Benzodiazepine binding sites and their modulators in hippocampus of violent suicide victims.

Benzodiazepine binding sites were studied by autoradiography in several hippocampic layers in brains of drug-free violent suicide victims (hanging) and matched controls. Kd was increased in suicides, and when brain sections from control subjects were incubated in the bath fluid that had previously served to incubate sections from suicides, Kd was increased in the same way. Results are discussed in terms of possible modulators of benzodiazepine binding sites, mainly tissue GABA and amino acid concentrations.

A key role for the caudoventral pontine tegmentum in the simultaneous generation of eye saccades in bursts and associated ponto-geniculo-occipital waves during paradoxical sleep in the cat.

Ponto-geniculo-occipital waves and rapid eye movements (eye saccades) are two prominent phasic events of paradoxical sleep which occur in conjunction. Although they have been studied intensively, the neuronal link between these two events is still poorly understood. On the basis of our previous results, combining brainstem transections and carbachol microinjections, we postulated that the oculomotor and ponto-geniculo-occipital systems do not work in series, but in parallel, and that the caudoventral pontine tegmentum might represent a structure controlling and/or co-ordinating the simultaneous production of the two phenomena. This hypothesis was further supported by the demonstration that, during paradoxical sleep, the instantaneous velocity of eye saccades in bursts is higher than that of isolated ones which, in turn, are more rapid than waking saccades. This acceleration of eye saccades in bursts also seems to be under the cholinergic control of the caudoventral pontine tegmentum. In order to test the hypothesis that this area may be a prime mover leading to the simultaneous appearance of these two phasic events as a whole, we investigated, in the present study, the effects of pharmacological stimulation (with carbachol) and inhibition (with atropine) of the caudoventral pontine tegmentum on the production and the characteristics of eye saccades and ponto-geniculo-occipital waves. Cats' eye movements were recorded using the technique of the scleral search coil in a magnetic field, together with sleep-waking parameters. We found that: (i) unilateral microinjections of carbachol (0.4 microg) induced, during waking, a majority of long bursts of ponto-geniculo-occipital waves (i.e. bursts containing at least five waves) which had intra-burst intervals similar to natural ones (48-259 ms) and decreased the frequency of isolated ponto-geniculo-occipital waves; (ii) unilateral microinjections of atropine (2.4 microg) strongly decreased, during paradoxical sleep, the frequency (number/min) of eye saccades in bursts directed contralaterally to the side of the injection (by 48-54%) and reduced the velocity of these saccades to that of isolated eye saccades. Atropine also significantly reduced the frequency (by 60%) of all types of bursts of ponto-geniculo-occipital waves, with a maximal effect (80% reduction) on long bursts of ponto-geniculo-occipital waves, while it increased the frequency of isolated ponto-geniculo-occipital waves. However, atropine did not change the value of intra-burst intervals. These findings support the hypothesis that eye saccades in bursts and associated ponto-geniculo-occipital waves are generated as a whole by a common structure and that this structure is at least partly defined by the caudoventral pontine tegmentum.

Effects of an acute immobilization stress upon proopiomelanocortin (POMC) mRNA levels in the mediobasal hypothalamus: a quantitative in situ hybridization study.

The aim of this study was to examine by quantitative in situ hybridization the effects of an acute stress on the expression of the POMC gene in the mediobasal hypothalamus (MBH) of the rat. In control animals, the highest levels of POMC mRNA were observed in the posterior periventricular region of the MBH. Lower levels were found in the anterior and posterior arcuate nucleus. At the end of a one hour immobilization, a small decrease (-8%) was observed in the periventricular region only. Four hours after the end of immobilization, increases in POMC mRNA levels were detected in the anterior part (7%), in the posterior part (25%) and in the periventricular region (13%) of the MBH. These results suggest that MBH POMC-derived peptides might be an important component in the central response to stress.

Neuron-specific enolase as a marker of neuronal lesions during various comas in man.

