A GABAergic habenulo-raphe pathway mediates both serotoninergic and hypnogenic effects of vasotocin in cats

Extremely small amounts (10(-4) pg) of the pineal nonapeptide arginine vasotocin (AVT), injected into the pineal recess of freely moving cats, decreased the levels of 5-hydroxyindole acetic acid (5-HIAA) in the raphe dorsalis nucleus and induced slow wave sleep. In cats with lesions in the lateral habenula, 10(-4) pg AVT injected into the pineal recess, failed to decrease raphe dorsalis 5-HIAA levels and to induce slow wave sleep. The GABA antagonist picrotoxin (1 ng) injected into the pineal recess 15 min before the administration of AVT (10(-4) pg), completely prevented AVT from decreasing raphe dorsalis 5-HIAA levels and from inducing slow wave sleep. A highly significant correlation could be established between the decrease of raphe dorsalis 5-HIAA levels and the induction of slow wave sleep. No changes in raphe dorsalis 5-HIAA levels could be detected in cats injected with 10(-4) pg AVT into the lateral or into the fourth ventricle. Neither arginine vasopressin nor oxytocin (10(-4) pg) injected into the pineal recess, could alter raphe dorsalis 5-HIAA levels. It is concluded that AVT induces slow-wave sleep in cats by activating an inhibitory GABAergic lateral habenula-raphe dorsalis pathway.     

Pineal vasotocin and sleep: Involvement of serotonin containing neurons

Extremely small amounts (10^?4 pg) of arginine vasotocin (AVT) injected into the third ventricle of unanesthetized cats induce non-rapid eye movement (NREM) sleep and suppress rapid eye movement (REM) sleep. The same amount (10^?4 pg) of intraventricularly injected AVT increased at 15 and 30 min 5-hydroxytryptamine (5-HT) and decreased at 30 and 60 min 5-hydroxyindole acetic acid (5-HIAA) levels of the brain. Fluoxetine, a specific 5-HT uptake inhibitor, greatly enhanced the NREM sleep induced by AVT. Neither arginine vasopressin, nor oxytocin at the doses used (10^?4 pg), was able to affect indole levels of the brain or to enhance NREM sleep and to suppress REM sleep after fluoxetine, demonstrating the high specificity of AVT effects. Methergoline, a selective central 5-HT receptor blocker, completely prevented AVT induction of NREM sleep. Extremely small amounts of AVT (10^?6 pg), which in some cats were unable to induce NREM sleep and to suppress REM sleep, become effective after pretreatment with small amounts of 5-hydroxytryptophan (5-HTP). It is suggested that AVT induces NREM sleep and suppresses REM sleep by interfering with 5-HT release at postsynaptic receptor sites. The present results strongly support the monoaminergic theory of sleep providing the first evidence that a peptide synthesized by the brain induces sleep by a serotoninergic mechanism.     

REM sleep suppression in cats by melatonin

The pineal indole melatonin, injected into the third ventricle of unanesthetized cats, induced NREM sleep within 11–15 min and suppressed REM sleep for about 3 hr. A dose-related effect could be established between 1, 10 and 100 ng melatonin. The reappearance of REM sleep at about 3 hr following melatonin administration occurred with a marked rebound. The present results further support the hypothesis that melatonin represents the releasing hormone for pineal arginine vasotocin.     

Pharmacological evidence that effects of vasotocin on brain maturation are mediated by GABA mechanisms

Active sleep, brain weight as well as total lipids and galactolipids were assayed in 8-day-old rat pups, after daily administration (between 2 and 8 days of postnatal age) of saline, arginine vasotocin (AVT) (10⁻⁷ mg), picrotoxin (5 × 10⁻⁵ mg), valproic acid (10⁻⁵ and 10⁻⁶ mg) or of AVT+picrotoxin and AVT+valproic acid. AVT increases active sleep and decreases the weight, total lipids and the galactolipids of the brain. Although picrotoxin alone was without any apparent effects on maturational parameters, when administered together with AVT, it completely prevented AVT from producing its effects. Valproic acid had AVT-like effects when administered in a higher dose (10⁻⁵ mg), but was without any apparent effects when administered in the lower dose (10⁻⁶ mg). However, both doses intensely potentiated the effects of AVT. Since both drugs specifically interfere with the GABAergic neurotransmission, it is concluded that AVT retards the accumulation of brain lipids by activating a GABAergic pathway.     

