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8-th Multidisciplinary International Conference of Biological Psychiatry "Stress and Behavior"
Interaction of the GABAA receptor agonist THIP with sleep homeostasis in mice. I. Tobler, C. Kopp, G. Bosch, Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland. Biochemical and neurophysiological evidence support a role of the main central inhibitory neurotransmitter Gamma-aminobutyric acid (GABA) in sleep regulation. In rats and humans the GABAA receptor agonist THIP is known to enhance slow-wave activity (SWA; EEG power 0.75-4 Hz) in non-REM sleep, mimicking the response to sleep deprivation (SD). Although pharmacological agents enhancing or mimicking the action of GABA on GABAA receptors influence sleep and the sleep electroencephalogram (EEG), it remains unclear whether GABAA receptors are involved in the physiological regulation of sleep. Our aim was to investigate the interaction between THIP and sleep homeostasis. Methods. Sleep was recorded for 12 h in two groups of C57BL/6 mice kept individually in Macrolon cages (36 x 20 x 35 cm), with food and water available ad libitum, and maintained on a 12 h light - 12 h dark cycle (light from 07:00-19:00 h) at 22-24° C ambient temperature. One group (SD group; n=10) was subjected to 3 h SD immediately followed by an injection of THIP (4 mg/kg) or vehicle. Group 2 (control; n=10) was treated with THIP or vehicle at the time of day corresponding to the end of the SD in the first group, i.e. 3 h after light onset. The vigilance states NREM sleep, REM sleep and waking were scored for 4-s epochs and EEG power spectra were computed for the corresponding epochs. Results and discussion. THIP significantly reduced non-REM sleep latency in both groups. The amount of non-REM sleep and waking were not affected by THIP, while REM sleep was initially reduced in both groups. A prominent increase of SWA in non-REM sleep occurred after THIP and after SD alone. However, also in the waking EEG SWA was increased after THIP. In the SD group, SWA was further increased by THIP, but reached similar levels as in the control group receiving THIP alone. After THIP the dynamics of SWA at the transitions of waking to non-REM sleep were unchanged, despite higher SWA levels in non-REM sleep and in waking. This contrasted to SD alone, which led to a faster build-up of SWA during the first 3 h of recovery. The changes in sleep and the sleep EEG induced by THIP in mice confirmed the hypnotic properties of this GABAergic compound. The enhancement of SWA induced by THIP in mice corresponds to the effects of the GABAA receptor agonist muscimol, and is in contrast to the typical SWA suppression induced by benzodiazepines. The rapid recovery of SWA after treatment with THIP is consistent with its rapid absorption. Conclusion. The results do not support the notion that THIP exerts its hypnotic and SWA-enhancing effect through mechanisms underlying the homeostatic regulation of sleep. The study was supported by the European Union grant QLK6-CT-2000-00499. (30 min)
The dynamics of spindles and delta EEG activity during sleep in mice: interhemispheric coherence and effects of sleep deprivation. V.Vyazovskiy, P. Achermann, A.A. Borbely, I. Tobler. Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland. The temporal and spatial dynamics of slow-wave activity (SWA, EEG power between 0.5-4.0 Hz) and spindle EEG activity during sleep may reflect complex regulatory processes. Vigilance state-related topographic variations of EEG activity have been reported in humans and animals. SWA is considered a marker of sleep homeostasis but little is known about the role of spindle activity in sleep regulation. Analysis of regional dynamics of EEG oscillations in specific frequency bands during sleep may provide a clue for elucidating the mechanisms underlying sleep regulation at a global level. Congenital dysgenesis of the corpus callosum in mice provides an opportunity to address the substrate of interhemispheric