The detection of neuron-specific enolase in biological fluids has been investigated as an indirect marker of neuronal damage in man. This protein was measured by a sandwich enzymoimmunoassay in serums and cerebrospinal fluids from patients with consciousness disorders of various aetiologies. Neuron-specific enolase level was significantly increased in sera from patients with comas resulting from anoxemia, head injury, septic state, cirrhosis and fulminant hepatitis. On the other hand, patients with meningitis (affection not normally accompanied with neuronal lesion) exhibited no change of this marker level. The statistical analysis of our results suggests that, in neurological disorders, the neuron-specific enolase levels in cerebrospinal fluid could have some prognostic value. The correlation between its level in cerebrospinal fluid and in serum was also demonstrated. Neuron-specific enolase increase in biological fluids thus represents a useful and promising marker to biochemically characterize various strokes possibly resulting in neuronal damage.

Waking selective neurons in the posterior hypothalamus and their response to histamine H3-receptor ligands: an electrophysiological study in freely moving cats.

Neurons which discharge selectively during waking (waking selective) have been found in the tuberomamillary nucleus (TM) and adjacent areas of the posterior hypothalamus. Although they share some electrophysiological properties with aminergic neurons, there is no direct evidence that they are histaminergic. We have recorded from posterior hypothalamic neurons during the sleep-wake cycle in freely moving cats, and investigated the effects on waking selective neurons of specific ligands of histaminergic H3-receptors, which autoregulate the activity of histaminergic neurons. Two types of neurons were seen. Waking selective neurons, termed "waking-on (W-on)," were located exclusively within the TM and adjacent areas, and discharged at a low regular rate during waking (1.71-2.97 Hz), decreased firing during light slow wave sleep (SWS), became silent during deep SWS and paradoxical sleep (PS) and resumed their activity on, or a few seconds before, awakening. "Waking-related" neurons, located in an area dorsal to the TM, displayed a similar, although less regular, low rate of firing (1.74-5.41 Hz) and a similar discharge profile during the sleep-wake cycle; however, unlike "W-on" neurons, they did not completely stop firing during deep SWS and PS. Intramuscular (i.m.) injection of ciproxifan (an H3-receptor antagonist, 1mg/kg), significantly increased the discharge rate of W-on neurons and induced c-fos expression in histamine-immunoreactive neurons, whereas i.m. injection of imetit (an H3-receptor agonist, 1mg/kg) or microinjection of alpha-methylhistamine (another H3-receptor agonist, 0.025-0.1 microg/0.2 microl) in the vicinity of these cells significantly decreased their discharge rate. Moreover, the effect of the antagonist was reversed by the agonists and vice versa. In contrast, "waking-related" neurons were unaffected by these H3-receptor ligands. These data provide evidence for the histaminergic nature of "W-on" neurons and their role in cortical desynchronization during waking, and highlight the heterogeneity of posterior hypothalamic neuronal populations, which might serve different functions during the wakefulness.

Role of the locus coeruleus in the sleep rebound following two different sleep deprivation methods in the rat.

The aim of the present study was to assess the involvement of the locus coeruleus in the paradoxical sleep rebound following sleep deprivation in the rat. Animals were sleep-deprived for 10 h before, and after, specific N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) lesioning of the noradrenergic-locus coeruleus system. Sleep deprivation was produced using either an instrumental (water tank) or pharmacological (methylamphetamine) method. After lesioning, the rats submitted to the instrumental method showed a significant decrease in the paradoxical and slow-wave sleep rebounds (-54% and -78%, respectively), while animals receiving metamphetamine did not. Our results suggest that the noradrenergic system of the locus coeruleus is a relevant component of the sleep rebound mechanisms. However, the extent of involvement is dependent on the sleep deprivation method used.

Influence of a 1 h immobilization stress on sleep states and corticotropin-like intermediate lobe peptide (CLIP or ACTH18-39, Ph-ACTH18-39) brain contents in the rat.

