Effect of drugs of the nootropic class on rat behavior after deprivation of the paradoxical stage of sleep

Pharmacological analysis was used for studying the influence of 24-hour deprivation of paradoxical sleep by Jouvet method on retention of conditioned reaction of passive avoidance in rats. Psychotropic substances of different action were used for the analysis: nootropes as anti-amnestic--pyracetam (400 mg/kg), kleregil (100 mg/kg), centrofenoxin (50 mg/kg) and watersoluble salt of 3-oxypiridin derivative (3-OP) (50 mg/kg) and tranquilizer of bensodiazepine series phenazepam (1 mg/kg) as antistress and antiphobic. It was established that 24-hour deprivation disturbed the elaborated reaction but did not change the rate of emotionality and orienting-investigating behaviour of rats in the open field. Nootropes effectively restored the conditioned passive avoidance reaction while phenazepam had no effect. This allows to suggest that Jouvet method of paradoxical sleep deprivation elicits amnesia and its cause is not only stress but deficit of paradoxical sleep.     

DEPRIVATION OF PARADOXICAL SLEEP AND BRAIN GLYCOGEN

Abstract— Rats deprived of paradoxical sleep (PS) for 72 hr showed a considerable fall of total glycogen in the subcortex and caudal brain stem. No important changes were discovered in the parts of the cerebral cortex which were examined. The stress of the experimental technique of PS deprivation did not lead to significant changes in glycogen content in any of the structures examined. It was concluded that changes in glycogen content corresponded specifically to PS deprivation stress.     

THE EFFECT OF PHYSOSTIGMINE AND NEOSTIGMINE ON THE CONCENTRATION OF GLYCOGEN IN VARIOUS BRAIN STRUCTURES OF THE RAT

Abstract—

• 1 Hypothalamus, mesencephalon, cerebral cortex, cerebellar cortex and medulla oblongata of the rat brain contain varying amounts of glycogen. The highest concentration was found in the medulla, and the lowest in the hypothalamus.
• 2 Low doses of physostigmine produced a significant decrease in the concentration of glycogen in mesencephalon, cerebral cortex, cerebellar cortex and medulla. Higher doses of physostigmine were necessary to produce glycogenolysis in the hypothalamus. In the first four structures glycogen stores were almost equally sensitive to the action of physostigmine. Neostigmine did not affect brain glycogen. The glycogenolytic effect of physostigmine was dose-dependent.
• 3 Both atropine and propranolol were found to block the glycogenolytic effect of physostigmine in brain.
• 4 It is concluded that probably both cholinergic and adrenergic processes participate in the glycogenolytic effect of physostigmine. It is suggested that physostigmine initiates the cholinergic processes which then trigger adrenergic processes.

     

Paradoxical sleep deprivation: effects on brain energy metabolism.

A study was made of brain nucleotides and glycolytic intermediates in paradoxical sleep (PS)-deprived and recovery-sleeping rats. It was observed that PS deprivation of 24 h produced a fall in glucose, glucose 6-phosphate and pyruvate in cerebral frontal lobes. After three hours of recovery sleep all values returned toward their predeprivational levels. In cerebellar hemispheres ATP was increased, while glucose 6-phosphate and pyruvate were decreased. After three hours of recovery sleep, glucose 6-phosphate was increased and pyruvate decreased, indicating restoration of glycogen and creatine phosphate respectively.     

