and finally on changes in the intrinsic properties of the vestibular neurons themselves. Similar strategies may be used by the mammalian brain to compensate for other types of deafferentations or environmental changes.

Vestibular compensation for the postural and oculomotor deficits induced by unilateral labyrinthectomy is a model of post-lesional plasticity in the central nervous system. Just after the removal of one labyrinth, the deafferented, ipsilateral vestibular nucleus neurons are almost silent, and the discharge of the contralateral vestibular nucleus neurons is increased. The associated static disorders disappear in a few days, as normal activity is restored in both vestibular nuclei. In this study, we searched for traces of vestibular compensation in isolated whole brains taken from adult guinea-pigs. The electrophysiological responses evoked in control brains were compared to those evoked in brains taken from animals that had previously been labyrinthectomized. Guinea-pigs compensated for an initial labyrinthectomy within three days. In vivo, subsequent deafferentation of vestibular nucleus neurons on the intact side triggered "Bechterew's phenomenon": a new postural and oculomotor syndrome appeared, similar to the one induced by the first lesion, but directed to the newly deafferented side. These disturbances would be caused by the new imbalance between the discharges of neurons in the two vestibular nuclei triggered by the second deafferentation. Experiments were designed to search for a similar imbalance in vitro in brains taken from labyrinthectomized animals, where the intact vestibular nerve is cut during the dissection. Isolated whole brains were obtained from young guinea-pigs at various times (one to seven days) following an initial labyrinthectomy. An imbalance between the resting activities of medial vestibular nucleus neurons on both sides of the brainstem was revealed in brains taken more than three days after the lesion: their discharge was higher on the compensated, initially lesioned side than on the newly deafferented side. In some cases, an oscillatory pattern of discharge, reminiscent of the spontaneous nystagmus associated in vivo with Bechterew's syndrome, appeared in both abducens nerves. These data demonstrate that most of the changes underlying vestibular compensation persist, and can thus be investigated in the isolated whole brain preparation. Brains removed only one day after the lesion displayed normal commissural responses and symmetric spinal inputs to vestibular nucleus neurons. However, an unusually large proportion of the neurons recorded on both sides of the preparation had very irregular spontaneous discharge rates. These data suggest that the first stages of vestibular compensation might be associated with transient changes in the membrane properties of vestibular nucleus neurons. Brains taken from compensated animals displayed a significant, bilateral decrease of the inhibitory commissural responses evoked in the medial vestibular nucleus by single-shock stimulation of the contralateral vestibular nerve. The sensitivity of abducens motoneurons on the initially lesioned, compensated side to synaptic activation from the contralesional vestibular nucleus neurons was also decreased. Both changes may explain the long-term, bilateral decrease of vestibular-related reflexes observed following unilateral labyrinthectomy. Spinal inputs to vestibular nucleus neurons became progressively asymmetric: their efficacy was increased on the lesioned side and decreased on the intact one. This last modification may support a functional substitution of the deficient, vestibular-related synergies involved in gaze and posture stabilization by neck-related reflexes.

The spiking behaviour of 66 second-order vestibular neurons was studied in alert, chronically prepared guinea-pigs during the horizontal vestibulo-ocular reflex (VOR). Among the 66 studied neurons, seven were held long enough (> 1 h) to compare their spiking behaviour before and after a training procedure inducing a decrease in the gain of the VOR. When tested in darkness following adaptation, five of them showed a significant decrease of their sensitivity to head rotation. However, the resting discharge of these five neurons remained unchanged. This suggests that VOR adaptation is mediated not only by changes in synaptic efficacities but also by modifications in the spike generator which transforms synaptic inputs into a pattern of action potentials.

The aim of the present study was to investigate whether the voltage-dependent inhibition of calcium currents by serotonin 5-HT1A agonists can be alleviated (facilitated) by action potential-like depolarizations. In dissociated cholinergic basal forebrain neurons using whole-cell recordings, it is shown that a selective serotonin 5-HT1A agonist (8-OH-DPAT) predominantly blocks N-type HVA calcium current, although a minor reduction of P-type current was also observed. The inhibition may principally occur through Gi-Go subtypes of G-proteins because it was prevented by N-ethylmaleimide, a substance known to block specifically pertussis-sensitive G-proteins. The inhibitory effect of 8-OH-DPAT on calcium currents is voltage-dependent because it was alleviated by long-lasting depolarizing prepulses. Interestingly, the inhibition could also be reversed by prepulses made-up of action potential-like depolarizations that were given at a frequency of 200 Hz. This observation may have important implications during periods of high-frequency rhythmic bursts, a firing pattern that is prevalent in cholinergic basal forebrain neurons.