EEG synchronization during sleep. Methods. Sleep was recorded in male mice with congenital callosal dysgenesis (B1, total dysgenesis, n=3; partial dysgenesis, n=6) and in normal C57BL/6 mice (n=7) during a 24-h baseline, 6-h sleep deprivation (SD) and subsequent 18-h recovery. A parietal and frontal derivation was recorded from each hemisphere. NREM sleep, REM sleep and waking were scored for 4-s epochs. The occurrence of spindles and their relationship to the EEG power spectrum (2s epochs) was analyzed and coherence between hemispheres was computed in C57BL/6 mice. Results and discussion. Distinct spindle events with a dominant frequency approximately at 11 Hz were present in the frontal derivation and recognized as EEG bursts. Spindles were most abundant in NREM sleep and increased before NREM-REM sleep transitions. Whereas spindles increased concomitantly with SWA at the beginning of a NREM sleep episode, these measures showed an opposite evolution prior to the transition to REM sleep. The 24-h timecourse of spindle density (n/1 h) showed a maximum at the end of the 12-h light period, and was a mirror image of SWA in NREM sleep. After 6 h SD the spindles in NREM sleep were initially suppressed, and showed a delayed rebound. In contrast, spindles occurring immediately before the transition to REM sleep were enhanced initially after SD. In C57 mice spindles occurred simultaneously in the left and right hemisphere. To assess their relationship, interhemispheric coherence spectra were computed between the left and right bipolar (fronto-parietal) derivations. Interhemispheric coherence of 12-s epochs in NREM sleep where spindles occurred was significantly higher between 10-12.5 Hz compared to epochs without spindles. To investigate the role of the corpus callosum in interhemispheric EEG synchronization, coherence spectra were compared between the mice with different degrees of congenital callosal dysgenesis. Interhemispheric EEG coherence in B1 mice was significantly lower than in control mice in all three vigilance states. In addition, the level of coherence in each of the three totally acallosal mice was lower than in the mice with only partial callosal dysgenesis. The largest strain difference was seen in NREM sleep where coherence values were reduced in acallosal mice compared to control mice for all frequency bins. In control mice, SD led to a rise of coherence in the delta band of NREM sleep in the first 2 h of recovery. This effect was absent in B1 mice with total callosal dysgenesis and attenuated in mice with partial dysgenesis. In REM sleep and waking, interhemispheric coherence was lower in B1 mice than in controls with the exception of two frequency bands (3-7 Hz). Conclusion. These results provide further insights into regional mechanisms of sleep regulation. The large-scale synchronization of major NREM sleep EEG rhythms - delta- and spindle-activity may indicate that interhemispheric interactions between homologous cortical regions mediated by the corpus callosum have a functional significance. In contrast, interhemispheric synchronization of theta oscillations in waking and REM sleep may be mediated by direct interhippocampal connections. In addition, this study provides the first quantitative assessment of sleep spindles across 24 h and after sleep deprivation in mice. The data suggest that spindles in NREM sleep may be involved in sleep maintenance, while spindles heralding the transition to REM sleep may be related to mechanisms of REM sleep initiation. Supported by the Swiss National Science Foundation grants 3100-053005.97 and 3100A0-100567 and Human Frontier Science Program RG00131/2000. (20 min)