The ontogeny and physiology confirms the dual nature of sleep states

In the Jouvet's laboratory, as early as 1960 the study of the ontogenesis of paradoxical sleep (PS) named "sleep 'with jerks" began in the kitten and led to the first publication in 1961. Then, several species were studied, lamb, rat, human neonates, etc. These works showed that at birth sleep with jerks was preponderant in altricial (immature) species (cat, rat) and the first to appear during the second half of gestation in precocious species (guinea pig). For Jouvet, sleep with jerks is a immature form of PS. Why PS is so important at birth? The maturation of the central nervous system, based on the myelinization, starts in the spinal cord then forwards to the brainstem and forebrain. So, PS mechanisms located in the brainstem are the first to mature and the only one to function. Then the slow wave sleep (SWS) and waking structures become mature. Phylogenetic studies showed that in mammals and birds PS was present even in marsupials and monotremes. Until now only the one exception is the dolphin with a voluntary breathing. To sleep and breath, dolphin has developed an unilateral sleep without classical PS. In other animals, reptiles, amphibians, fishes, PS was not observed with the parameters used in mammals. The study at birth (not yet done) of reptiles would allow perhaps the observation of a temporary PS. Based on these findings, a schematic model of the sleep regulation can be elaborated. Haeckel's aphorism "Ontogeny recapitulates phylogeny" seems true for PS which appears in birds and mammals i.e. at the end of evolution as it appears at the end of gestation when PS cerebral structures are present and mature.     

Neurophysiological analysis of the effects of partial paradoxical sleep deprivation

A neurophysiological study was made of the effects of partial and complete paradoxial sleep deprivation by substituting episodes of active wakefulness for spells of paradoxical sleep (PS) of the same duration in the sleep-wake cycle. Neither accumulated need for paradoxical sleep (culminating in increased onset of PS during deprivation), PS rebound during the post-deprivation period, nor dissociation of the stages of paradoxical sleep resulting in their intervening individually at unaccustomed points in the sleep-wake cycle were observed during our experimental procedure. The phenomenon of self-deprivation, increased heart rate, eye movements, and pontogeniculooccipital (PGO) action potentials also failed to occur during the post-deprivation period. It is postulated that PS requirement and the need for periods of wakefulness stem from the same neurochemical alterations.I. S. Beritashvili Institute of Physiology, Academy of Sciences of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 20–28, January–February, 1988.     

Paradoxical (REM) sleep genesis: the switch from an aminergic-cholinergic to a GABAergic-glutamatergic hypothesis.

In the middle of the last century, Michel Jouvet discovered paradoxical sleep (PS), a sleep phase paradoxically characterized by cortical activation and rapid eye movements and a muscle atonia. Soon after, he showed that it was still present in "pontine cats" in which all structures rostral to the brainstem have been removed. Later on, it was demonstrated that the pontine peri-locus coeruleus alpha (peri-LCalpha in cats, corresponding to the sublaterodorsal nucleus, SLD, in rats) is responsible for PS onset. It was then proposed that the onset and maintenance of PS is due to a reciprocal inhibitory interaction between neurons presumably cholinergic specifically active during PS localized in this region and monoaminergic neurons. In the last decade, we have tested this hypothesis with our model of head-restrained rats and functional neuroanatomical studies. Our results confirmed that the SLD in rats contains the neurons responsible for the onset and maintenance of PS. They further indicate that (1) these neurons are non-cholinergic possibly glutamatergic neurons, (2) they directly project to the glycinergic premotoneurons localized in the medullary ventral gigantocellular reticular nucleus (GiV), (3) the main neurotransmitter responsible for their inhibition during waking (W) and slow wave sleep (SWS) is GABA rather than monoamines, (4) they are constantly and tonically excited by glutamate and (5) the GABAergic neurons responsible for their tonic inhibition during W and SWS are localized in the deep mesencephalic reticular nucleus (DPMe). We also showed that the tonic inhibition of locus coeruleus (LC) noradrenergic and dorsal raphe (DRN) serotonergic neurons during sleep is due to a tonic GABAergic inhibition by neurons localized in the dorsal paragigantocellular reticular nucleus (DPGi) and the ventrolateral periaqueductal gray (vlPAG). We propose that these GABAergic neurons also inhibit the GABAergic neurons of the DPMe at the onset and during PS and are therefore responsible for the onset and maintenance of PS.     

Brainstem structures responsible for paradoxical sleep onset and maintenance

This paper is dedicated to our mentor, Michel Jouvet who inspired our career and transmitted to us his passion for the study of the mechanisms responsible for paradoxical sleep genesis and also that of its still mysterious functions. We expose in the following the progresses in the knowledge in this field brought during 40 years by Michel Jouvet and his team and more recently by the members of a new CNRS laboratory in which we aim to pursue in the path opened by Michel Jouvet.     