Intraseptal administration of vasopressin and oxytocin affects hippocampal electroencephalogram in rats

Two peptides, arginine⁸-vasopressin and oxytocin were tested in rats for their effects on the function of the septum, the pacemaker for hippocampal theta rhythm. The peptides or the placebo were injected in small quantities into the septum or other brain structures. The spectra computed from hippocampal EEGs recorded during paradoxical sleep and slow-wave sleep episodes which followed the injections of peptide, were compared. The peptides in doses of 10^?3 pg or more injected into the septum altered the hippocampal EEG during paradoxical sleep episodes in a similar fashion. The EEG alterations were confined to frequencies of the theta band (4.1 to 12.0 Hz) and consisted of an increase in peak and mean frequencies in this band. The hippocampal EEG during slow-wave sleep was little affected by intraseptal injections of the peptides. Microinjections of AVP in the same or higher (to 10 pg) amounts of the peptide into the frontal cortex or the subicular region of the dorsal hippocampus had little effect on hippocampal activity during either sleep stages. The arginine⁸-vasopressin-like effect of intraseptally administered oxytocin on hippocampal synchronization during paradoxical sleep contrasted with the effect found in previous studies in which oxytocin had an effect opposite that of arginine⁸-vasopressin. The present study demonstrates that the septal mechanism pacing hippocampal theta rhythm is affected by these peptides. Because the septum receives vasopressinergic innervation, we suggested that the role of this peptidergic innervation includes regulation of processes which are related to generation of the theta rhythm.     

Opposite effects of vasotocin and of a specific vasotocin antiserum on active sleep of kittens

Summary The effects of intracerebroventricularly administered synthetic arginine vasotocin (AVT) or undiluted AVT, vasopressin (AVP) and oxytocin (OT) antisera on active sleep (AS) of newborn kittens have been investigated in comparison with rabbit serum control. In contrast to AVP and OT antisera, AVT antiserum has produced opposite effects on AS as AVT itself. Since after 10 l of undiluted AVT antiserum the percentage of AS did not decrease under 20% and even after 100 l AS did not decrease under 5%, it is concluded that, at least during perinatal life, AVT could be considered as a neuromodulator with AS-promoting effect.     

Vasoactive intestinal polypeptide (VIP) and cerebrospinal fluid (CSF) of sleep-deprived cats restores REM sleep in insomniac recipients

In the past few years a steadily increasing number of substances have been suggested to qualify as sleep-inducing factors. Most ‘sleep factors’ appear to exert their effects on slow-wave sleep. Recently, however, it has been shown that the cerebrospinal fluid (CSF) of sleep-deprived cats may contain a rapid eye movement (REM) sleep factor, and that vasoactive intestinal peptide (VIP) may be a specific REM sleep inducer. The purpose of this study is to determine whether the CSF of sleep-deprived cats and VIP can reverse insomnia produced by parachlorophenylalanine (PCPA). Donor cats were sleep-deprived for 24 h and their CSF extracted. Some donor cats were additionally pre-treated with chloraphenicol, and some extracted CSF was heated. Recipient cats were injected with 400 mg/kg i.p. of PCPA on two consecutive days. Twenty-four h after the second injection, the recipient cats were intraventricularly injected with a 100-μl of the various CSF types or 200 ng of VIP. The results showed that only CSF from sleep-deprived cats and VIP were capable of restoring REM sleep in the otherwise PCPA insomniac cats. Since the return of REM sleep was through an increase in its frequency, it is suggested that the CSF of sleep-deprived cats contains a VIP-like sleep factor possibly involved in triggering REM sleep.     