The potential influence of GABAergic input to cholinergic basalis neurons was studied in guinea-pig basal forebrain slices. GABA and its agonists were applied to electrophysiologically-identified cholinergic neurons, of which some were labelled with biocytin and confirmed to be choline acetyltransferase-immunoreactive. Immunohistochemistry for glutamate decarboxylase was also performed in some slices and revealed GABAergic varicosities in the vicinity of the biocytin-filled soma and dendrites of electrophysiologically-identified cholinergic cells. From rest (average - 63 mV), the cholinergic cells were depolarized by GABA. The depolarization was associated with a decrease in membrane resistance and diminution in firing. The effect was mimicked by muscimol, the specific agonist for GABA(A) receptors, and not by baclofen, the specific agonist for GABA(B) receptors, which had no discernible effect. The GABA- and muscimol-evoked depolarization and decrease in resistance were found to be postsynaptic since they persisted in the presence of solutions containing either high Mg2+/low Ca2+ or tetrodotoxin. They were confirmed as being mediated by a GABA(A) receptor, since they were antagonized by bicuculline. The reversal potential for the muscimol effect was estimated to be approximately -45 mV, which was -15 mV above the resting membrane potential. Finally, in some cholinergic cells, spontaneous subthreshold depolarizing synaptic potentials (average 5 mV in amplitude), which were rarely associated with action potentials, were recorded and found to persist in the presence of glutamate receptor antagonists but to be eliminated by bicuculline. These results suggest that GABAergic input may be depolarizing, yet predominantly inhibitory to cholinergic basalis neurons.

The contributions made by low- (LVA) and high-voltage-activated (HVA) calcium currents to afterhyperpolarizations (AHPs) of nucleus basalis (NB) cholinergic neurons were investigated in dissociated cells. Neurons with somata >25 μmwere studied because 80% of them stained positively for choline acetyltransferase and had electrophysiological characteristics identical to those of cholinergic NB neurons previously recorded in basal forebrain slices. Calcium currents of cholinergic NB neurons first were dissected pharmacologically into an amiloride-sensitive LVA and at least five subtypes of HVA currents. Approximately 17% of the total HVA current was sensitive to nifedipine (3 μm), 35% to ω-conotoxin-GVIA (200–400 nm), 10% to ω-Agatoxin-IVA (100 nm), and 20% to ω-Agatoxin-IVA (300–500 nm), suggesting the presence of L-, N-, P-, and Q-type channels, respectively. A remaining current (R-type) resistant to these antagonists was blocked by cadmium (100–200 μm). We then assessed pharmacologically the role that LVA and HVA currents had in activating the apamin-insensitive AHP elicited by a long train of action potentials (sAHP) and the AHP evoked either by a short burst of action potentials or by a single action potential (mAHP) that is known to be apamin-sensitive. During sAHPs, ∼60% of the hyperpolarization was activated by calcium flowing through N-type channels and ∼20% through P-type channels, whereas T-, L-, and Q-type channels were not involved significantly. In contrast, during mAHPs, N- and T-type channels played key roles (∼60 and 30%, respectively), whereas L-, P-, and Q-type channels were not implicated significantly. It is concluded that in cholinergic NB neurons various subtypes of calcium channels can differentially activate the apamin-sensitive mAHP and the apamin-insensitive sAHP.