Paradoxical (REM) sleep genesis: The switch from an aminergic–cholinergic to a GABAergic–glutamatergic hypothesis

In the middle of the last century, Michel Jouvet discovered paradoxical sleep (PS), a sleep phase paradoxically characterized by cortical activation and rapid eye movements and a muscle atonia. Soon after, he showed that it was still present in “pontine cats” in which all structures rostral to the brainstem have been removed. Later on, it was demonstrated that the pontine peri-locus coeruleus α (peri-LCα in cats, corresponding to the sublaterodorsal nucleus, SLD, in rats) is responsible for PS onset. It was then proposed that the onset and maintenance of PS is due to a reciprocal inhibitory interaction between neurons presumably cholinergic specifically active during PS localized in this region and monoaminergic neurons. In the last decade, we have tested this hypothesis with our model of head-restrained rats and functional neuroanatomical studies. Our results confirmed that the SLD in rats contains the neurons responsible for the onset and maintenance of PS. They further indicate that (1) these neurons are non-cholinergic possibly glutamatergic neurons, (2) they directly project to the glycinergic premotoneurons localized in the medullary ventral gigantocellular reticular nucleus (GiV), (3) the main neurotransmitter responsible for their inhibition during waking (W) and slow wave sleep (SWS) is GABA rather than monoamines, (4) they are constantly and tonically excited by glutamate and (5) the GABAergic neurons responsible for their tonic inhibition during W and SWS are localized in the deep mesencephalic reticular nucleus (DPMe). We also showed that the tonic inhibition of locus coeruleus (LC) noradrenergic and dorsal raphe (DRN) serotonergic neurons during sleep is due to a tonic GABAergic inhibition by neurons localized in the dorsal paragigantocellular reticular nucleus (DPGi) and the ventrolateral periaqueductal gray (vlPAG). We propose that these GABAergic neurons also inhibit the GABAergic neurons of the DPMe at the onset and during PS and are therefore responsible for the onset and maintenance of PS.     

The Inhibition of the Dorsal Paragigantocellular Reticular Nucleus Induces Waking and the Activation of All Adrenergic and Noradrenergic Neurons: A Combined Pharmacological and Functional Neuroanatomical Study

GABAergic neurons specifically active during paradoxical sleep (PS) localized in the dorsal paragigantocellular reticular nucleus (DPGi) are known to be responsible for the cessation of activity of the noradrenergic neurons of the locus coeruleus during PS. In the present study, we therefore sought to determine the role of the DPGi in PS onset and maintenance and in the inhibition of the LC noradrenergic neurons during this state. The effect of the inactivation of DPGi neurons on the sleep-waking cycle was examined in rats by microinjection of muscimol, a GABA_A agonist, or clonidine, an alpha-2 adrenergic receptor agonist. Combining immunostaining of the different populations of wake-inducing neurons with that of c-FOS, we then determined whether muscimol inhibition of the DPGi specifically induces the activation of the noradrenergic neurons of the LC. Slow wave sleep and PS were abolished during 3 and 5 h after muscimol injection in the DPGi, respectively. The application of clonidine in the DPGi specifically induced a significant decrease in PS quantities and delayed PS appearance compared to NaCl. We further surprisingly found out that more than 75% of the noradrenergic and adrenergic neurons of all adrenergic and noradrenergic cell groups are activated after muscimol treatment in contrast to the other wake active systems significantly less activated. These results suggest that, in addition to its already know inhibition of LC noradrenergic neurons during PS, the DPGi might inhibit the activity of noradrenergic and adrenergic neurons from all groups during PS, but also to a minor extent during SWS and waking.     

