Michel Muhlethaler

Identification of sleep-promoting neurons in vitro

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 γ-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.

Elevated Tribbles homolog 2–specific antibody levels in narcolepsy patients

Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness and attacks of muscle atonia triggered by strong emotions (cataplexy). Narcolepsy is caused by hypocretin (orexin) deficiency, paralleled by a dramatic loss in hypothalamic hypocretin-producing neurons. It is believed that narcolepsy is an autoimmune disorder, although definitive proof of this, such as the presence of autoantibodies, is still lacking. We engineered a transgenic mouse model to identify peptides enriched within hypocretin-producing neurons that could serve as potential autoimmune targets. Initial analysis indicated that the transcript encoding Tribbles homolog 2 (Trib2), previously identified as an autoantigen in autoimmune uveitis, was enriched in hypocretin neurons in these mice. ELISA analysis showed that sera from narcolepsy patients with cataplexy had higher Trib2-specific antibody titers compared with either normal controls or patients with idiopathic hypersomnia, multiple sclerosis, or other inflammatory neurological disorders. Trib2-specific antibody titers were highest early after narcolepsy onset, sharply decreased within 2-3 years, and then stabilized at levels substantially higher than that of controls for up to 30 years. High Trib2-specific antibody titers correlated with the severity of cataplexy. Serum of a patient showed specific immunoreactivity with over 86% of hypocretin neurons in the mouse hypothalamus. Thus, we have identified reactive autoantibodies in human narcolepsy, providing evidence that narcolepsy is an autoimmune disorder.

Orexins (hypocretins) directly excite tuberomammillary neurons

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.

The Isolated and Perfused Brain of the Guinea‐pig In Vitro

We describe here an isolated and perfused in vitro adult guinea‐pig whole brain preparation which is an extension of the previously described in vitro brainstem–cerebellum preparation, Viability was tested by the analysis of trans‐synaptic responses along the visual pathways following the electrical stimulation of the optic nerve or the optic radiations. The evoked field potentials were recorded in the dorsal lateral geniculate, the superior colliculus and the visual cortex. The distribution of extracellular currents was studied using current source density analysis, in order to determine the amplitude, time course and spatial organization of the synaptic activity at these sites. The study indicates that field potentials were very similar to those described in vivo. These data demonstrate the survival of a complex adult sensory system in vitro and suggest that this preparation can be used for the analysis of multisynaptic circuits in the mammalian brain.

Medial vestibular nucleus in the guinea-pig

SummaryIntracellular 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 subthreshold 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).

Cholinergic nucleus basalis neurons are excited by histamine in vitro

Considerable evidence has shown that both cholinergic and histaminergic neurons in the brain may act to facilitate processes of cortical activation that occur during wakefulness. In the present study, the potential influence of histaminergic neurons upon cholinergic neurons of the basal forebrain was investigated in guinea-pig basal forebrain slices. We found that electrophysiologically identified and immunohistochemically verified cholinergic neurons of the nucleus basalis were depolarized and excited by histamine, as manifested by an increase in tonic firing. The depolarization was associated with an increase in membrane input resistance. The effect of histamine persisted in the presence of either tetrodotoxin or a high-magnesium/low-calcium solution, indicating that it is postsynaptic. By a process of elimination, the participation in this response of the three described histamine receptors was examined. Involvement of H3 receptors was excluded on the basis that the H3 agonist (R)-alpha-methyl-histamine had no direct effect, and the H3 antagonist, thioperamide, did not block the effect of histamine. In contrast, the presence of a small response to impromidine, a selective agonist of H2 receptors, and the partial block of the response to histamine by the H2 receptor antagonist, cimetidine, indicated the participation of H2 receptors. Finally, the complete elimination of histamines effect occurred when low doses of the H1 antagonist, mepyramine, were added to the H2 antagonist, cimetidine, indicating the involvement and predominance of H1 receptors in the response. Our data thus suggest that histamine excites nucleus basalis cholinergic neurons by a concomitant activation of H1 and H2 receptors. Histaminergic tuberomammillary neurons may accordingly facilitate tonic firing of cholinergic neurons during wakefulness. Cholinergic basalis neurons could thus act in tandem with histaminergic neurons during periods of arousal to collectively promote widespread cortical activation.