Failure to detect radioimmunoassayable arginine vasotocin in mammalian pineals

The presence of arginine vasopressin (AVP) and arginine vasotocin (AVT) in pineal and neural lobes of a variety of species was evaluated by radioimmunoassay using two antisera. One antiserum recognizes only AVP, while the other cross-reacts 100% with both AVP and AVT. Only traces of AVP were detected in toad and chicken pituitaries and chicken pineals, tissues found to contain abundant AVT. On the other hand, pineals from rats and rabbits, while containing measurable quantities of AVP, did not contain detectable levels (less than 6 pg) of AVT. These results fail to demonstrate the presence of AVT in rat and rabbit pineals and strongly question the role of AVT as a physiological pineal hormone in mammals.     

Sleep suppression induced by intravenous and intraventricular infusions of methoxamine in the dog

Central adrenergic mechanisms have been implicated in behavioral arousal. We studied the effects of methoxamine, a long-acting direct α-adrenergic agonist. Cortical and hippocampal electroencephalograms, nuchal electromyograms, and electrooculograms were recorded from beagle-type dogs in an isolation chamber while their behavior was observed on a videomonitor. Following intravenous saline, total sleep occurred during 61 ± 6.0% of the 2-h recording period. Of that time, 35 ± 4.5% was in light sleep, 54 ± 3.3% was in slow-wave sleep, and 11 ± 1.2% was in paradoxical sleep. An intravenous dose of 0.33 mg/kg methoxamine caused no significant changes, whereas a 0.66 mg/kg dose significantly reduced total sleep (25 ± 5.7%) and paradoxical sleep (3 ± 1.2%). When infused into the dorsal aspect of the third ventricle, methoxamine (100, 400, or 1200 μg) produced no significant effects. However, these doses of methoxamine injected into the ventral third ventricle caused a significant dose-related decrease in total sleep, slow-wave sleep, and paradoxical sleep and an increase in light sleep. Phenoxybenzamine (4 mg/kg, intravenously) pretreatment significantly antagonized the arousal effects of methoxamine (400 μg) administered into the ventral third ventricle. The distribution of infusions of a contrast medium and bromphenol blue into the dorsal and ventral third ventricle differed in that infusions at the former site did not reach anterior hypothalamic structures. In other experiments, tritium-labeled methoxamine was found to cross the blood-brain barrier in the rat. These results suggest that methoxamine is a centrally active α-adrenergic agonist and support the concept that hypothalamic adrenergic mechanisms are involved in electroencephalographic and behavioral arousal.     

Restoration of paradoxical sleep cerebrospinal fluid transfer to PCPA pretreated insomniac cats

In p-chlorophenylalanine (PCPA) pretreated insomniac cats, the intraventricular (i.v.t.) injection of artificial cerebrospinal fluid (CSF) did not impair the insomnia. CSF transer from normal cats was followed 4 out of 9 cats by the restoration of paradoxical sleep (PS). However, CSF transfer from paradoxical sleep deprived cats did result in 12 out of 13 experiments in a significant increase in slow wave sleep (SWS) and the induction of PS. Biochemical analysis of the CSF from normal or PS deprived cat has shown that the highest quantity of indolamines was at least 1000 times smaller than the threshold dose of 5-HTP (200 μg) which has been shown to be able to restore sleep by i.v.t. injection in PCPA pretreated insomniac cats. These experiments provide evidence that the transfer of a small quantity of CSF (250 μl) from a previously paradoxical sleep deprived cat can restore paradoxical sleep in an insomniac PCPA pretreated cat in bypassing the biosynthesis of serotonin (5-HT). These results suggest that a ‘paradoxical sleep inducing factor’ may be stored in the central nervous system during sleep deprivation.     

