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.

Chronic neuropathy with IgM anti-ganglioside antibodies: lack of long term response to rituximab.

Two patients with chronic motor neuropathy, high antiganglioside antibody (AGA) titers, and a declining response to IV immunoglobulins were treated with rituximab at a standard dose. The drug was well tolerated and effectively eliminated peripheral B cells (CD20+), but AGA titers continued significantly high. No clinical improvement was detected during the 1-year follow-up.

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.

Sleep-waking states develop independently in the isolated forebrain and brain stem following early postnatal midbrain transection in cats.

We report the effects of permanently separating the immature forebrain from the brain stem upon sleeping and waking development. Kittens ranging from postnatal 9 to 27 days of age sustained a mesencephalic transection and were maintained for up to 135 days. Prior to postnatal day 40, the electroencephalogram of the isolated forebrain and behavioral sleep-wakefulness of the decerebrate animal showed the immature patterns of normal young kittens. Thereafter, the isolated forebrain showed alternating sleep-wakefulness electrocortical rhythms similar to the corresponding normal patterns of intact, mature cats. Olfactory stimuli generally changed forebrain sleeping into waking activity, and in cats with the section behind the third nerve nuclei, normal correlates of eye movements-pupillary activity with electrocortical rhythms were present. Behind the transection, decerebrate animals showed wakefulness, and after 20 days of age displayed typical behavioral episodes of rapid eye movements sleep and, during these periods, the pontine recordings showed ponto-geniculo-occipital waves, which are markers for this sleep stage, together with muscle atonia and rapid lateral eye movements. Typically, but with remarkable exceptions suggesting humoral interactions, the sleep-waking patterns of the isolated forebrain were dissociated from those of the decerebrate animal. These results were very similar to our previous findings in midbrain-transected adult cats. However, subtle differences suggested greater functional plasticity in the developing versus the adult isolated forebrain. We conclude that behavioral and electroencephalographic patterns of non-rapid eye movement sleep and of rapid eye movement sleep states mature independently in the forebrain and the brain stem, respectively, after these structures are separated early postnatally. In terms of waking, the findings strengthen our concept that in higher mammals the rostral brain can independently support wakefulness/arousal and, hypothetically, perhaps even awareness. Therefore, these basic sleeping-waking functions are intrinsic properties of the forebrain/brain stem and as such can develop autochthonously. These data help our understanding of some normal/borderline sleep-waking dissociations as well as peculiar states of consciousness in long term patients with brain stem lesions.

Cathepsins are upregulated by IFN-gamma/STAT1 in human muscle culture: a possible active factor in dermatomyositis.

The aim of this work was to study which genes upregulated by the IFN-gamma/STAT1 system in human muscle might be involved in the process of muscle fiber atrophy in dermatomyositis (DM). These proteins included proteases (cathepsins B and L, calpain), proteins implicated in apoptosis and cell cycle (Bcl-x(l), Fas, p21), structural proteins (beta-actin, utrophin, desmin), and other proteins whose expression is known to be modified by IFN-gamma (neural cell adhesion molecule (N-CAM), major histocompatibility complex-I (MHC-I)). We performed immunocytochemistry, Western blot, and semiquantitative reverse transcriptase-polymerase chain reaction using human muscle cultures. We found upregulation of cathepsins B and L, bcl-x(l) and p21 while N-CAM, calpain, utrophin, desmin, beta-actin and Fas remained at basal levels. Immunohistochemistry on frozen sections from biopsies of patients with different muscle diseases showed upregulation of cathepsin L and calpain in perifascicular muscle fibers in DM. In view of these results, the increased expression of cathepsins L and B after IFN-gamma stimulation in muscle cultures and its inhibition using fludarabine, a STAT1 blocker, further support our previous studies and suggest that the increased expression of cathepsins detected in perifascicular muscle fibers in DM is mediated by IFN-gamma/STAT1 and contributes to their atrophy.

Centralis superior raphe, reticularis pontis nuclei, and sleep-wakefulness cycle in cats.

