Hypocretin/Orexin neuropeptides: participation in the control of sleep-wakefulness cycle and energy homeostasis.

Hypocretins or orexins (Hcrt/Orx) are hypothalamic neuropeptides that are synthesized by neurons located mainly in the perifornical area of the posterolateral hypothalamus. These hypothalamic neurons are the origin of an extensive and divergent projection system innervating numerous structures of the central nervous system. In recent years it has become clear that these neuropeptides are involved in the regulation of many organic functions, such as feeding, thermoregulation and neuroendocrine and cardiovascular control, as well as in the control of the sleep-wakefulness cycle. In this respect, Hcrt/Orx activate two subtypes of G protein-coupled receptors (Hcrt/Orx1R and Hcrt/Orx2R) that show a partly segregated and prominent distribution in neural structures involved in sleep-wakefulness regulation. Wakefulness-enhancing and/or sleep-suppressing actions of Hcrt/Orx have been reported in specific areas of the brainstem. Moreover, presently there are animal models of human narcolepsy consisting in modifications of Hcrt/Orx receptors or absence of these peptides. This strongly suggests that narcolepsy is the direct consequence of a hypofunction of the Hcrt/Orx system, which is most likely due to Hcrt/Orx neurons degeneration.The main focus of this review is to update and illustrate the available data on the actions of Hcrt/Orx neuropeptides with special interest in their participation in the control of the sleep-wakefulness cycle and the regulation of energy homeostasis. Current pharmacological treatment of narcolepsy is also discussed.

Relationship between the perifornical hypothalamic area and oral pontine reticular nucleus in the rat. Possible implication of the hypocretinergic projection in the control of rapid eye movement sleep.

The perifornical (PeF) area in the posterior lateral hypothalamus has been implicated in several physiological functions including the regulation of sleep-wakefulness. Some PeF neurons, which contain hypocretin, have been suggested to play an important role in sleep-wake regulation. The aim of the present study was to examine the effect of the PeF area and hypocretin on the electrophysiological activity of neurons of the oral pontine reticular nucleus (PnO), which is an important structure in the generation and maintenance of rapid eye movement sleep. PnO neurons were recorded in urethane-anesthetized rats. Extracellular recordings were performed by means of tungsten microelectrodes or barrel micropipettes. Electrical stimulation of the ipsilateral PeF area elicited orthodromic responses in both type I (49%) and type II (58%) electrophysiologically characterized PnO neurons, with a mean latency of 13.0 +/- 2 and 8.3 +/- 5 ms, respectively. In six cases, antidromic spikes were evoked in type I PnO neurons with a mean latency of 3.2 +/- 0.4 ms, indicating the existence of PnO neurons that projected to the PeF area. Anatomical studies showed retrogradely labeled neurons in the PeF area from the PnO. Some of these neurons projecting to the PnO contained hypocretin (17.8%). Iontophoretic application of hypocretin-1 through a barrel micropipette in the PnO induced an inhibition, which was blocked by a previous iontophoretic application of bicuculline, indicating that the inhibitory action of hypocretin-1 may be due to activation of GABA(A) receptors. These data suggest that the PeF area may control the generation of rapid eye movement sleep through a hypocretinergic projection by inhibiting the activity of PnO neurons.

Firing activity and postsynaptic properties of morphologically identified neurons of ventral oral pontine reticular nucleus.

The ventral part of the oral pontine reticular nucleus (vRPO) is an important region for the generation and maintenance of REM sleep. Firing activity and synaptic response properties of morphologically identified vRPO neurons have been investigated in urethane-anaesthetized cats. Extracellular recordings were performed through recording micropipettes and neurons were extracellularly stained with biocytin. Two types of neurons were identified under spontaneous conditions: type I neurons (77%) are characterized by non-rhythmic firing; type II neurons (23%) display single spikes firing rhythmically at between 7 and 22 Hz. Type I neurons displayed ellipsoid somata with thick dendritic trunks and axons that arose from either the soma or the initial dendritic segment; these axons could not be clearly followed. Type II neurons showed polygonal somata with radial dendrites; their axons branched at a small distance from the soma. Electrical stimulation of the contralateral vRPO elicited responses in both neuron types (57% and 31%, respectively); this effect was blocked by the non-NMDA glutamatergic receptor antagonist CNQX. Electrical stimulation of the PpT evoked orthodromic responses in type I neurons (41%) and inhibited the firing rate of all type II neurons for 50-100 ms. Both effects were blocked by the muscarinic receptor antagonist atropine. The cholinergic agonist, carbachol, increased the firing rate in most type I neurons and inhibited most type II neurons in these animals. The results demonstrated that the activity of vRPO neurons is modulated through the postsynaptic activation exerted by extrinsic afferents on cholinergic and glutamatergic receptors.