A 1 h immobilization stress (IS) was imposed to rats at the beginning of the dark period, i.e., when the animals start to be active. The IS was accompanied by an intense polygraphic waking and followed, over 12 h of the dark period, by a significant rebound of slow-wave sleep (SWS, +17%) and paradoxical sleep (PS, +57%). In order to estimate the IS-related changes in the endogenous concentrations of corticotropin-like intermediate lobe peptide (CLIP, ACTH18-39) and related compounds, a specific radioimmunoassay (RIA) was used. Assays performed in cerebral biopsies taken from arcuate (AN) and raphe dorsalis (nRD) nuclei led to the obtention of 2 main immunoreactive peaks, corresponding to CLIP and its phosphorylated form Ph-CLIP. Just after end of the IS and within the nRD. Ph-CLIP immunoreactivity increased by about 95%. Four hours later, i.e., when PS rebound was maximal, a 37% increase in Ph-CLIP immunoreactivity was measured in the AN. These observations have never been described before. In the blood, at the end of the restraint, CLIP/ACTH1-39 total immunoreactivity was increased by 330%. It returned to baseline level 4 h later. Blood concentration of corticosterone was also increased by 56% at the end of the IS and was close to baseline level 4 h later. Data reported here indicate that the IS first triggers an increase in Ph-CLIP within the nRD. Since the nRD contains sleep permissive components, this increase might be determinant for the SWS and PS rebound induction. The changes observed in the blood as regards CLIP/ACTH1-39 total immunoreactivity and corticosterone concentration testify to the efficacy of the IS and are part of the conventional picture accompanying such a situation. Finally, the increase in Ph-CLIP, occurring in the AN 4 h after the end of the restraint, might be part of the restorative processes necessary to compensate the stress overshoot.

Effects of tianeptine, sertraline and clomipramine on brain serotonin metabolism: a voltammetric approach in the rat.

Tianeptine is a substance enhancing the serotonir uptake while sertraline and clomipramine inhibit it. By means of 5-hydroxyin-doleacetic acid (5-HIAA) voltammetric measurements, this study investigated their influence on serotonin metabolism which depends mainly upon the activity of monoamine oxidase type A. After tianeptine injection the 5-HIAA signal increased by about 60%. This effect was maintained when the animals were pre-treated with MDL 72145 (an inhibitor of monoamine oxidase type B) but reduced when clorgyline (an inhibitor of monoamine oxidase type A) was administered after tianeptine. Administration of sertraline or clomipramine reduced the 5-HIAA signal by about 30-50%, whether the animals were pre-treated with MDL 72145 or not. It is to be concluded that tianeptine, sertraline and clomipramine can regulate the 5-HT fraction present in the synaptic cleft, not only by acting at the level of the serotoninergic neurons, but also by favoring or reducing the access of the amine to monoamine oxidase type A which is synthesized within non-serotoninergic neurons and glial cells.

Influence of a 1-h immobilization stress on sleep and CLIP (ACTH(18-39)) brain contents in adrenalectomized rats.

Basal sleep amounts in adrenalectomized rats (AdX), as compared to intact animals, exhibit a significant increase in slow-wave sleep (SWS), a tendency towards an increase in paradoxical sleep (PS), and circadian rhythms (SWS and PS) flattened in amplitude. An immobilization stress (IS) of 1 h, imposed on AdX rats at the beginning of the dark period, is accompanied by an intense polygraphic waking. Just after the IS, SWS amount become significantly higher than in control rats (+44%/11 h of darkness) whereas significant increases of PS occur only 5-10 h after the IS (+24%/11 h of darkness). A specific radioimmunoassay for CLIP (corticotropin-like intermediate lobe peptide or ACTH(18-39)) was performed in biopsies taken either from the nucleus raphe dorsalis (nRD) or the arcuate nucleus (AN). In the nRD, just after the IS, phosphorylated CLIP (Ph-CLIP) concentration exhibits a decreasing tendency, but 4 h later, it increases significantly (+22%, p<0.05). In the AN, Ph-CLIP concentration remains unchanged after the IS as well as 4 h later. These results differ from those previously reported in intact animals also submitted to a 1-h IS, that is, a SWS rebound less marked (+27%/11 h of darkness), a PS rebound more important starting immediately after the IS (+46%/11 h of darkness) and a significant increase in Ph-CLIP occurring just after the end of the restraint. In conclusion, data obtained after a restraint stress either in AdX or in control rats point out the dependence of the PS rebound on the nRD Ph-CLIP concentration.