Decreased muscarinic receptor binding in rat brain after paradoxical sleep deprivation: an autoradiographic study

Previous work demonstrated that paradoxical sleep deprivation (PSD) leads to a decrease in yawning behavior elicited by cholinergic agonists, suggesting that a downregulation of cholinergic muscarinic receptors may occur after PSD. More recent work using intracerebral injections of muscarinic agonists has suggested a critical role for M2 receptors in paradoxical sleep. In this study [³H]AF-DX 384 was used to investigate the effects of PSD on M2-type cholinergic receptors throughout the brain using quantitative autoradiography. After 96 h of paradoxical sleep deprivation, [³H]AF-DX 384 binding was generally reduced throughout the brain, and significantly so in the olfactory tubercle (−20%), n. accumbens (−23%), frontal caudate-putamen (−16%), islands of Callejas (−20%), piriform cortex (−24%), lateral (−26%) and medial (−24%) septum, anteromedial (−19%), ventrolateral (−22%), and lateral geniculate (−15%) nuclei of thalamus, deep layers of the superior colliculus (−15%), entorhinal cortex (−12%) and subiculum (−23%). [³H]AF-DX 384 binding was reduced in pontine structures, but not to a higher degree than in other brain areas. The observed downregulation of M2-type muscarinic receptors after PSD may be causally related to the previously reported decrease in cholinergically induced behaviors after PSD.     

Time-dependent effects of actinomycin S3 on sleep and brain concentrations of 5-hydroxytryptamine in the rat

Changes in the sleep pattern were investigated in rats given actinomycin S3 (AcS3, 0.25 mg/kg, intraventricularly), an inhibitor of RNA synthesis, at 0000, 0600, 1200, and 1800. The amounts of slow-wave (SS) and paradoxical sleep (PS) were increased during the dark period (1800 to 0600) in the groups treated at 0000, 0600, and 1200. In the 1800 injected group, PS was decreased. 5-Hydroxytryptamine (5-HT) was determined in the 0600 injected group. The 5-HT concentration in the AcS3-treated group was twofold higher than in the control group at 1800 (12 h after administration) in the cerebral cortex and lower brain stem but returned to control values at 0000 (18 h after) while sleep time continued to increase. Thus, AcS3 affected the circadian rhythm of the sleep-wake cycle. The increase of sleep time in the dark period was independent of changes in brain 5-HT content.     

An arginine vasotocin-like neuropeptide is present in the nervous system of the marine mollusc Aplysia californica

Radioimmunoassays and high pressure liquid chromotography have been used to demonstrate the presence of an arginine-vasotocin-like peptide (AVT) in the anterior ganglia of Aplysia. Previously, AVT, using similar methods, was found to be present only in vertebrates. AVT when perfused over the abdominal ganglion (10⁻⁶–10⁻¹²M) was found to increase the bursting activity of R₁₅, to decrease the bursting activity of L₃–L₆ and to increase the CNS's suppressive influence over the gill withdrawal reflex evoked by siphon stimulation. The AVT present in the nervous system of Aplysia may mediate long-term suppression of gill reflex behaviors induced by factors such as satiation and, as well, regulate the activity of certain neurosecretory neurons.     

Oxytocin acts at V1 receptors to excite sympathetic preganglionic neurones in neonate rat spinal cord in vitro