Serotonergic connections to the ventral oral pontine reticular nucleus: implication in paradoxical sleep modulation.

Cholinergic microstimulation of the ventral part of the oral pontine reticular nucleus (vRPO) in cats generates and maintains paradoxical sleep. The implication of rostral raphe nuclei in modulating the sleep-wakefulness cycle has been based on their serotonergic projections to the pontine structures responsible for the induction of paradoxical sleep. However, serotonergic neurons have also been described in brainstem structures other than the raphe nuclei. The aim of the present work is to trace the origin of the serotonergic afferents to the vRPO and to the locus coeruleus alpha and perilocus coeruleu alpha nuclei, closely related with different paradoxical sleep events. Anterograde and retrograde horseradish peroxidase conjugated with wheat germ agglutinin tracer injections in these nuclei in cats were combined with serotonin antiserum immunohistochemistry. Our results demonstrate that reciprocal connections linking the rostral raphe nuclei to those oral pontine nuclei are scarce. The percentage of double-labeled neurons after injections in the vRPO averaged 18% in rostral raphe nuclei, while a level of 82% was estimated in mesopontine tegmentum structures other than the raphe nuclei. These results showed that the main source of serotonin to the vRPO, implicated in generation and maintenance of paradoxical sleep, arises from these mesopontine tegmentum structures. This indicates that the serotonin modulation of paradoxical sleep could be the result of activation in non-raphe mesopontine tegmentum structures. The existence of a complicated network in the vRPO, which maintains a balance between different neurotransmitters responsible for the generation and alternance of paradoxical sleep episodes, is discussed.

Electrophysiological properties and cholinergic responses of rat ventral oral pontine reticular neurons in vitro.

In order to characterize the electrophysiological properties of morphologically identified neurons of the ventral part of the oral pontine reticular (vRPO) nucleus and the effects of cholinergic agonists on them, intracellular recordings were obtained from 45 cells in a rat brain-slice preparation. Intracellular staining was performed with 2% biocytin in potassium acetate (1 M)-filled micropipettes. Results demonstrated the presence of two types of vRPO neurons. Type I cells (n = 12, 24%) were characterized by a break with a decrease of the depolarizing slope following hyperpolarizing pulses which delayed the return to the resting Vm and subsequent spike-firing. The delay was antagonized by 4-AP (200-500 microM) which specifically blocks the transient outward K+-mediated current I(A). Type II neurons (n = 38, 76%) displayed a typical depolarizing sag during hyperpolarizing current pulses which was blocked by Cs+. This behavior is characteristic of the hyperpolarization-activated current I(Q). These two neuronal types displayed different morphological features. Most type I and II cells (100 and 73.7%, respectively) were depolarized by acetylcholine (1-15 microM), carbachol (0.5-1 microM) and muscarine (1-10 microM) through the activation of post-synaptic muscarinic receptors. The remaining type II cells (26.3%) were hyperpolarized (1-10 min, 3-15 mV) through the activation of post-synaptic muscarinic receptors. Results are consistent with the hypothesis that the vRPO could be a neuronal target of Cch in eliciting paradoxical sleep because most of its neurons are activated by muscarinic agonists.

Afferent connections of the lateralis medialis thalamic nucleus in the cat.