Known to exert an important modulatory influence on the cerebral cortex, the cholinergic neurons of the basal forebrain are modulated in turn by neurotransmitters which may include acetylcholine released from processes of brainstem or forebrain neurons. In the present study, we examined the effect of carbachol, a non-specific cholinergic agonist, either alone or in the presence of N-methyl-D-aspartate upon electrophysiologically identified cholinergic basalis neurons in guinea-pig basal forebrain slices. Carbachol produced a direct postsynaptic hyperpolarization, accompanied by a decrease in membrane resistance. Muscarine could mimic this hyperpolarizing effect, whereas nicotine produced a direct postsynaptic membrane depolarization. The interaction of carbachol with N-methyl-D-aspartate was subsequently tested since, in a prior study, N-methyl-D-aspartate was shown to induce rhythmic bursting in cholinergic cells when they were hyperpolarized by continuous injection of outward current. Applied simultaneously with N-methyl-D-aspartate in the absence of current injection, carbachol was also found to promote rhythmic bursting in half of the cells tested. Since the bursts under these conditions were markedly longer in duration than those observed in the presence of N-methyl-D-aspartate alone, it was hypothesized that carbachol might have another action, in addition to the membrane hyperpolarization. Using dissociated cells, it was found that brief applications of carbachol could indeed diminish the slow afterhyperpolarizations that follow single spikes, short bursts or long trains of action potentials in cholinergic basalis neurons. These results indicate that, through its dual ability to hyperpolarize cholinergic neurons and to reduce their afterhyperpolarizations, acetylcholine can promote the occurrence of rhythmic bursting in the presence of N-methyl-D-aspartate. Accordingly, whether derived from brainstem or local sources, acetylcholine may facilitate rhythmic discharge in cholinergic basalis neurons which could in turn impose a rhythmic modulation upon cortical activity during particular states across the sleep-waking cycle.

The presence of theta rhythm (5-10 Hz) in the hippocampus has been shown to enable long-term potentiation, a synaptic mechanism which has been proposed to underlie learning and memory. Medial septum cholinergic and GABAergic neurons that project to the hippocampus have been hypothesized to play conjointly a major role in the genesis of this rhythm. Building upon previous studies that have established the electrophysiological criteria for distinguishing cholinergic and non-cholinergic neurons in this area, it is demonstrated here that medial septum non-cholinergic neurons, putatively GABAergic, have the ability to discharge in rhythmic clusters of action potentials occurring at frequencies ranging from 1 to 8 Hz. Within the clusters, the firing frequency of action potentials varied between 13 and 57 Hz in a voltage-dependent manner. In addition, small voltage-dependent subthreshold membrane potential oscillations (16-54 Hz) were observed between clusters. Both subthreshold oscillations and clusters were eliminated by tetrodotoxin at 1 microM. These results indicate that non-cholinergic medial septum neurons could convey to the hippocampus not only theta but also higher frequency rhythmicity in the beta-gamma range (20-60 Hz).

Gigantocellular neurons of the medullary nucleus gigantocellularis represent a major source of reticulospinal pathways. Among other roles, they have been involved in the processing of vestibular information. The aim of the present study was to describe the major intrinsic membrane properties of these cells in guinea-pig brainstem slices. We found nucleus gigantocellularis neurons to be segregated in two cell types. Type A nucleus gigantocellularis neurons were characterized by the presence of a single large afterhyperpolarization and a potent transient 4-aminopyridine-sensitive rectification likely due to the presence of a transient outward potassium current. In contrast, type B nucleus gigantocellularis neurons had a narrower and faster rising action potential followed by an early fast and a delayed slower after-hyperpolarization. In contrast to type A neurons, type B neurons were, in addition, endowed with subthreshold tetrodotoxin-sensitive sodium-dependent plateau potentials. Whereas both cell types were endowed with high-threshold calcium-dependent action potentials, only type B nucleus gigantocellularis neurons also displayed long-lasting calcium-dependent plateau potentials. These results show that nucleus gigantocellularis neurons can be segregated by their intrinsic membrane properties it two cell types which are very similar to those that we have previously described in the medial vestibular nucleus. The possibility that these differences between type A and B neurons might play a role in the segregation between tonic and kinetic cells is discussed.