Change of wakefulness-sleep cycle in rabbits following dimethyldipyrazolelselenid administration. Chichinadze O.N., Kosenko P.O., Shmalko N.Y. Rostov State Pedagogical University, Rostov-on-Don, Russia. The important role of selenorganic compounds has been shown by pioneer studies of V. Vernadsky. It has been established later that most of selenium in animal proteins is in the form of selenometionin and selenocysteine, replacing methionine in various proteins fibers, and forming active center of many enzymes (glutathioneperoxidases, iodothyroninedeiodinases and selenoproteins), respectively. The minimal daily need of selenium is 1mkg/kg. It has a very narrow physiological dose range, with a little gap between insufficiency and toxicity. Selenorganic compound dimethyldipyrazolelselenid (DMDPS) was synthesized in Rostov State University (Russia). It possesses strongly pronounced biological activity with marked antioxidants properties. Toxicity of DMDPS is lower than toxicity of sodium selenite. In line with the hypotheses that wakefulness-sleep cycle can be based on cyclicity of free-radical processes , we studied the effect of DMDPS on wakefulness-sleep cycle in rabbits (chinchilla strain, 3 kg), with preliminary implanted cortico-afferent electrodes in the frontal, somatosensory and visual zones. Following 10 ml water solution of DMDPS injected i.p. (70 mkg/kg), we registered ECoG, EMG (cervical muscles), ECG, and body temperature. Single injection of DMDPS resulted in 2-fold increase in wakefulness, with almost unaltered duration of sleep, during the first 5 h after treatment. However, the increased duration of the second and third stages of slow sleep and their depths, and sleepy spindles of greater amplitude and frequency, and greater synchronism at different locations, were seen. During the next hour there was a gradual return to initial duration of the wakefulness. At chronic doses, DMDPS (3 times per for 5 days) also resulted in similar increase in total wakefulness, while the number of sleep cycles was slightly reduced. In contrast to controls, increased slow sleep, especially the third stage, was observed, as well as some increase of intra-spindle frequency, together with lesser frequency of delta of waves (1,5-4 to 0,2-2 Hz). In control animals no changes of a cycle wakefulness-sleep was observed. Literature: 1. Dorofeenko A.I. et al. Patent 2185819 of 27.07.02. 2. Ananjan A.A. et al. Free-radical processes of an organism of the person in the cycle "wakefulness-sleep". In "Sleep - a window in the world of wakefulness". Moscow 2003. (10 min)
Neuronal activity in thalamus and the big hemispheres in rabbits during the slow sleep. Burikov A.A., Svetlova N.V., Chichinadze O.N., Chuguev O.I., Shmalko N.J. Rostov State Pedagogical University, Rostov-on-Don, Russia. Analysis of the role of different brain structures in the organization of sleep usually is based on the information on when and at which phase the neurons become excited, i.e. have a high level of pulse activity (AP) and on what are inactive - pulse categories are absent or their frequency low. Our attention was focused on structures of the forebrain which are crucial for the genesis of rhythms (EcîG), and also, in the organization of sleep [1,2]. However, AP analysis of neurons at different structures in slow sleep has not been yet performed. Our experiments used adult rabbits, fixed and under artificial breathing (temperature 37.0 oC) with continuous evacuation of contents of bladder. Mono ECîG was registered from the surface of cortex. AP of neurons was assessed extracellularly (only bodies of neurons, initital-positive solderings by amplitude >0,5mV). We analysed neuronal AP in 123 neurons of sensomotory cortex, 112 neurons of the central medial nucleus (nCm), 83 neurons of the reticular nucleus (nRt), 71 neurons of ventroptoslateralis nucleus (nVPL). Results. Episodes of wakefulness (irregular, low-amplitude ECîG) alternated with longer episodes of slow sleep (sleepy spindles and delta-activity). We noted short transitions from wakefulness to sleep, and back. However, neurons did not stop the activity at one stage of a cycle wakefulness-sleep. Overall, thalamus in general had the maximal activity of neurons in slow sleep. Thus, the majority of neurons are unloaded to packs of pulses which (infrascpindles to