Genetic studies of daily variations of first-step enzymes of monoamines metabolism in the brain of inbred strains of mice and hybrids. II. Daily variations of tyrosine hydroxylase activity in the locus coeruleus.

Daily variations of tyrosine hydroxylase (TH) activity in the locus coeruleus of 3 inbred strains of mice (BALB/c; C57BL6; C57Br) and the F1 hybrids obtained from BALB/c and C57BL/6 are discussed. Precise characteristics of the circadian rhythms were observed in each strain. They were found significantly different in two genetically pure parents (BALB/c and C57BL6). In their two F1 hybrids the daily variation of TH activity was similar to that observed in one of the parents (C57BL6). This strongly suggests selective and genetically controlled mechanisms of regulation responsible for the daily variation of TH activity in the locus coeruleus of mice.

Role of catecholamines in the modafinil and amphetamine induced wakefulness, a comparative pharmacological study in the cat.

Seventeen adult cats were chronically implanted with electrodes for polygraphic recordings in order to assess the role of catecholamines in the arousal effects of oral administrations of modafinil, a presumed noradrenergic agonist, and amphetamine, a well-known catecholamine-releasing agent. Whereas both modafinil (1, 2.5 and 5 mg/kg) and amphetamine (0.25, 0.5 and 1 mg/kg) caused a significant and dose-dependent increase in wakefulness and brain temperature, amphetamine, but not modafinil, elicited marked signs of behavioral excitation. Pretreatments with alpha-methyl-DL-p-tyrosine methyl ester (50 mg/kg, i.p.), an inhibitor of catecholamine synthesis, almost completely prevented the effects of amphetamine (0.25 and 1 mg/kg), but only slightly reduced the duration of the waking effect of modafinil (2.5 and 5 mg/kg). Pretreatments with phentolamine (10 mg/kg, i.p.), prazosin (1.5 mg/kg, per os) and propranolol (5 mg/kg, i.p.), an alpha-, alpha 1- and beta-receptor antagonist, respectively, attenuated significantly the arousal effect of modafinil (1 mg/kg, the same as below) but not of amphetamine (0.25 mg/kg, the same as below). Intraperitoneal injections of haloperidol (0.5 mg/kg), a dopamine-receptor antagonist, blocked significantly the arousal of amphetamine but not of modafinil. The effects of both modafinil and amphetamine were enhanced by a pretreatment with yohimbine (1 mg/kg, i.p.), an alpha 2-receptor antagonist. These results suggest that the arousal effect of modafinil does not depend on the availability of the endogenous catecholamines but results from an enhancement of alpha 1- and beta-receptor activity and that the waking and behavioral effects of amphetamine may be mainly due to an increase in dopamine release.

Hypoprolactinemic rats under conditions of constant darkness or constant light. Effects on the sleep-wake cycle, cerebral temperature and sulfatoxymelatonin levels.