1. Neuronal activity was investigated in the left superior vestibular nucleus (SVN), lateral vestibular nucleus (LVN), and rostral part of the medial vestibular nucleus (MVN) in the alert guinea pig after a unilateral (left) labyrinthectomy was performed. Vestibular neurons were recorded either immediately (just-postoperative group, n = 6) or 1 wk after labyrinthectomy (1-wk-postoperative group, n = 6) and compared with the activity recorded in intact animals (control group, n = 6). 2. Animals were prepared for extracellular recording of single-unit activity and for eye movement recording (scleral search coil technique). To enable stimulation of the left vestibular nerve, bipolar silver ball electrodes were chronically implanted either in contact with the bony labyrinth in the control group or close to the stump of the vestibular nerve after labyrinthectomy. Complete labyrinthectomy was performed under halothane anesthesia. 3. The criterion used to select vestibular neurons for analysis was their recruitment by an electric shock on the vestibular nerve. Of the 589 recorded neurons, 424, defined as second-order vestibular neurons, were recruited at monosynaptic latencies (0.85-1.15 ms) and 165 were recruited at polysynaptic latencies. One hundred three second-order vestibular neurons were recorded in the control group, 173 in the just-postoperative group, and 148 in the 1-wk-postoperative group. 4. The activity of the electrically recruited neurons was recorded during sinusoidal horizontal head rotation in the dark (0.3 Hz, 40 degrees/s peak velocity). The behavior of the neurons was analyzed by plotting their firing rate against head velocity. The Y-intercept of the regression line was used to express spontaneous firing rate (resting discharge), and its slope was used to express the sensitivity of the neuron-to-head velocity. 5. In the absence of statistically significant difference between the characteristics of the neuronal discharge of the second-order vestibular neurons recorded in the SVN, LVN, and rostral MVN, the data were pooled. The Resting discharge of these cells amounted to 41.0 +/- 24.7 (SD) spikes/s in the control state, fell to 7.2 +/- 13.9 spikes/s just after labyrinthectomy, and completely returned to normal values 1 wk after surgery (42.5 +/- 21.6 spikes/s). Among the monosynaptically recruited neurons, the percentage of silent units was 0% in the control group, 69% in the just-postoperative group, and 0% in the 1-wk-postoperative group. 6. By contrast, the sensitivity to head velocity of the second-order vestibular neurons, which was 0.69 +/- 0.48 (SD) spikes.s-1/deg.s-1 in the control state and which fell to 0.03 +/- 0.11 spikes.s-1/deg.s-1 just after labyrinthectomy, remained low 1 wk after injury (0.21 +/- 0.26 spikes.s-1/deg.s-1). Moreover, the slight recovery of sensitivity to head rotation was due only to units behaving as type II neurons. 7. The mean resting discharge of the polysynaptically recruited neurons (pooled from the 3 explored nuclei) was 31.6 +/- 19.3 spikes/s in the control group. It decreased to 11.6 +/- 12.1 spikes/s in the just-postoperative group and recovered to 39.8 +/- 20.2 spikes/s in the 1-wk-postoperative group. No neuron was silent at rest either in the control group or in the 1-wk-postoperative group. Just after labyrinthectomy, 35% of the neurons had a null resting activity. The mean sensitivity to head velocity of these neurons was 0.55 +/- 0.42 spikes.s-1/deg.s-1 in the control group. It decreased to 0.05 +/- 0.12 spikes.s-1/deg.s-1 in the just-postoperative group and recovered to 0.22 +/- 0.17 spikes.s-1/deg.s-1 in the 1-wk-postoperative group. 8. We conclude that, at least in the guinea pig, the restoration of the spontaneous activity of the deafferented neurons is complete 1 wk after a unilateral labyrinthectomy and thus probably plays an important role in vestibular compensation...

Using intracellular recordings of medial vestibular nucleus neurones (MVNn) in guinea-pig brainstem slices, the effects of baclofen, a specific agonist of the metabotropic GABAB receptors, were tested on the three main types of MVNn (A, B and B + LTS MVNn) that were previously identified in this nucleus. Regardless of their type, almost all MVNn were hyperpolarized and inhibited by baclofen. These hyperpolarizing effects persisted following either the addition of tetrodotoxin (TTX) in the perfusion medium, or in the presence of a high Mg2+/low Ca2+ solution known to block synaptic transmission. These results demonstrate that all types of MVNn are endowed with postsynaptic GABAB receptors.

Acetylcholine has long been known to play an important role in the cortical activation that accompanies the states of wakefulness and paradoxical sleep (for review, see Refs 17, 21) when this neurotransmitter is released from the cerebral cortex at the highest rates. The major supply of acetylcholine to the cerebral cortex arises from the cholinergic neurons of Meynert's Basal-ganglion or nucleus basalis of the forebrain. Lying in the substantia innominata within the major ascending pathway from the brain stem reticular formation, magnocellular basalis neurons project upon the cerebral cortex as the important ventral, extrathalamic relay of the ascending reticular activating system. Although the cholinergic basalis nucleus neurons have been shown to be important for cortical activation, the precise manner in which they influence cortical activity has not as yet been elucidated, in part because the cholinergic cells of this nucleus have not been identified in electrophysiological studies. Using intracellular recording in guinea-pig brain slices, we were able to record and fill with biocytin nucleus basalis neurons which were subsequently revealed by immunohistochemical staining to be choline acetyltransferase-positive and thus cholinergic. The cholinergic cells displayed rhythmic bursting activity mediated by a low-threshold calcium spike in vitro, which would endow them with a capacity for phasic (in addition to tonic) firing in vivo. By virtue of these different modes, cholinergic basalis neurons may accordingly deter or facilitate the cortical response to sensory input and may furthermore modulate the major frequencies of cortical activity across the different states of the sleep-waking cycle.