This study examines the influence of lesions in the centralis superior raphe nucleus (CeSR) and adjacent paramedial pontine tegmentum on the sleep/wakefulness cycle (SWC) in cats. Sixteen cats had electrodes implanted for electro-oculogram (EOG), electromyogram (EMG), electroencephalogram (EEG) and ponto-geniculo-occipital (PGO) recordings. There were 10 experimental animals: seven animals received diathermocoagulation lesions destroying between 7 and 27% of the CeSR; the remaining three cats suffered bilateral lesions in the paramedial portion of the reticularis pontis oralis (RPO) and caudalis (RPC) nuclei. Six sham-operated animals were used as controls. Recordings were taken of all animals in continuous 23-h sessions once a week for 12 weeks. Results indicated that the threshold for SWC state changes (increase of wakefulness (W) and drowsiness (D), and decrease of slow wave sleep (SWS)) after CeSR lesion is approximately 11.3% following volumetric destruction of the nucleus. The amount of CeSR damage (CeSR-D) only correlated significantly with the amount of W (positive correlation) and SWS (negative correlation) during the first week post-lesion. The changes in W over the course of the study were different in the two experimental groups. In both groups, total W was increased with respect to the controls, however, these increases were observed earlier in the CeSR-D group. The return to near control values in SWC state over days 15-28 of the study does not represent a definitive recovery by the CeSR-D cats. All the SWC states returned to control values by the tenth week in the cats with paramedial reticular pontine damage.

Sleep patterns after carbachol delivery in the ventral oral pontine tegmentum of the cat.

This study examines dose-related effects on sleep produced by low-volume and low-dose carbachol microinjections in the ventral part of the cat nucleus reticularis pontis oralis. Carbachol microinjections (0.04, 0.08, 0.8 or 4 microg; volume 20 nl) in this location triggered paradoxical sleep with a very short dose-unrelated latency. The four carbachol doses effectively generated all the polygraphic and behavioral signs of paradoxical sleep when microinjected at any level within the ventral part of the nucleus reticularis pontis oralis (AP 0.5 to -3.5, L 0.5-3.5, V 3.5-5.0, on the Reinoso-Suárez atlas [Topographischer Hirnatlas der Katze (1961); Merck, Darmstadt]). The dose-related increase of total paradoxical sleep time was due to the increase in both the duration and number of paradoxical sleep episodes. This paradoxical sleep increase was associated with a dose-related decrease in the amount of time spent in both slow wave sleep and drowsiness, but not with any decrease in total wakefulness. The lengthening of the latency to slow wave sleep onset was dose related. These results show that the ventral oral pontine tegmentum is a very sensitive site for the induction and maintenance of paradoxical sleep.

Neocortical and hippocampal electrical activities are similar in spontaneous and cholinergic-induced REM sleep.

Neocortical and hippocampal EEG power spectra obtained during REM-like sleep induced by unilateral carbachol microinjections (0.01 M, 0.02 M and 0.2 M; volume 20 nl) into the ventral part of the nucleus reticularis pontis oralis have been compared with EEG power spectra obtained during spontaneous REM sleep. Our findings indicate that neocortical and hippocampal electrical activities during the REM-like state generated by carbachol delivery in this pontine region mimic those present in spontaneous REM sleep.

Opiate microinjections in the locus coeruleus area of the cat enhance slow wave sleep.

The effects on sleep/wakefulness states of morphine, morphiceptin (specific mu agonist), DPDPE (delta agonist) and U-50,488H (kappa agonist) microinjections in the Locus coeruleus area (LC) were studied in cats. Morphine (0.8-1.75 nmols in 50 nl of saline) and morphiceptin (1.75 nmols) in LC significantly increased the total time spent in slow wave sleep (SWS) and the mean duration of SWS episodes. Prior naloxone administration blocked the morphine hypnogenic effects. The total time spent in SWS was unaffected by delivery of equimolar doses of DPDPE or U-50,488H in LC; however, the mean duration of the SWS episodes increased significantly after U-50,488H microinjections in LC. Thus, when acting in the LC, opiates have a SWS-enhancing effect and this effect appears to be mediated by mu receptors, although kappa receptors may have a subsidiary action.

Effects of opioid microinjections in the nucleus of the solitary tract on the sleep-wakefulness cycle states in cats.