Exclusive Postsynaptic Action of Hypocretin-Orexin on Sublayer 6b Cortical Neurons

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-OX2 receptors 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.

Noradrenergic modulation of cholinergic nucleus basalis neurons demonstrated by in vitro pharmacological and immunohistochemical evidence in the guinea-pig brain.

The effects of noradrenalin were tested upon electrophysiologically characterized cholinergic nucleus basalis neurons in guinea‐pig brain slices. According to their previously established intrinsic membrane properties, the cholinergic cells were distinguished by the presence of low‐threshold Ca2+ spikes and transient outward rectification that endowed them with the capacity to fire in low‐threshold bursts in addition to a slow tonic discharge. A subset of the electrophysiologically identified cholinergic cells that responded to noradrenalin had been filled with biocytin (or biotinamide) and documented in previously published reports as choline acetyltransferase (ChAT)‐immunoreactive. The noradrenalin‐responsive, biocytin‐filled/ChAT+ cells were mapped in the present study and shown to be distributed within the substantia innominata amongst a large population of ChAT+ cells. Slices from another subset of noradrenalin‐responsive, electrophysiologically identified cholinergic cells were stained for dopamine‐β‐hydroxylase to visualize the innervation of the biocytin‐filled neurons by noradrenergic fibres. These biocytin‐filled neurons were surrounded by a moderate plexus of varicose noradrenergic fibres and were ostensibly contacted by a small to moderate number of noradrenergic boutons abutting their soma and dendrites. Applied in the bath, noradrenalin produced membrane depolarization and a prolonged tonic spike discharge. This excitatory action was associated with an increase in membrane input resistance, suggesting that it occurred through reduction of a K+ conductance. These effects persisted when synaptic transmission was eliminated (by tetrodotoxin or low Ca2+/high Mg2+) and were therefore clearly postsynaptic. The excitatory effect of noradrenalin was blocked by the α1‐adrenergic receptor antagonist prazosin and not by the α2‐antagonist yohimbine, and it was mimicked by the α1‐agonist L‐phenylephrine but not by the α2‐agonists clonidine and UK14.304, indicating mediation by an α1‐adrenergic receptor. There was also evidence for a contribution by a β‐adrenergic receptor to the effect, since the β‐antagonist propranolol partially attenuated the effect of noradrenalin, and the β‐agonist isoproterenol produced, like noradrenalin, alone or when applied in the presence of the α1‐antagonist prazosin, membrane depolarization and an increase in tonic spike discharge. These results indicate that through a predominant action upon α1‐adrenergic receptors, but with the additional participation of β‐adrenergic receptors, noradrenalin depolarizes and excites cholinergic neurons. This action would tend to drive the cholinergic cells into a tonic mode of firing and to stimulate or increase the rate of repetitive spike discharge for prolonged periods. The noradrenergic locus coeruleus neurons could thereby recruit the cholinergic basalis neurons to act in tandem with them in facilitating cortical activation during wakefulness.

Pharmacological and Immunohistochemical Evidence for Serotonergic Modulation of Cholinergic Nucleus Basalis Neurons

Identified electrophysiologically by low threshold bursts and transient outward rectification, cholinergic nucleus basalis neurons were recorded and labelled intracellularly in guinea‐pig basal forebrain slices. By means of a triple labelling immunofluorescent technique, serotonin‐immunoreactive fibres were visualized in close proximity to the soma and dendrites of the biocytin‐labelled, choline acetyl transferase (ChAT)‐immunoreactive cells. By bath application, 5‐hydroxytryptamine (5‐HT) produced a direct hyperpolarization of the identified cells which was mimicked by 5‐HT1A receptor agonists, suggesting that it may inhibit the tonic firing but also modulate the low threshold bursting of the cholinergic nucleus basalis neurons.

Opposite effects of noradrenaline and acetylcholine upon hypocretin/orexin versus melanin concentrating hormone neurons in rat hypothalamic slices.

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.