We have used anterograde and retrograde horseradish peroxidase tracing methods in this study. Peroxidase injections in the lateralis medialis thalamic nucleus (LM) of the cat resulted in labeled neurons in cortical and subcortical structures that averaged 71% and 29%, respectively. Every LM sector receives abundant projections from the polymodal sylvian anterior cortical area, the reticular thalamic nucleus, and the stratum opticum and intermediate layer of the superior colliculus. Less abundant but consistent projections were detected in cingular, auditory II, lateral suprasylvian and anterior ectosylvian visual cortices, and cortical area 7. A topographical distribution of afferent connections to different LM sectors arising from other cortical and subcortical structures could be established. The ventromedial sector receives connections mainly from the insular agranular, limic and prefrontal cortical areas, as well as from brain stem structures and the contralateral pretectal region. The dorsolateral sector is mainly related to cortical areas and subcortical structures processing visual information. The existence of overlap among neuronal LM populations receiving and sending connections to and from various cortical areas suggests that this nucleus is an appropriate substrate for effective interaction between different and distant cortical areas.

Location and anatomical connections of a paradoxical sleep induction site in the cat ventral pontine tegmentum.

The brainstem mechanisms for the generation of paradoxical sleep are under considerable debate. Previous experiments in cats have demonstrated that injections of the cholinergic agonist carbachol into the oral pontine tegmentum elicit paradoxical sleep behaviour and its polygraphic correlates. The different results on the pontine structures that mediate this effect do not agree. We report here that limited microinjections of a carbachol solution into the ventral part of the oral pontine reticular nucleus in the cat induce, with a short latency, a dramatic, long-lasting increase in paradoxical sleep. Moreover, neuronal tracing experiments show that this pontine site is connected with brain structures responsible for the different bioelectric events of paradoxical sleep. These two facts suggest that the ventral part of the oral pontine reticular nucleus is a nodal link in the neuronal network underlying paradoxical sleep mechanisms.

[Involvement of pontine tegmentum in wakefulness and sleep states]. / Implicación del tegmento pontino en los estados de vigilia y de sueño.

Some of our personal contributions in the last years on pontine sleep mechanisms are presented. The results after specific pontine lesions and cholinergic microstimulation of brain stem areas are reviewed. These results are discussed in relation to current concepts about the role of the pontine tegmentum in sleep mechanisms.

Connections to the lateral posterior-pulvinar thalamic complex from the reticular and ventral lateral geniculate thalamic nuclei: a topographical study in the cat.

The projections from the reticular thalamic nucleus and the ventral lateral geniculate nucleus to the lateral posterior-pulvinar thalamic complex were studied in the adult cat using the retrograde transport of horseradish peroxidase. Small, stereotaxically guided injections of the enzyme were placed in the various nuclei of this complex, including the pulvinar, lateralis intermedius oralis, lateralis intermedius caudalis, lateralis posterior lateralis, lateralis posterior medialis and lateralis medialis nuclei. The distribution of labeled neurons indicates that these nuclei receive topographically organized projections from the reticular and ventral lateral geniculate nuclei. The pulvinar nucleus receives only very scarce projections from the reticular thalamic nucleus originating in its posterodorsal and posteroventral sectors. The reticular projection to the nucleus lateralis intermedius oralis is even sparser. The nuclei lateralis intermedius caudalis, lateralis posterior lateralis and lateralis posterior medialis receive substantial projections from the suprageniculate sector of the reticular thalamic nucleus. The nucleus lateralis medialis receives an abundant projection from the three sectors (suprageniculate, pregeniculate and infrageniculate) of the reticular thalamic nucleus. Except for the lateralis intermedius caudalis, all nuclei of the lateral posterior-pulvinar complex receive consistent projections from the ventral lateral geniculate nucleus, the nucleus lateralis medialis receiving the densest one. Our findings suggest that visual, auditory, somatosensory, motor and limbic impulses from thalamic nuclei and from primary sensory and association cortical areas modulate the activity of the nucleus lateralis medialis via the reticular thalamic nucleus. The remaining nuclei of the lateral posterior-pulvinar complex are mainly modulated by sectors of the reticular thalamic nucleus that receive afferent connections from visual structures. The intrathalamic projections arising from the ventral lateral geniculate nucleus may be the way through which visuomotor inputs reach the different components of the lateral posterior-pulvinar thalamic complex.