Intracellular recordings were obtained from medial vestibular nuclei neurones (MVNn) in guinea-pig brainstem slices. Two main distinct neuronal classes were encountered. Type A MVNn (32.3%) were characterized by a broad action potential followed by a deep single afterhyperpolarization, a transient A-like rectification, and a single range of firing in response to current injection. Type B MVNn (47.1%), in contrast, were distinguished by the presence of a thin action potential followed first by a fast and then by a delayed and slower afterhyperpolarization. In addition, they displayed a secondary range of firing in their response to current injection. A majority of B MVNn also had either sub-threshold plateau potentials or low threshold spike bursts or a combination thereof. A third, non-homogeneous class of cells, could not be fitted into either one of the two main classes (20.6%, type C MVNn).

Intracellular recordings from nucleus gigantocellularis (NGC) neurones were obtained in isolated and perfused whole brains of guinea pigs in vitro. A majority of cells (90%) were characterized by an action potential of short duration (0.3 ms) followed first by a fast and then by a slower afterhyperpolarization (AHP). Their firing pattern was mostly irregular. These cells were shown to have high threshold calcium spikes and plateau potentials. The other cell type represented only 10% of the recorded cells in the NGC. It was characterized by a wider (0.6 ms) action potential, a large single AHP, the presence of a transient rectification presumably due to an A-current and a rather regular resting discharge. Using Lucifer yellow injections in brainstem slices, both cell types were shown to correspond to gigantocellular neurones.

Intracellular recordings were obtained from pedunculopontine neurones in guinea-pig brainstem slices. These cells were characterized by a broad action potential, an A-like conductance and fired spontaneously in a regular manner. These neurones were inhibited by bath-application of both carbachol and serotonine at concentrations of 10(-4) M. Opioid peptides induced a dose-dependent hyperpolarization and a reduction in the spontaneous firing. These latter effects could be blocked by the opiate antagonist naloxone and were direct as they persisted in presence of tetrodotoxine or high magnesium/low calcium-containing salines. They were mediated by an opiate receptor of the mu type since they were obtained with the mu-preferring enkephalin analogues FK 33-824 and DAGO, but neither with the delta nor the kappa analogues such as DPLPE or U-50,488.

As the major brain circadian pacemaker, the suprachiasmatic nucleus (SCN) is known to influence the timing of sleep and waking. We thus investigated here the effect of SCN stimulation on neurons of the ventrolateral preoptic nucleus (VLPO) thought to be involved in promoting sleep. Using an acute in vitro preparation of the rat anterior hypothalamus/preoptic area, we found that whereas single-pulse stimulations of the SCN evoked standard fast ionotropic IPSPs and EPSPs, train stimulations unexpectedly evoked a long-lasting inhibition (LLI). Such LLIs could also be evoked in VLPO neurons by pressure application of NMDA within the SCN, indicating the specific activation of SCN neurons. This LLI was shown to result from the presynaptic facilitation of noradrenaline release, because it was suppressed in presence of yohimbine, a selective antagonist of alpha2-adrenoreceptors. The LLI depended on the opening of a potassium conductance, because it was annulled at E(K) and could be reversed below E(K). These results show that the SCN can provide an LLI of the sleep-promoting VLPO neurons that could play a role in the circadian organization of the sleep-waking cycle.

The neurons of the lateral hypothalamus that contain hypocretin/orexin (hcrt/orx) are thought to promote arousal through the excitatory action they exert on the multiple areas to which they project within the CNS. We show here that the hcrt/orx peptides can also exert a strong action on the amygdala, a structure known for its implication in emotional aspects of behavior. Indeed, the hcrt/orx peptides, applied in acute rat brain slices, excite a specific class of "low threshold burst" neurons in the central medial (CeM) nucleus which is considered as a major output of the amygdala. These excitatory effects are postsynaptic, mediated by Hcrt2/OX2 receptors and result from the closure of a potassium conductance. They occur on a class of neurons that are also excited by vasopressin acting through V1a receptors. These results suggest that the hcrt/orx system can act through the amygdala to augment arousal and evoke the autonomic and behavioral responses associated with fear, stress or emotion.

