Mauro Serafin

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.

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.

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).

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.

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.

Cholinergic nucleus basalis neurons display the capacity for rhythmic bursting activity mediated by low-threshold calcium spikes

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 Meynerts 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.

Central vestibular networks in the guinea-pig: functional characterization in the isolated whole brain in vitro

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.

Nicotinic enhancement of the noradrenergic inhibition of sleep-promoting neurons in the ventrolateral preoptic area.

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.

Distinct contributions of high- and low-voltage-activated calcium currents to afterhyperpolarizations in cholinergic nucleus basalis neurons of the guinea pig.

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 μm were 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.

The Wake-Promoting Hypocretin/Orexin Neurons Change Their Response to Noradrenaline after Sleep Deprivation

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 phenomenon in vitro by 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.