waves). Frequency of categories in packs reached 300 imp/s. Neuronal activity in nRt had duration up to 300 ms, while in other thalamic structures the prevailing duration was <30 ms (Burikov, 1985, 1989). Separate delta-waves or their groups were not accompanied by altered frequency of AP or MT of cells unless these waves preceded spindles, as though starting the last. Then changes in AP correspond to what we observed for the spindles. During sleep with generalized delta-activity, neurons again had lower frequency of AP. Notably, the greatest dynamics of reorganization of AP was observed in "slow" neurons. Neurons of nVPL changed frequency of AP vs. neurons of nCm and nRt, at transition from active wakefulness to sleep. Transition from wakefulness to Slow sleep did not change neuronalAP, except for a stage of spindles. Neuronal cells in the cortex showed high level of AP developing slow sleep with regular delta-activity, showing expressed communication of packs, and is more often - groups of pulses with phases of deltas-waves. At awakening, numerous neurons are being activated. In wakefulness, thalamic neurons generally showed lower frequency AP. Literature: 1. Burikov A.A. Neurophysiological basis, strategy and tactics of management by a functional condition of a brain. Problems of neurokibernetics: diagnostics and correction of a functional condition. Rostov-on-Don, 1989. 2. Steriade M., McCarley R. In: Brainstem control of wakefulness and sleep. New York: Plenum, 1990. (15 min for the two)
Behavioral and neurophysiological characteristics of a cycle activity-sleep of turtles. Burikov A.A., Sevostjanova M.V., Eryomenko E.A., Kosenko P.O., Balytskaja A.V. Rostov State Pedagogical University, Rostov-on-Don, Russia. Currently behavioral and neurophysiological studies of cycle activity-sleep in poikilothermic animals are becoming increasingly important to assess the general mechanisms of activity and its inhibition during sleep. Since reptiles represent the lower animal taxons, than mammal and birds, possessing "real" sleep, their sleep is a very interesting object to study. Sleep has distinct division into stages and phases (2), and differs from sleep-like conditions at which, according to widely accepted views, such division is absent. Analysis of bioelectric activity of a forebrain and thalamus in superficial and deep sleep is a key aspect of sleep research. Our present study aimed to reveal features of these forms of sleep using "SAGURA" polygraph. Analysis of sleep in turtles revealed several forms of sleep and activity, which differ electrophysiologically. In the transition from low activity to superficial sleep, the reduction of frequency of EEG rhythms can be generally observed. Here we show that in turtles, several periods (usually 2,5-5 s) can be found, including: 1) retraction of eyeballs; 2) twitching; 3) series of fast movements of eyeballs. These periods appear irregularly, each 3-60 s. Overall, these features are very similar to sleep with fast eyes movements in mammal and, especially, birds. In the deep sleep, there were episodes of high-amplitude low-frequency activity (0,5-5 Hz) with smoothed waves up to 30s - the patterns which are never seen in activity, except for partial sleep and deep sleep as intermediate conditions. It is necessary to note that turtles' response to external stimuli (sound, light) is opposite to those in mammals, and is characterized by increased frequency of EEG components and EEG synchronization. (1). Thus, we can suggest that sleep in turtles is not a homogenous condition, and their sleep stages differ not only quantitatively but also qualitatively. In reptiles, at least in turtles, it is possible to divide sleep into two phases which can be considered as predecessors of sleep of the superior animals (with its traditional 2 phases with and without fast eyes movements). Literatures. 1. Belichova M.G. Thalamo-telencephalic system of reptiles. Moscow, 1977. 2. Kovalzon V.Ì. Mag. Evol. Biochem. Physiol., 1993, 29, 627-634.