Numerous models of the oculomotor neuronal integrator located in the prepositus hypoglossi nucleus (PHN) involve both highly tuned recurrent networks and intrinsic neuronal properties; however, there is little experimental evidence for the relative role of these two mechanisms. The experiments reported here show that all PHN neurons (PHNn) show marked phasic behavior, which is highly oscillatory in ∼25% of the population. The behavior of this subset of PHNn, referred to as type D PHNn, is clearly different from that of the medial vestibular nucleus neurons, which transmit the bulk of head velocity-related sensory vestibular inputs without integrating them. We have investigated the firing and biophysical properties of PHNn and developed data-based realistic neuronal models to quantitatively illustrate that their active conductances can produce the oscillatory behavior. Although some individual type D PHNn are able to show some features of mathematical integration, the lack of robustness of this behavior strongly suggests that additional network interactions, likely involving all types of PHNn, are essential for the neuronal integrator. Furthermore, the relationship between the impulse activity and membrane potential of type D PHNn is highly nonlinear and frequency-dependent, even for relatively small-amplitude responses. These results suggest that some of the synaptic input to type D PHNn is likely to evoke oscillatory responses that will be nonlinearly amplified as the spike discharge rate increases. It would appear that the PHNn have specific intrinsic properties that, in conjunction with network interconnections, enhance the persistent neural activity needed for their function.

Sleep deprivation is accompanied by the progressive development of an irresistible need to sleep, a phenomenon whose mechanism has remained elusive. Here, we identified for the first time a reflection of that phenomenonin vitroby showing that, after a short 2 h period of total sleep deprivation, the action of noradrenaline on the wake-promoting hypocretin/orexin neurons changes from an excitation to an inhibition. We propose that such a conspicuous modification of responsiveness should contribute to the growing sleepiness that accompanies sleep deprivation.

Hypocretin/orexin (Hcrt/Orx) and melanin concentrating hormone (MCH) are peptides contained in overlapping cell groups of the lateral hypothalamus and commonly involved in regulating sleep-wake states and energy balance, though likely in different ways. To see if these neurons are similarly or differentially modulated by neurotransmitters of the major brainstem arousal systems, the effects of noradrenaline (NA) and carbachol, a cholinergic agonist, were examined on identified Hcrt/Orx and MCH neurons in rat hypothalamic slices. Whereas both agonists depolarized and excited Hcrt/Orx neurons, they both hyperpolarized MCH neurons by direct postsynaptic actions. According to the activity profiles of the noradrenergic locus coeruleus and cholinergic pontomesencephalic neurons across the sleep-waking cycle, the Hcrt/Orx neurons would be excited by NA and acetylcholine (ACh) and thus active during arousal, whereas the MCH neurons would be inhibited by NA and ACh and thus inactive during arousal while disinhibited and possibly active during slow wave sleep. According to the present pharmacological results, Hcrt/Orx neurons may thus stimulate arousal in tandem with other arousal systems, whereas MCH neurons may function in opposition with other arousal systems and thus potentially dampen arousal to promote sleep.

According to multiple lines of evidence, neurons in the ventrolateral preoptic area (VLPO) that contain GABA promote sleep by inhibiting neurons of the arousal systems. Reciprocally, transmitters used by these systems, including acetylcholine (ACh) and noradrenaline (NA), exert an inhibitory action on the VLPO neurons. Because nicotine, an agonist of ACh, acts as a potent stimulant, we queried whether it might participate in the cholinergic inhibition of these sleep-promoting cells. Indeed, we found that ACh inhibits the VLPO neurons through a nicotinic, as well as a muscarinic, action. As evident in the presence of atropine, the non-muscarinic component was mimicked by epibatidine, a nonselective nicotinic ACh receptor (nAChR) agonist and was blocked by dihydro-β-erythroidine, a nonselective nAChR antagonist. It was not, however, blocked by methyllycaconitine, a selective antagonist of the α7 subtype, indicating that the action was mediated by non-α7 nAChRs. The nicotinic inhibition was attributed to a presynaptic facilitation of NA release because it persisted in the presence of tetrodotoxin and was blocked by yohimbine and RS 79948, which are both selective antagonists of α2 adrenergic receptors. Sleep-promoting VLPO neurons are thus dually inhibited by ACh through a muscarinic postsynaptic action and a nicotinic presynaptic action on noradrenergic terminals. Such dual complementary actions allow ACh and nicotine to enhance wakefulness by inhibiting sleep-promoting systems while facilitating other wake-promoting systems.