A neuroanatomical gradient in the pontine tegmentum for the cholinoceptive induction of desynchronized sleep signs.

Microinjection of cholinergic agonists into the pontine tegmentum was used to evoke a state which was polygraphically and behaviorally similar to desynchronized (D) sleep. This study was designed to test the hypothesis that the production of this pharmacologically induced D sleep-like state (D-ACh) was dependent upon the anatomical locus of drug administration within the pontine tegmentum. Four dependent variables of D sleep were measured: D latency, D percentage, D duration and D frequency. Multiple regression analysis and analysis of variance were performed to evaluate the relationship between the three-dimensional coordinates of the injection site (posterior, vertical and lateral) and these 4 dependent measures. The intrapontine site of drug administration accounted for a statistically significant amount of the variance in D latency, D percentage and D duration. There was no significant relationship between the anatomical site of saline injection and the dependent measures of D sleep. A significant increase in D frequency following microinjection of cholinergic agonists was found to be independent of injection site. Pontine injection sites which yielded the shortest D latencies were found to be the same sites from which the highest D percentages were evoked. Rostrodorsal pontine tegmental injection sites were most effective in producing the highest percentages of D-ACh with the shortest latencies to onset. Injections made more caudally and ventrally within the pontine tegmentum produced lower percentages of D-ACh with longer latencies to onset. These data suggest the existence of an anatomical gradient within the pontine tegmentum for the cholinoceptive evocation of a D sleep-like state, and further support the concept that D sleep is generated, in part, by pontine cholinergic mechanisms.

Physiological studies of brainstem reticular connectivity. II. Responses of mPRF neurons to stimulation of mesencephalic and contralateral pontine reticular formation.

The connectivity between medial pontine reticular formation (mPRF) and the contralateral mPRF and between mPRF and the mesencephalic reticular formation (MRF) was studied by intracellular recordings of mPRF neuronal responses to microstimulation of the contralateral gigantocellular field (cFTG) portion of mPRF and ipsilateral MRF in unanesthetized, undrugged cats. There was a very high percentage (75-86%) of monosynaptic latency postsynaptic potentials (PSPs) in mPRF neurons in response to microstimulation of cFTG and MRF, and most PSPs (72-82%) were excitatory ones (EPSPs). The initial EPSPs from cFTG stimulation were characterized by a rapid rise time and a relatively constant latency, while those from MRF had a less rapid rise time and a longer plateau; EPSPs from both sites frequently led to spike potential generation. In contrast, the percentage of initial monosynaptic inhibitory PSPs (EPSPs) was less than 4% from each of these regions, statistically significantly less than that from bulbar FTM and bulbar FTG stimulation (about 12%) reported in the companion paper. Injection of depolarizing current in mPRF neurons unmasked hyperpolarizing PSP responses to stimulation that followed initial depolarizing PSPs. Intracellular HRP labeling indicated that these data were from recordings from neurons with 20-100 microns diameters, with 80% greater than 40 microns. Neurons with a different discharge pattern for this area of the pons, a stereotyped burst pattern, were recorded just ventral to mPRF; this discharge pattern resembled that found in inhibitory interneurons in other central nervous system regions. There were no differences in the density and pattern of orthodromic PSPs between those mPRF neurons that were antidromically activated from cFTG and the general population that was not antidromically activated from cFTG or other stimulated sites; this suggests, when combined with data of the companion paper, an identity of input and output elements in mPRF with respect to synaptic response properties. The high degree of connectivity between reticular regions may furnish a substrate for functional interaction.

Cerebellar projections to the lateral posterior-pulvinar thalamic complex in the cat.

Stereotaxic injections of horseradish peroxidase in the subdivisions of the lateral posterior-pulvinar thalamic complex were made in adult cats. Labeled neurons were found in the ventrolateral part of the contralateral lateral cerebellar nucleus. Cerebellar-positive neurons were more abundant after injections situated in the lateralis intermedius oralis, lateralis intermedius caudalis and rostral pulvinar nuclei, while they were scarce or absent when the injections were located in the extra-geniculate visual subdivision of the lateral posterior-pulvinar complex (lateralis posterior lateralis and lateralis posterior medialis nuclei). These projections give an anatomical substrate to support the cerebellar modulation of the lateral posterior-pulvinar complex motor activities as well as its dinamogenic cortical functions.