Intracellular recordings were made from sympathetic preganglionic neurones (SPNs) in transverse slices of thoraco-lumbar spinal cord of young rats (12–20 days old). A small group of SPNs generally having higher membrane potentials (− 70mV) compared to a remaining group (− 66mV) showed spontaneous oscillations of their membrane potential. Oxytocin superfused in concentrations of 0.1–30 μM had four effects on SPNs, inducing slow depolarisation, EPSPs, IPSPs and brief rhythmic oscillations. The slow depolarisation was unaffected by TTX whereas this abolished the other changes. The oxytocin-induced depolarisation was associated with a slow inward current and was not reversed at membrane potentials negative toE_K, it increased at more positive potentials and was still present in low Ca²⁺ and high Mg²⁺ solutions. These features of the oxytocin induced current are similar to those of the TTX resistant voltage dependent Na⁺ current described in brainstem autonomic neurones. Vasopressin superfused at concentrations of 0.1 μM to 30 μM had similar effects on SPNs to those of oxytocin. A comparison of the effects of oxytocin and vasopressin on the same neurones revealed that oxytocin was almost 10 times less potent than vasopressin. The effects of oxytocin were not mimicked by a selective oxytocin agonist but were mimicked by a selective vasopressin V_1a agonist and blocked by a selective V_1a antagonist. Therefore it is concluded that the effects of oxytocin on SPNs are mediated by the vasopressin V_1a receptor. It is suggested that oxytocin and vasopressin terminals in the lateral horn are part of a descending system controlling oscillating networks of SPNs in the spinal cord.     

A novel effect of MPTP: the selective suppression of paradoxical sleep in cats

We studied the effect of MPTP on sleep-wakefulness cycle in cats. Five mg/kg n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was administered i.p. for 5 consecutive days. Electrocorticographic, electrooculographic and electromyographic recordings were performed before (5 days), during (5 days) and after (14 days) the treatment. Total selective paradoxical sleep deprivation was observed from the first injection. This effect lasted 6-9 days after the last dose, while the relative amount of slow wave sleep increased. The Berg-Fourier analysis showed no significant change in the EEG power spectra of slow-wave sleep during the paradoxical sleep deprivation compared to control period. Recurrence of paradoxical sleep was parallel to the disappearance of the motor symptoms. Histopathological investigation showed neuronal loss mainly in the substantia nigra. Our present study suggests a complex behavioral effect of MPTP.     

Delta sleep inducing peptide (DSIP) stimulates the release of LH but not FSH via a hypothalamic site of action in the rat

Long term ovariectomized (OVX) Sprague-Dawley rats were injected intraventricularly (3rd ventricle) with 5 μg (2 μl) of DSIP. This caused a significant elevation (p=0.01) of LH levels within 30 min. The values remained elevated for 2 hr; however, FSH levels remained unchanged. The minimal effective dose of DSIP to evoke this effect was 1 μg. If plasma LH was lowered by pretreatment of the animals with estradiol, the 5 μg dose evoked an even greater effect to elevate LH significantly at 30 and 60 min following its intraventricular injection. To determine the site of action of DSIP, dispersed, overnight cultured pituitary cells from OVX rats were incubated with varying concentrations (10⁻⁷ to 10⁻¹² M) of DSIP in an in vitro system. There was no response to DSIP from the cells in the above system. To evaluate its possible action on the hypothalamus, median eminence (ME) fragments from male rats were incubated in vitro with DSIP in varying concentrations from 10⁻⁷ to 10⁻¹⁰ M. There was a significant (p<0.001) increase in LHRH released from the ME at a concentration of DSIP of 10⁻⁷ M. A sleep-related increase in LH release is seen during puberty in man. It is possible that DSIP released within the hypothalamus may play a physiological role in sleep-related LH release.     

Paroxysmal discharges during paradoxical sleep anticipating the occurrence of focal motor seizures in cats

Sleep recordings were made some days before the occurrence of focal motor seizures in cats injected with alumina cream in the motor cortex. The wakefulness EEG was completely normal. Slow-wave sleep showed spike-spindles and occasional isolated spikes. During paradoxical sleep, abortive seizures appeared. They were only electrographic and never produced arousal of the animals. The precipitating role of phasic paradoxical sleep on focal motor seizures is proposed.     