In genetic hypoprolactinemic rats under light-dark (LD) conditions, the circadian rhythms of slow-wave (SWS) and paradoxical (PS) sleep display an alteration of their phase relationship. The aim of our study was to investigate the effects of constant darkness (DD) or constant light (LL) on the daily distribution and amounts of sleep-wake stages, cerebral temperature and concentrations of the urinary melatonin metabolite, 6-sulfatoxymelatonin, in prolactin-deficient rats. After 3 weeks of DD, the SWS period was 24 h 8+/-6 min and the acrophase occurred at 15:44+/-1:35, while for PS, the period was more stable than during LD (24 h 10+/-8 min vs. 24 h 55+/-43 min) and the acrophase occurred at 16:44+/-1:54. Under LL conditions, circadian sleep rhythms persisted during the first 3 days, then completely disappeared during the third week, to be replaced by ultradian rhythms (period of 4-6 h). Time-series analysis showed that the two sleep states became synchronous as early as the second day under constant conditions. The total amount of PS was increased under both conditions (LL and DD) at the expense of duration of waking. Under LD and constant conditions, the pattern of changes in cerebral temperature was similar to that for wakefulness (W). Sulfatoxymelatonin was rhythmically secreted under both LD and DD conditions, whereas, under LL conditions, its rhythm was abolished. The results show that, in IPL rats in the absence of a zeitgeber, the PS and SWS rhythms recover a synchronous phase relationship and PS amounts are increased.

Daily variations of various parameters of serotonin metabolism in the rat brain. I. Circadian variations of tryptophan-5-hydroxylase in the raphe nuclei and the striatum.

Daily changes in tryptophan-5-hydroxylase (TrH) activity have been studied in six different 5-HT-containing cell groups (B3, B4, B5, B6, B7, B8,) in the brain stem of rats maintained in a regular cycle of 12 h of light and 12 h of darkness. Significant circadian alternations of TrH activity were observed in most of the raphe nuclei tested, although the changes were not identical from one structure to another. The striatum showed daily variations in TrH activity in opposite phase to the nucleus raphe dorsalis which projects specific terminals into this area. Monoamine oxidase (MAO) activity was simultaneously estimated in these nuclei but did not exhibit and significant rhythm, suggesting a specific regulation of TrH. Total protein levels were also subject to daily changes.

RU 24722, a new eburnamine derivative, induces selective alterations in cerebral glucose utilization in freely moving rat.

The effect of a new eburnamine derivative, RU 24722, a putative phasic activator of catecholaminergic systems on local cerebral glucose utilization was studied in freely moving rats 15 min, 90 min and 6 h after the intraperitoneal administration of the drug (25 mg/kg). Of the 53 brain regions examined, 9 exhibited significant time-dependent increases of glucose utilization (up to 45-55%). Some changes were early and transient, as in the substantia nigra reticulata and the paraventricular nuclei. Other areas showed sustained (median septal nucleus) or delayed increases of glucose utilization (lateral septum, dorsal subiculum, hippocampal fimbria, fronto-parietal motor cortex and ventral cochlear nucleus). No significant alterations of glucose utilization could be elicited in the locus coeruleus and raphe nuclei, and none of the brain regions showed a decrease in glucose consumption. Our findings suggest that RU 24722 preferentially stimulates the activity in some brain areas involved in cognitive, vegetative and locomotor functions.

Noradrenaline neurotoxin DSP-4 effects on sleep and brain temperature in the rat.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) has a selective degenerative effect on noradrenergic fibers originating from locus coeruleus (LC) neurons. In the present study, we studied its effect on vigilance states and brain temperature by continuous recordings for periods of 1-5 days and 2-4 weeks following DSP-4 treatment. On the first day, paradoxical sleep duration was significantly decreased (-67%, P < 0.05), slow-wave sleep (SWS) duration increased (+16%, P < 0.05) up to 48 h after DSP-4 treatment (+8%, P < 0.05) and the wake period decreased (-8%, P < 0.05). The vigilance states returned to control values 4-5 days later. The brain temperature was decreased during the first night (-2 degrees C) and then recovered the control values. Two and 4 weeks after DSP-4 treatment, paradoxical sleep was still decreased (-18% and -23%, respectively, P < 0.05), while SWS was significantly increased only at night during the fourth week (+23%, P < 0.05). These results therefore provide evidence for a differential involvement of the noradrenergic LC system in sleep mechanisms depending on the light-dark cycle. Different hypotheses are proposed.