Influence of Ultram on integrative activity of the brain. I. Rukhadze, T.Basishvili, I.Gvilia, N. Darchia, M. Eliozishvili, N. Oniani, Department of Neurobiology of Sleep-Wakefulness Cycle, I.Beritashvili Institute of Physiology, Tbilisi, Georgia. Investigation of basic mechanisms of Ultram-induced deterioration of integrative activity of the brain may play an important role in search for ways of correction during disorders concerned with substance intake. The aim of our investigation was study of influences of Ultram on general behavior and structure of sleep-waking cycle (SWC) in animals. Methods: Experiments were performed in chronic preparations of adult cats (n=4) of either sex, 2.5-3 kg. In order to make polygraph records of SWC, metal electrodes were implanted into various structures. Different doses (0.25 mg/kg and 0.5 mg/kg) of Ultram were injected intraperitoneally. The data obtained were evaluated statistically by the Student's t-test. Results and Discussion: General pharmacological impact of Ultram was manifested in total deprivation of sleep and development of "hallucinatory" type behaviors. These effects were dose-dependent. Under action of the preparation solely waking state represents the cycle and active waking stage is characterized with variety of hallucinatory type of behavioral acts - vigorous play with imaginary items in the cage, and deteriorated coordination of movements. An animal was non-responding to external sounds and visual stimuli. Normalization of quantitative and qualitative alterations in electrical activity of the brain begins following termination of the period of active action of the injected substance, which is a fairly long time. In the post-deprivation period rebound of deep slow sleep and increase of total paradoxical sleep does occur. Complete recovery of SWC develops in about 24 hr after post-deprivation period. Regular waking recovers first and only than sleep, with normal alternations of all its phases and stages. Analysis of above changes points at multi-facet character of Ultram action. In generation of its effects both opioid and non-opioid mechanisms are involved. Conclusions: In causation of SWC structure deterioration a leading part must belong to the non-opioid mechanism of Ultram. In this aspect the Ultram-induced effects of monoamines (especially of serotonine) re-uptake blockage and respective increase of their concentration in the brain, are most evident. (15 min)
Effectiveness of Ivadal for the Treatment of Sleep Disturbances in Patients with Posttraumatic Stress Disorder. Sukiasyan S, Manasyan N, Babakhanyan A, Kirakosyan A. Centre of Mental Health "Stress", Yerevan, Armenia. Sleep disorder is one of the most widespread symptoms, which occurs frequently both in healthy population and patients with mental disorders. Sleep disturbances occur in 50% of the population, and have a major impact on the psychological, social and economic functioning of the society. Insomnia is polygenic and multifactorial disorder, and it always develops secondary. The main causes of sleep disturbances are anxiety and depression. Insomnia is one of the most important manifestation in the clinical structure of Posttraumatic Stress Disorder (PTSD). Due to the increased level of anxiety and irritability, in the patients with PTSD we observe all types of sleep disorders, i.e. difficulties in falling asleep, interrupted sleep, awakening in the early morning, and, besides this, they suffer from nightmares. Correction of sleep disturbances is very important part of the complex treatment of PTSD. In the present study we wanted to assess the effectiveness of Ivadal (Zolpidem) in the treatment of sleep disorders in patients with PTSD. Ivadal is a non-benzodiazepine hypnotic drug (derivate of imidazopyridin) designed and introduced in 1988 by Sanofi-Synthelabo (France). Ivadal is one of the popular hypnotics of last years, because pharmacological characteristics of Ivadal are near to the "ideal" hypnotic drug. Material and methods: 18 patients, ex-combatants of Karabakh war, who met the DSM-111 criteria of PTSD, where included into the study. All patients had different sleep disturbances: difficulties in falling asleep interrupted sleep, awakening in the early morning, nightmares. Patients were receiving 10 mg Ivadal at the bedtime during 4 weeks - 28 days. Assessment of patients' condition was undertaken three times: before the treatment (basic examination), at tha 14th day (middle examination), and at the 28th day (last examination). In order to evaluate the duration and quality of sleep, as well as the well-being and activity of patients during the period of treatment, several experimental-psychological tests had been used, such as SCL-90; "number arrangement" test; the test for evaluation of "imagination memory"; " Subjective evaluation of satisfaction with sleep" questionnaire (self-elaborated). Results and discussion: Initial examination showed that most of patients had pronounced sleep disturbances, including difficulties with falling asleep (12 cases), interrupted sleep (12 cases), awakening in the early morning (5 cases), as well as the combination of all mentioned types (5 cases). 