The Effect of an Osmotic Stimulus on the Release of Neurohypophysial Hormones in the Cat: Preferential Release of Vasopressin with a Possible Involvement of the Area Postrema

In cats anaesthetized with intravenous chloralose, the injection of 0.05 to 0.4 ml 1.54 M NaCl solution (hypertonic saline, HS) into a lateral cerebral ventricle caused a large release of vasopressin. The concentration of vasopressin greatly exceeded that of oxytocin in the same samples of plasma. Vasopressin was also released when HS was injected into the fourth ventricle and into the cisterna magna from which there is no access in the cat to the ventricles, but it was less effective by these routes than when injected into a lateral ventricle in the same cat. This suggests a possible action of HS on circumventricular organs related to the third ventricle but also indicates an additional site of action reached from the subarachnoid space which would give access to the ventral and dorsal surfaces of the brainstem. Vasopressin was not released on topical application of HS to the ‘nicotine sensitive area’ on the ventral surface of the brainstem where nicotine acts to release vasopressin without oxytocin. Vasopressin, however, was released without detectable oxytocin on topical appliction of HS to the dorsal surface of the brainstem either outside the fourth ventricle or to the floor of the ventricle at its distal extremity, in the region of the obex. A possible site where HS acts to cause a preferential release of vasopressin on injection into a lateral ventricle is the area postrema, a circumventricular organ which impinges on the walls of the fourth ventricle at the obex. Preferential release of vasopressin might then be mediated by a selective neural input, possibly through the nucleus of the tractus solitarius, from osmoreceptors in the area postrema to the vasopressin-secreting cells in the supraoptic and paraventricular nuclei.     

A Quantitative Study of Electroencephalography, Eye Movements and Neck Electromyography Characterizing the Sleep—Wake Cycle of the Guinea-pig

The qualitative and quantitative characteristics of cerebral cortex electrical activity, ocular motility and muscular activity were studied in six head-restrained guinea-pigs during wakefulness, slow-wave and paradoxical sleep. Animals were chronically implanted with bipolar electrodes in the obliquus capitis muscle for electromyographic recordings and epidurally through the parietal bones for electroencephalographic (EEG) recordings. Eye movements were recorded using the scleral search-coil technique. After postoperative recovery and a short period of habituation to immobilization, head-restrained animals exhibited a polyphasic sleep–wake cycle similar to what has already been described in the unrestrained guinea-pig. Paradoxical sleep periods of mean duration 110 ± 42 s occurred at a mean interval of 32.2 ± 7.2 min. Amplitude and frequency components of EEG activity were different for each state of vigilance. EEG amplitude was highest and frequency range lowest—with two well-defined peaks at 4 and 10 Hz—during slow-wave sleep. During paradoxical sleep, frequencies were higher and amplitudes lower than during wakefulness. Three types of eye movement intermingled with periods of ocular fixation were recorded: saccadic movements during wakefulness and paradoxical sleep, slow drifts during slow-wave sleep and paradoxical sleep, and a new type of eye movement—bursts of high-velocity eye oscillations during paradoxical sleep. Saccadic eye movements during paradoxical sleep were more frequent and showed higher velocities and amplitudes than during wakefulness. During paradoxical sleep the episodes of eye oscillation (8–14 Hz) occurred quite regularly every 1.6 s and had a mean duration of 1.4 s. During wakefulness, the obliquus muscle activity displayed a burst-tonic pattern. Bursting components were closely related to saccadic eye movements directed to the side of the recorded muscle. The muscle activity was predominantly tonic during slow-wave sleep and was completely absent during paradoxical sleep except for small bursts or twitches. These twitches were tightly synchronized with the occurrence of the rapid eye movements oriented towards the side of the recorded obliquus muscle, as during wakefulness. These results strongly suggest that paradoxical sleep is characterized by the oscillatory discharge of at least two neuronal populations: the brainstem saccadic generators and the tecto-reticular spinal network which underlies gaze-orienting behaviour during wakefulness. The occurrence of rhythmic discharges at ?11 Hz may explain the spinal motoneurons’ inhibition during paradoxical sleep in order to avoid anarchic motor behaviour. Whether these neuronal oscillations are simply an epiphenomenon or have functional implications remains to be determined.