BACKGROUND: Previous studies have shown that the region of the nucleus of the solitary tract (NST) is involved in the control of electrocortical activity and in sleep mechanisms. It also is well known that this region contains the highest concentration of opioid receptors within the medullary brainstem. The involvement of the NST opioid system in sleep-wakefulness states were evaluated. METHODS: Ten cats were implanted with electrodes for chronic polygraphic recordings of their sleep-wakefulness states and provided with an implanted guide cannula stereotaxically aimed at the NST region. Microinjections of saline, morphine sulfate, morphiceptin (specific mu agonist), D-pen-2-D-pen-5-enkephalin (delta agonist), and U-50488H (kappa agonist) were given to the freely moving animals (doses 0.8-2.4 x 10(-9) M, in a volume of 0.05 microliters of saline). After microinjections, sleep-wakefulness recordings were obtained for 8 h. RESULTS: Morphine microinjections in NST provoked a dose-dependent enhancement of all the polygraphic and behavioral manifestations of slow wave sleep. This effect was blocked by the prior intraperitoneal administration of naloxone. The mu and delta agonists also produced a hypnotic effect by enhancing slow wave sleep. By contrast, the kappa agonist caused no changes in sleep-wakefulness states. CONCLUSIONS: These results indicate that endogenous opioids could be involved in controlling electrocortical activity generated by NST and that activation of mu and delta NST opioid receptors enhanced the electroencephalographic synchronization associated with behavioral slow wave sleep in cats.

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.

Re-examination of the effects of raphe lesions on the sleep/wakefulness cycle states in cats.

To study the specific effects of central superior raphe nucleus (CeSR) lesions on the different sleep/wakefulness cycle states of the cat, nine animals with implanted electrodes for EOG, EMG and EEG recordings were used. Seven cats received diathermocoagulation lesions that destroyed between 13 and 100 percent of the CeSR; the remaining two cats, which suffered lesions in the paramedial region of the oral pontine reticular nucleus (RPO), were used to determine the effects on sleep/wakefulness states caused by damage to adjacent CeSR structures and/or passage fibres. Three prelesion and five postlesion weekly 24 h recordings were obtained from each cat. Recordings were scored according to the polygraphic criteria for wakefulness (W), drowsiness (D), slow wave sleep (SWS) and paradoxical sleep (PS). Results indicated that insomnia is not produced exclusively by CeSR lesions, since adjacent paramedial RPO lesions also decrease both SWS and PS; however, increased W occurred after the former while increased D occurred after the latter. Correlation coefficient analyses showed that W is the only state that correlates significantly with the volume of CeSR destroyed. The following correlations between different states of the sleep/wakefulness cycle were, however, significant: W-D, W-SWS and SWS-PS. Disinhibition of W, therefore, and not sleep loss seems to be the primary effect of CeSR lesions. Thus, the CeSR nucleus appears to be involved in arousal mechanisms rather than in direct sleep promotion.

Morphine effects in brainstem-transected cats: I. EEG and 'sleep-wakefulness' in the isolated forebrain.

In order to examine the prosencephalic mechanisms that might sustain the effects of opiates on EEG and sleep-wakefulness, the actions of morphine sulfate on the EEG and the pupil size were examined in the chronically isolated forebrain of brainstem-transected cats. Single morphine doses (0.5, 2.0 or 3.0 mg/kg, i.p.) administered to these animals produced a long-lasting EEG desynchronization in the isolated forebrain which was associated with pupil mydriasis. The specificity of these morphine effects was shown by the fact that naloxone blocks both the EEG and pupillary effects of the drug. After morphine, spontaneous synchronized EEG with delta waves normally seen in the isolated forebrain preparation was suppressed for 6-18 h, followed by a strong rebound. Both the suppression and rebound in synchronization with delta waves occurred in a dose-dependent manner. The duration of these effects closely paralleled previously reported morphine effects on non-rapid eye movement (NREM) sleep in intact cats. Therefore, in relation to the effects of morphine on EEG and sleep-wakefulness in intact animals, this study suggests that: (1) Morphine suppression of NREM sleep and the subsequent arousal state of the animal are mediated by prosencephalic structures; (2) the generation of the typical neocortical EEG slow burst activity produced by opiates depends on lower brainstem structures.

Morphine effects in brainstem-transected cats: II. Behavior and sleep of the decerebrate cat.