Self-deprivation of paradoxical sleep in cats

The phenomenon of paradoxical sleep (PS) self-deprivation has been detected and described. The self-deprivation is acquired just as a classical conditioned reflex during enforced PS deprivation both by water tank procedure and by the animal's awakenings in response to sensory stimuli or direct electric stimulation of activating structures of the midbrain and diencephalon, following the transition of slow-wave sleep to PS. In this situation the transition of the brain from one physiological state to another is a conditioned signal, and sensory stimulation or brain stimulation, resulting in arousal reaction, serves as an unconditioned stimulus. It is suggested that the detection and analysis of PS self-deprivation are of a great importance, on the one hand, for correct understanding of the functional significance of this physiological brain state, and, on the other hand, for accurate analysis and assessment of the dissociative processes, observed during PS deprivation and postdeprivation period.     

Induction of paradoxical sleep (PS) by infusion of cerebrospinal fluid from PS-deprived rats

In the rat, alpha-Methyldopa (alpha-MD, 50 mg/kg i.p.) induced during 8 hrs. an important reduction of paradoxical sleep (PS) and an increase of light slow waves sleep (SWS1). These effects were reversed by intraventricular infusion of cerebrospinal fluid (CSF) from PS-deprived donor rats, and PS restauration depended directly on the duration of the deprivation in the donor. It is probable that hypnogenic substances accumulate in the CSF during PS-deprivation, and that these factors can by-pass the noradrenergic step in the chain of biochemical events normally leading to the appearance of PS.     

Central temperature is the principal factor of regulation of paradoxical sleep in pontile cats

In chronic po?kilothermic pontile cats whose central temperature is artificially regulated, the quantity of paradoxical sleep (PS), its ultradian periodicity and the duration of PS episodes are dependent upon central temperature level and periodicity.     

An attempt to influence by drugs recovery sleep after sleep deprivation.

Rats were deprived of sleep by placing them for 36 hours in a slowly moving drum. After this procedure, during recovery sleep, the latency of onset of the first rhombencephalic - paradoxical sleep period decreased and the proportion of telencephalic/rhombencephalic - slow wave sleep reversed (during the first hour of recovery sleep). Repeated administration during the deprivation period of physostigmine (0,5 mg/kg i. p. in 30 min intervals 20-30 times) inducing in waking animals in EEG pattern close to that of rhombencephalic sleep, or atropine (1 mg/kg i. p. in 60 min intervals 10-15 times) evoking an activity resembling telencephalic sleep, did not change the above measures of recovery sleep. Pharmacologically induced sleep-like patterns did not substitute for the sleep the rats were deprived off.     

Auditory deprivation modifies biological rhythms in the golden hamster

To assess to what extent auditory sensory deprivation affects biological rhythmicity, sleep/wakefulness cycle and 24 h rhythm in locomotor activity were examined in golden hamsters after bilateral cochlear lesion. An increase in total sleep time as well as a decrease in wakefulness (W) were associated to an augmented number of W episodes, as well as of slow wave sleep (SWS) and paradoxical sleep (PS) episodes in deaf hamsters. The number of episodes of the three behavioural states and the percent duration of W and SWS increased significantly during the light phase of daily photoperiod only. Lower amplitudes of locomotor activity rhythm and a different phase angle as far as light off were found in deaf hamsters kept either under light-dark photoperiod or in constant darkness. Period of locomotor activity remained unchanged after cochlear lesions. The results indicate that auditory deprivation disturbs photic synchronization of rhythms with little effect on the clock timing mechanism itself.     

Paradoxical REM sleep promoting and permitting neuronal networks

Since its electrophysiological identification in the 1950's, the state of REMS or PS has been shown through multiple lines of evidence to be generated by neurons in the oral pontine tegmentum. The perpetration of this paradoxical state that combines cortical activation with the most profound behavioral sleep occurs through interplay between PS-promoting (On) and PS-permitting (Off) cell groups in the pons. Cholinergic cells in the LDTg and PPTg promote PS by initiating processes of both forebrain activation and peripheral muscle atonia. Bearing alpha1-adrenergic receptors, cholinergic cells, which likely project to the forebrain, are excited by NA and active during both W and PS (W/PS-On), when they promote cortical activation. Bearing alpha2-adrenergic receptors, other cholinergic cells, which likely project to the brainstem, are inhibited by NA and thus active selectively during PS (PS-On), when they promote muscle atonia. Noradrenergic, together with serotonergic, neurons, as PS-Off neurons, thus permit PS in part by lifting their inhibition upon the cholinergic PS-On cells. The noradrenergic/serotonergic neurons are inhibited in turn by local GABAergic PS-promoting neurons that may be excited by ACh. Other similarly modulated GABAergic neurons located through the brainstem reticular formation become active to participate in the inhibition of reticulo-spinal and raphe-spinal neurons as well as in the direct inhibition of motor neurons. In contrast, a select group of GABAergic neurons located in the oral pontine reticular formation and possibly inhibited by ACh turn off during PS. These GABAergic PS-permitting neurons release from inhibition the neighboring large glutamatergic neurons of the oral pontine reticular formation, which are likely concomitantly excited by ACh. In tandem with the cholinergic neurons, these glutamatergic reticular neurons propagate the paradoxical forebrain activation and peripheral inactivation that characterize PS.     