Wakefulness depends on the activity of hypocretin–orexin neurons because their lesion results in narcolepsy. How these neurons maintain their activity to promote wakefulness is not known. Here, by recording for the first time from hypocretin–orexin neurons and comparing their properties with those of neurons expressing melanin-concentrating hormone, we show that hypocretin–orexin neurons are in an intrinsic state of membrane depolarization that promotes their spontaneous activity. We propose that wakefulness and associated energy expenditure thus depend on that property, which allows the hypocretin–orexin neurons to maintain a tonic excitatory influence on the central arousal and peripheral sympathetic systems.

International audience; The neurons responsible for the onset of sleep are thought to be located in the preoptic area and more specifically, in the ventrolateral preoptic nucleus (VLPO). Here we identify sleep-promoting neurons in vitro and show that they represent an homogeneous population of cells that must be inhibited by systems of arousal during the waking state. We find that two-thirds of the VLPO neurons are multipolar triangular cells that show a low-threshold spike. This proportion matches that of cells active during sleep in the same region. We then show, using single-cell reverse transcriptase followed by polymerase chain reaction, that these neurons probably contain gamma-aminobutyric acid (GABA). We also show that these neurons are inhibited by noradrenaline and acetylcholine, both of which are transmitters of wakefulness. As most of these cells are also inhibited by serotonin but unaffected by histamine, their overall inhibition by transmitters of wakefulness is in agreement with their relative inactivity during waking with respect to sleep. We propose that the reciprocal inhibitory interaction of such VLPO neurons with the noradrenergic, serotoninergic and cholinergic waking systems to which they project is a key factor for promoting sleep.

Using intracellular recordings in guinea pig brain slices, the pharmacology of electrophysiologically identified and immunohistochemically confirmed non-cholinergic nucleus basalis neurons was studied to determine their response to the major neurotransmitters of the subcortical afferents to this region. The cells were differentiated into three types: Type A cells (approximately 44%) were depolarized by noradrenaline (NA) and muscarine, Type B cells (approximately 23%) were depolarized by NA but hyperpolarized by muscarine, and Type C cells (approximately 15%) were hyperpolarized by both agonists. These cell types were also differentially responsive to serotonin (hyperpolarizing B, C) and histamine (depolarizing A, B). Accordingly, the non-cholinergic neurons share certain discharge properties but appear nonetheless to comprise distinct types which respond differentially to the major modulatory neurotransmitters and thus play potentially different roles in cortical modulation across the sleep-wake cycle.

Voltage-dependent inhibition of high voltage-activated (HVA) calcium currents by G-proteins can be transiently relieved (facilitated) by strong depolarizing prepulses. However, with respect to the physiological significance of facilitation, it remains to be established if it can be induced by action potentials (AP) in central neurons. With the use of whole-cell recordings of dissociated cholinergic basal forebrain neurons of the guinea pig, it is shown that the GTPγS-inhibited HVA currents that occur throughN-ethylmaleimide (NEM)-sensitive Gi–Gosubtypes of G-proteins can be facilitated. Furthermore, although different types of HVA channels are present in these neurons, facilitation occurred mostly through disinhibition of the N-type current. On the basis of data indicating that the recovery from facilitation was relatively slow, we tested if more physiological stimuli that crudely mimicked APs (2 msec long depolarizations to 40 mV from a holding of −50 mV) potentially could induce facilitation of HVA currents inhibited by GTPγS and cholinergic agonists. Indeed, evidence is provided that the extent of facilitation is dependent on both the number and frequency of AP-like depolarizations. These results suggest that firing rates and patterns of discharge of neurons could influence their responsiveness to transmitters acting on N-type HVA calcium channels.

To understand the cellular mechanisms underlying behaviours in mammals, the respective contributions of the individual properties characterizing each neuron, as opposed to the properties emerging from the organization of these neurons in functional networks, have to be evaluated. This requires the use, in the same species, of various in vivo and in vitro experimental preparations. The present review is meant to illustrate how such a combined in vivo in vitro approach can be used to investigate the vestibular-related neuronal networks involved in gaze and posture stabilization, together with their plasticity, in the adult guinea-pig. Following first a general introduction on the vestibular system, the second section describes various in vivo experiments aimed at characterizing gaze and posture stabilization in that species. The third and fourth parts of the review deal with the combined in vivo-in vitro investigations undertaken to unravel the physiological and pharmacological properties of vestibulo-ocular and vestibulo-spinal networks, together with their functional implications. In particular, we have tried to use the central vestibular neurons as examples to illustrate how the preparation of isolated whole brain can be used to bridge the gap between the results obtained through in vitro, intracellular recordings on slices and those collected in vivo, in the behaving animal.