Sleep increase after immobilization stress: role of the noradrenergic locus coeruleus system in the rat.

In a preliminary study we showed that the sleep rebound occurring after sleep deprivation is decreased in rats treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), a neurotoxic agent specific for the noradrenergic cells of the locus coeruleus (LC). Sleep deprivation methods not only involve sleep loss, but also stress, which per se may induce an increase in sleep duration. Extensive research showed that the locus coeruleus is involved in stress. To evaluate the participation of LC in this mechanism, the effect of DSP-4 treatment was studied on sleep duration following a short intense stress in the absence of sleep loss. The results showed that the augmentation of sleep after 1 h of immobilization stress is lower in DSP-4-treated rats (slow-wave sleep duration, -24%; paradoxical sleep duration, -52%). These findings suggest that the increase in sleep induced by such a stressor is mediated, at least in part, by the noradrenergic LC.

The caudo ventral pontine tegmentum is involved in the generation of high velocity eye saccades in bursts during paradoxical sleep in the cat.

Cat eye movements were recorded in the head restrained condition, with the technique of the scleral search coil in a magnetic field, and the maximum velocity/amplitude relationships were analyzed for saccades in the following conditions, (1) during waking (W); (2) during paradoxical sleep (PS); and (3) during W following carbachol microinjections in the medioventral part of the caudal pontine tegmentum. These findings indicate that (1) the neurophysiological mechanisms underlying carbachol induced events are similar to those acting during PS and that the caudal pontine tegmentum might be the generator of high velocity eye saccades in bursts accompanied by ponto geniculo occipital waves (PGOw) during PS, and (2) caudal pontine tegmentum neurons show 'state-dependent' responsiveness to cholinergic inputs, suggesting that a change in the synaptic inputs and/or the membrane properties of these neurons during PS may be responsible for the induction of saccadic eye movements in bursts and associated PGOw.

Effect of noradrenergic denervation of the amygdala upon recovery after sleep deprivation in the rat.

We previously showed that the noradrenergic locus coeruleus (NA-LC) was involved in the regulatory mechanisms of the paradoxical sleep rebound following a 10 h sleep deprivation by using a systemic injection of a specific neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). Given that rebound mechanisms are mainly located in the forebrain, we planned to study the role of the forebrain structures receiving LC afferences. In this study we evaluated the involvement of noradrenergic afferences to the central nucleus of the amygdala in the sleep rebound by DSP-4 microinjections into the central nucleus of the rat amygdala. The results showed that during the first recovery day, the paradoxical sleep rebound is lower in DSP-4 treated rats (-67.28%). These findings indicate that the amygdala, through its NA afferents, contributes to the sleep rebound mechanisms.

Effect of noradrenergic denervation of medial prefrontal cortex and dentate gyrus on recovery after sleep deprivation in the rat.

The noradrenergic-locus coeruleus (LC) system has a regulatory influence on forebrain neuronal networks. We have previously shown that the amygdala is strongly implicated in the mechanism of rebound seen after a 10 h sleep deprivation (SD). In the present study, our objective was to determine whether the medial prefrontal cortex and dentate gyrus (DG) which receive an important innervation from the LC, play a role in the rebound mechanisms. We found that microinjection of the specific noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, into these regions had no effect on the increase in paradoxical sleep duration seen after SD, suggesting that noradrenergic (NA) innervation of the prefrontal cortex and DG are not involved in sleep rebound regulation.

Local cerebral glucose utilization during waking and slow wave sleep in the cat. A [14C]deoxyglucose study.

Local cerebral glucose utilization (LCGU) was measured during spontaneous waking and slow wave sleep (SWS) by an adaptation of the [14C]deoxyglucose method to the free moving cat. In awake animals heterogeneous rates of LCGU were observed throughout the brain with values extending from 8 mumol x 100 g-1 x min-1 for white matter, to 113 for the inferior colliculus. There was no significant difference for LCGU in SWS compared with awake animals.