Previous studies have shown that opiates suppress both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Furthermore, during the induced insomnia period, characteristic species-specific behaviors occur which are associated with high voltage slow waves in the EEG. This paper investigates the lower brainstem mechanisms involved in the generation of these effects, and describes the action of single morphine doses (0.5, 2.0 or 3.0 mg/kg, i.p.) on the behavior and REM sleep of chronic decerebrate cats. The effects of morphine in the decerebrate cat followed a 3-stage time course similar to that seen in intact cats: (1) autonomic manifestations (3-8 min postdrug); (2) a quiet state (10-60 min postdrug) with behavioral signs of NREM; and (3) a state of activated behavior (1-6 h postdrug), including motor activity and variations in muscle tone. The decerebrate cats also showed a dose-dependent suppression of REM sleep. The present results indicate that: (1) the lower brainstem provides the basic mechanisms for the behavioral deactivation-activation and the autonomic effects of the drug; (2) hypnogenic and synchronizing influences arising from the brainstem might induce the high voltage, slow burst EEG produced by opiates; (3) REM sleep suppression originates only partially in the lower brainstem; (4) the subsidiary action of the prosencephalon seems to be required for the full expression of the drug's effect on behavior and the EEG.

Relationships of nucleus reticularis pontis oralis neuronal discharge with sensory and carbachol evoked hippocampal theta rhythm.

The activity of 72 neurons recorded in the reticularis pontis oralis nucleus (RPO) was examined in anesthetized and curarized rats during hippocampal theta (theta) rhythm elicited by either sensory stimulation or carbachol microinjections. During hippocampal theta rhythm evoked by sensory stimulation, 63.9% of RPO neurons increased their discharge rate while the firing rate decreased in 20.8%. In all cases, the RPO neurons maintained a non-rhythmic discharge pattern. In 44% of the neurons the discharges tended to occur on the positive wave of the theta rhythm. Similar firing patterns were seen in 18 RPO neurons recorded during theta rhythm elicited by both, sensory stimulation and a carbachol microinjection; this effect was blocked by atropine. These results indicate that the RPO region contributes to the generation of hippocampal theta rhythm with a tonic and nonrhythmic outflow through a cholinergic system which may be muscarinic.

Chronic morphine administration in cats: effects on sleep and EEG.

Sleep-wakefulness and EEG responses to a chronic morphine treatment (2 mg/kg/day, IP, during 15 days) were studied in 8 cats provided with electrodes for EEG, EMG and EOG records. Results indicated that, in contrast to a resistance of the cats to exhibit overt signs of tolerance in the immediate behavioral and EEG responses to morphine, tolerance developed in sleep since: 1) there was a reduction in its onset latency after the initial insomnia period; 2) despite that the initial insomnia period was present throughout the treatment, compared to the effects of the first MS day, the total amount of both NREM sleep and REM sleep significantly increased in subsequent drug days, the total amount of REM sleep reached similar placebo values from day 5; 3) the restoration in the total amount of both sleep states was due to significant increases that occurred from day 5 after the first 6 hours of the MS injection. During the 19-24 hours after MS injections, increases of NREM and REM also resulted statistically significant compared to placebo values. A biphasic depressed and aroused response occurred during early withdrawal. REM sleep rebound was present after MS discontinuation and in the following week. Similarities with effects of opiate chronic administration in other species are discussed. These results support the potential use of the cat for the study of neural mechanisms involved in sleep chronic effects of opiates.

[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.

Unilateral lesions in locus coeruleus area enhance paradoxical sleep.

To clarify the effect of locus coeruleus (LC) lesions in sleep mechanisms, modifications in the amount of wakefulness (W), drowsiness (D), slow sleep (SS) and paradoxical sleep (PS) were examined in 9 cats with unilateral lesions in the dorso-lateral pontine tegmentum and in 3 sham-operated controls. In 4 animals the LC area was unilaterally destroyed--affecting structures that have previously been proposed as PGO-off elements--while the remaining 5 cats had lesions situated close to but sparing the LC area. Analysis of variance among baseline values of all cats, 'postlesion' values of the sham-operated controls and the postlesion values of the 2 groups of operated animals, indicated that the variations of W, D and SS among the different groups were not statistically significant. Only variations in the time spent in PS reached statistically significant values. Individual comparisons between PS values of the 4 groups showed that only an increase of PS in the animals with lesions in the LC area was statistically significant, in comparison with the PS values of the remaining groups. These results indicate that the dorso-lateral pontine area, which is considered to exert a tonic inhibitory influence in the generation of the phasic activity during PS, also mediates in the sleep-wakefulness cycle as an inhibitory region for controlling proportions of PS.