The isolated, in vitro whole brain of guinea-pig was used to assess some of the main physiological and pharmacological properties of the vestibulo-ocular pathways in this species. Extracellular and intracellular recordings were obtained from the vestibular, abducens and oculomotor nuclei, as well as from the abducens and oculomotor nerves, while inputs from the vestibular afferents, the visual pathways and the spinal cord were activated. The three main types of medial vestibular nucleus neurons (A, B and B+LTS), previously described on slices, were also identified in the isolated brain. They had similar membrane properties in both preparations. Eighty-five per cent of cells recorded in the vestibular nucleus responded with monosynaptic, excitatory postsynaptic potentials (latency 1.05-1.9 ms) to stimulation of the ipsilateral vestibular nerve, and were thus identified as second-order vestibular neurons. In addition, stimulation of the contralateral vestibular afferents revealed in most cases a disynaptic or trisynaptic, commissural inhibition. Second-order vestibular neurons displayed in the isolated brain a high degree of variability of their spontaneous activity, as in alert guinea-pigs. Type A neurons always exhibited a regular firing, while type B and B+LTS cells could have very irregular patterns of spontaneous discharge. Thus, type A and type B neurons might correspond, respectively, to the tonic and phasic vestibular neurons described in vivo. The regularity of spontaneous discharge was positively correlated with the amplitude of spike after hyperpolarization, and there was a trend for irregular neurons to be excited from ipsilateral vestibular afferents at shorter latencies than regular units. Synaptic activation could trigger subthreshold plateau potentials and low-threshold spikes in some of the second-order vestibular neurons. As a second step, the pharmacology of the synaptic transmission between primary vestibular afferents and second-order neurons was assessed using specific antagonists of the glutamatergic receptors. Both the synaptic field potentials and excitatory postsynaptic potentials elicited in the medial vestibular nucleus by single shock stimulation of the ipsilateral vestibular nerve were largely or, sometimes, totally blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, indicating a dominating role of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated glutamatergic transmission. The remaining component of the responses was completely or partially suppressed by DL-2-amino-5-phosphonovaleric acid in 35% of the cases, suggesting a concomitant, moderate involvement of N-methyl-D-asparate receptors. In addition, a synaptic response resistant to both antagonists, but sensitive to a zero Ca2+/high Mg(2+)-containing solution, was often observed. Finally, recordings from abducens and oculomotor complexes confirmed the existence in the guinea-pig of strong bilateral, disynaptic excitatory and inhibitory inputs from vestibular afferents to motoneurons of extraocular muscles, which contribute to generation of the vestibulo-ocular reflex. The functional integrity of vestibular-related pathways in the isolated brain was additionally checked by stimulation of the spinal cord and optic tract. Stimulation of the spinal cord evoked, in addition to antidromic responses in the vestibular nucleus, short-latency synaptic responses in both the vestibular nucleus and abducens motoneurons, suggesting possible recruitment of spinal afferents. Activation of visual pathways at the level of the optic chiasm often induced long latency responses in the various structures under study. These results demonstrate that the in vitro isolated brain can be readily used for detailed, functional studies of the neuronal networks underlying gaze and posture control.

Cloning and pharmacological studies have shown that glutamatergic receptors can be divided in two classes (refer to Table 1): ionotropic receptors including N-methyl-D-aspartate (NMDA) and non-NMDA subtypes, and the G-protein-coupled metabotropic receptors (glutamate metabotropic receptor). There are two types of non-NMDA receptors: the AMPA/low-affinity kainate receptor type (the AMPA receptors) activated by a specific agonist, the alpha-amino-3-hydroxy-5-methyl-4-iso-xalone propionate (AMPA), and the high affinity kainate receptors. The vestibular nuclei neurones are endowed with all these types of glutamatergic receptors, which fits well with the fact that various afferents, including the primary vestibular afferents, most probably use glutamate or aspartate as a neurotransmitter. This article is aimed at summarising several past studies of our group and some more recent data obtained in the in vitro whole-brain preparation concerning the NMDA receptors of the central vestibular neurones. In that process, we will detail also many valuable studies of other groups that had been devoted to the same topic.