The neuronal network responsible for paradoxical sleep (PS) onset and maintenance has not previously been identified in the rat, unlike the cat. To fill this gap, this study has developed a new technique involving the recording of sleep-wake states in unanaesthetized head-restrained rats whilst locally administering pharmacological agents by microiontophoresis from glass multibarrel micropipettes, into the dorsal pontine tegmentum and combining this with functional neuroanatomy. Pharmacological agents used for iontophoretic administration included carbachol, kainic acid, bicuculline and gabazine. The injection sites and their efferents were then identified by injections of anterograde (phaseolus vulgaris leucoagglutinin) or retrograde (cholera toxin B subunit) tracers through an adjacent barrel of the micropipette assembly and by C-Fos immunostaining. Bicuculline, gabazine and kainic acid ejections specifically into the pontine sublaterodorsal nucleus (SLD) induced within a few minutes a PS-like state characterized by a continuous muscle atonia, low voltage EEG and a lack of reaction to stimuli. In contrast, carbachol ejections into the SLD induced wakefulness. In PHA-L, glycine and C-Fos multiple double-labelling experiments, anterogradely labelled fibres originating from the SLD were seen apposed on glycine and C-Fos positive neurons (labelled after 90 min of pharmacologically induced PS-like state) from the ventral gigantocellular and parvicellular reticular nuclei. Altogether, these data indicate that the SLD nuclei contain a population of neurons playing a crucial role in PS onset and maintenance. Furthermore, they suggest that GABAergic disinhibition and glutamate excitation of these neurons might also play a crucial role in the onset of PS.

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.

Paradoxical sleep (PS) is a state characterized by cortical activation, rapid eye movements and muscle atonia. Fifty years after its discovery, the neuronal network responsible for the genesis of PS has been only partially identified. We recently proposed that GABAergic neurons would have a pivotal role in that network. To localize these GABAergic neurons, we combined immunohistochemical detection of Fos with non-radioactive in situ hybridization of GAD67 mRNA (GABA synthesis enzyme) in control rats, rats deprived of PS for 72 h and rats allowed to recover after such deprivation. Here we show that GABAergic neurons gating PS (PS-off neurons) are principally located in the ventrolateral periaqueductal gray (vlPAG) and the dorsal part of the deep mesencephalic reticular nucleus immediately ventral to it (dDpMe). Furthermore, iontophoretic application of muscimol for 20 min in this area in head-restrained rats induced a strong and significant increase in PS quantities compared to saline. In addition, we found a large number of GABAergic PS-on neurons in the vlPAG/dDPMe region and the medullary reticular nuclei known to generate muscle atonia during PS. Finally, we showed that PS-on neurons triggering PS localized in the SLD are not GABAergic. Altogether, our results indicate that multiple populations of PS-on GABAergic neurons are distributed in the brainstem while only one population of PS-off GABAergic neurons localized in the vlPAG/dDpMe region exist. From these results, we propose a revised model for PS control in which GABAergic PS-on and PS-off neurons localized in the vlPAG/dDPMe region play leading roles.

Extracellular electrophysiological recordings in freely moving cats have shown that serotonergic neurons from the dorsal raphe nucleus (DRN) fire tonically during wakefulness, decrease their activity during slow wave sleep (SWS), and are nearly quiescent during paradoxical sleep (PS). The mechanisms at the origin of the modulation of activity of these neurons are still unknown. Here, we show in the unanesthetized rat that the iontophoretic application of the GABA(A) antagonist bicuculline on dorsal raphe serotonergic neurons induces a tonic discharge during SWS and PS and an increase of discharge rate during quiet waking. These data strongly suggest that an increase of a GABAergic inhibitory tone present during wakefulness is responsible for the decrease of activity of the dorsal raphe serotonergic cells during slow wave and paradoxical sleep. In addition, by combining retrograde tracing with cholera toxin B subunit and glutamic acid decarboxylase immunohistochemistry, we demonstrate that the GABAergic innervation of the dorsal raphe nucleus arises from multiple distant sources and not only from interneurons as classically accepted. Among these afferents, GABAergic neurons located in the lateral preoptic area and the pontine ventral periaqueductal gray including the DRN itself could be responsible for the reduction of activity of the serotonergic neurons of the dorsal raphe nucleus during slow wave and paradoxical sleep, respectively.

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We recently determined in rats that iontophoretic application of bicuculline or gabazine [two GABAa antagonists] and kainic acid (a glutamate agonist) in the sublaterodorsal nucleus (SLD) induces with a very short latency a paradoxical sleep-like state. From these results, we proposed that GABAergic and glutamatergic inputs to the SLD paradoxical sleep (PS)-executive neurons gate the onset of PS [R. Boissard et al. (2002) Eur. J. Neurosci., 16, 1959-1973]. We therefore decided to determine the origin of the GABAergic and non-GABAergic inputs to the SLD combining ejection of a retrograde tracer [cholera-toxin B subunit (CTb)] with glutamate decarboxylase (GAD) immunohistochemistry. The presence of GAD-immunoreactive neurons in the SLD was confirmed. Then, following CTb ejections centred on the SLD, combined with GAD and CTb immunohistochemistry, double-labelled cells were observed in the mesencephalic and pontine reticular nuclei and to a lesser extent the parvicellular reticular nucleus. A large number of GAD-negative retrogradely labelled cells was also seen in these structures as well as in the primary motor area of the frontal cortex, the central nucleus of the amygdala, the ventral and lateral bed nucleus of the stria terminalis, the lateral hypothalamic area, the lateral and ventrolateral periaqueductal grey and the lateral paragigantocellular reticular nucleus. From these results, we propose that the activation of PS-executive neurons from the SLD is due to the removal of a tonic inhibition from GABAergic neurons localized in the SLD, and the mesencephalic and pontine reticular nuclei. Strong non-GABAergic inputs to the SLD could be excitatory and responsible for the tonic glutamatergic input on the PS-on neurons we have previously described. They could also terminate on SLD GABAergic interneurons and be indirectly responsible for the inhibition of the PS-on neurons during waking and slow-wave sleep.

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