Mass spectrometry-assisted study reveals that lysine residues 1967 and 1968 have opposite contribution to stability of activated factor VIII

The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys(1967) and Lys(1968) of region Thr(1964)-Tyr(1971) are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys(1967)-Lys(1968) within region Thr(1964)-Tyr(1971) has an opposite contribution to the stability of FVIIIa.     

Regional organisation of brain activity during paradoxical sleep (PS)

Human brain function is regionally organised during paradoxical sleep (PS) in a very different way than during wakefulness or slow wave sleep. The important activity in the pons and in the limbic/paralimbic areas constitutes the key feature of the functional neuroanatomy of PS, together with a relative quiescence of prefrontal and parietal associative cortices. Two questions are still outstanding. What neurocognitive and neurophysiological mechanisms may explain this original organization of brain function during PS? How the pattern of regional brain function may relate to dream content? Although some clues are already available, the experimental answer to both questions is still pending.     

Analysis of the action of the sleep neuropeptide (DSIP) on sleep organization in cats and rats

After administration of delta-sleep inducing peptide to cats and albino rats the decrease of total duration of paradoxical phase of sleep is more significant than prolongation of slow-wave sleep. Similar disturbances in the behaviour of animals were observed during deprivation of paradoxical sleep. This data strongly suggest that the DSIP influences the most ancient mechanisms of sleep regulation.     

Tuberal Hypothalamic Neurons Secreting the Satiety Molecule Nesfatin-1 Are Critically Involved in Paradoxical (REM) Sleep Homeostasis

The recently discovered Nesfatin-1 plays a role in appetite regulation as a satiety factor through hypothalamic leptin-independent mechanisms. Nesfatin-1 is co-expressed with Melanin-Concentrating Hormone (MCH) in neurons from the tuberal hypothalamic area (THA) which are recruited during sleep states, especially paradoxical sleep (PS). To help decipher the contribution of this contingent of THA neurons to sleep regulatory mechanisms, we thus investigated in rats whether the co-factor Nesfatin-1 is also endowed with sleep-modulating properties. Here, we found that the disruption of the brain Nesfatin-1 signaling achieved by icv administration of Nesfatin-1 antiserum or antisense against the nucleobindin2 (NUCB2) prohormone suppressed PS with little, if any alteration of slow wave sleep (SWS). Further, the infusion of Nesfatin-1 antiserum after a selective PS deprivation, designed for elevating PS needs, severely prevented the ensuing expected PS recovery. Strengthening these pharmacological data, we finally demonstrated by using c-Fos as an index of neuronal activation that the recruitment of Nesfatin-1-immunoreactive neurons within THA is positively correlated to PS but not to SWS amounts experienced by rats prior to sacrifice. In conclusion, this work supports a functional contribution of the Nesfatin-1 signaling, operated by THA neurons, to PS regulatory mechanisms. We propose that these neurons, likely releasing MCH as a synergistic factor, constitute an appropriate lever by which the hypothalamus may integrate endogenous signals to adapt the ultradian rhythm and maintenance of PS in a manner dictated by homeostatic needs. This could be done through the inhibition of downstream targets comprised primarily of the local hypothalamic wake-active orexin- and histamine-containing neurons.