Microinjection of neostigmine into the pontine reticular formation of cats enhances desynchronized sleep signs.

Microinjection of the acetylcholinesterase inhibitor neostigmine into the dorsal pontine tegmentum of intact, freely moving cats produced significant changes in electrographic desynchronized (D) sleep signs and D sleep-like behavior. The percentage, frequency and duration of D sleep signs were increased and the latency to onset of D sleep signs was significantly reduced after neostigmine administration. The effects of neostigmine were dose-dependent and could be blocked by centrally administered atropine. This is the first demonstration that microinjection of an acetylcholinesterase inhibitor into the pons enhances D sleep signs. These data suggest that endogenously released acetylcholine can initiate and maintain the state of D sleep and strongly support the cholinergic hypothesis of D sleep generation.

Site-specific enhancement and suppression of desynchronized sleep signs following cholinergic stimulation of three brainstem regions.

The behavioral effects of carbachol microinjection into the pontine, midbrain and medullary reticular formation (RF) have been studied. Enhancement of electrographic desynchronized (D) sleep signs and D sleep-like behavior was specific to the pons; these parameters were contrastingly suppressed following cholinergic stimulation of the midbrain and medullary RF. Polygraphically defined wakefulness (W) was increased from the midbrain and medulla and was characterized by stereotyped motor activity, whereas slow wave (S) sleep was reduced or eliminated when carbachol was administered into all 3 brainstem regions. In addition, as a function of the site of carbachol administration within the pons, some qualitative electrographic differences between carbachol-induced D sleep and the physiological D sleep state were observed. These results are consistent with the hypotheses that the pontine RF is involved in the cholinergic initiation and/or maintenance of D sleep and that the midbrain RF mediates arousal via cholinoceptive reticular cells. The hypothesis that the medullary RF plays a major role in the generation of S sleep and the atonia of D sleep is not supported by these data, but non-cholinergic mechanisms of these proposed roles remain to be investigated. Preliminary reports of these data have been published.

Topographical organization of the brainstem afferents to the lateral posterior-pulvinar thalamic complex in the cat.

Following stereotaxic injections of horseradish peroxidase in the dorsal thalamus of the cat which were restricted to the lateralis posterior-pulvinar complex, labelled neurons were found in the superficial layers of the superior colliculus and in the brainstem. The retrogradely-filled cells of the brainstem were situated principally in the nucleus tegmenti pedunculopontinus, the locus coeruleus complex, the parabrachial nuclei and the dorsal tegmental nucleus of Gudden; in each case, labelled cells were more numerous on the ipsilateral side. In addition, some scattered neurons were observed in the central grey matter, the mesencephalic reticular formation, the central superior and dorsal raphe nuclei, the cuneiform nucleus reticularis gigantocellularis, the nucleus praepositus hypoglossi and the oculomotor nuclei. The differential organization of these projections were observed. It is concluded that the rostrointermediate subdivision of the lateralis posterior-pulvinar complex receives most of its connections from the nucleus tegmenti pedunculopontinus, from the deep layers of the superior colliculus and from the other brainstem nuclei, while the caudal subdivision (extrageniculate visual subdivision) receives its main projection from the superficial layers of the superior colliculus. The findings may have functional implications for the role of the complex in oculomotor control.

The reversible effect of colchicine on the taste bud cells of the central circumvallate papilla in the rat.

It is believed that differentiation and maintenance of taste buds in vertebrates is dependent on the trophic function of their sensory nerve supply. In the present work, colchicine was injected into the circumvallate papilla of the rat. This produced a reversible blockade of neuroplasmic transport and disappearance of taste buds. Colchicine inhibited the further differentiation of bud cells, but apparently did not change the life cycle of the cells present already at the time of injection. It is speculated that the neurotrophic factors in this particular cell system are effective to induce cell differentiation only.