78% of patients were unable to sleep at least for 1 h after going to bed. The sleep for <3 h. over night was observed in 86% of patients. Moreover, most of the patients showed pathological characteristics of attention, learning ability, imagination memory, etc. During the treatment with Ivadal, all these parameters had stable tendency to improvement. After 14 days of receiving Ivadal only 14% of patients were dissatisfied with sleep yet, since other parameters of testing were still maintaining at the same level. After 28 days of treatment the positive dynamics of all parameters had been observed. Thus, following 4-week period, 93% of patients showed the improvement of night sleep. In 93% of patients, parameters of imagination memory became normal. At 28th day of treatment, only 14% of patients had high level of intellectual lability (in vs. 36% at the beginning examination). Positive dynamics of SCL-90 parameters also had been observed. At 14th day of treatment pathological levels of "somatization", " aggression", "trouble falling asleep" and " disturbed sleep " had reached approximately normal levels. No one of patients stopped the treatment before the end of 4-week period, because there were no unpleasant effects of Ivadal during the whole treatment period. Conclusions: We observed the high effectiveness of Ivadal for the treatment of sleep disorders in the patients with PTSD, showing improvement of both sleep and physical well-being. However, noone wished to continue the treatment, because, as we suggest, Ivadal did not eliminate other symptoms of PTSD, such as nightmares, flashbacks, personal disorders, etc. References: 1. Avedisova A, Alexandrovski Yu, Psych. Psychopharmacother., 2000, 2 (2). 2. Alexandrovski, Yu, Avedisova A. Clin. Pharmacol. 1995, (4). 3. Alexandrovski Yu, Avedisova A, Contemp. Psychiatry, 1 (3), 1998. (10 min)
Chronic carotid occlusion and sleep in rodents. V.M.Kovalzon, V.V.Loginov, V.B.Dorokhov, G.N.Fesenko, Inst. Ecology and Evolution, Inst. Immunopathology, Inst. Higher Nervous Activity and Neurophysiology, RAS, Moscow, Russia. Classical morphological studies of the cerebral ischemia and hypoxia revealed not only degenerative but also restorative processes in the brain tissue, including stem cells proliferation . On the other hand, classical somnological studies of I.Oswald in 1970s clearly demonstrated an important role of the natural sleep in tissue restoration . While studing sleep structure changes in healthy humans and patients after influences such as physical and mental exercises during the day, starvation, sleep deprivation and acute intoxication of psychotropic drugs, Oswald et al. suggested that general anabolic processes in the brain and body cells are realized mostly during slow wave sleep (SWS) periods, though specific synthetic processes into the central nervous system took place during fast (paradoxical) sleep (PS) phase. In accordance to this, objective qualitative changes of sleep structure should be anticipated in survived post-ischemic rats, related to a sharp decrease in cerebral energetic and synthetic potential which is followed by activation of intrinsic reparative functions of the brain . To examine this hypothesis, young male Wistar rats (180-200 g) were implanted (under pentobarbital anesthesia, 40 mg/kg, i.p.) with electrodes for the cerebral cortex and hippocampal EEG, and the EMG of neck muscles; after this one or both carotid arteries were completely and permanently ligated. On each day (09-12.00 h), free-moving animals were subjected to polygraphic recording until the 40th day since operation. From the total amount of more than 100 rats, only 30 animals have survived (30%). Survival level was the same after unilateral and bi-lateral occlusion. EEG revealed a tremendous increase in PS percentage, reaching almost 20% of the recording time (RT), or 30% of total sleep time (TST) 1-2 days after occlusion, then gradually decreasing day by day, finally reaching the control level of 2.5% of RT (6% of TST) by the end of the observation period (40 days). SWS percentage did not show significant changes within the observation period. Unilateral and bilateral occlusion resulted in the same PS increase. The same procedure has been performed in aged male chinchilla rabbits (2-2.5 years, 4-5 kg) preliminary implanted (under local procain anesthesia) with the same electrodes for polygraphic recording. Several weeks later when the animals were completely rehabilitated and fully adapted to experimental chambers, they were subjected to uni-lateral complete and permanent carotid occlusion under chloral-hydrate anesthesia (0.5 g/kg, i.p.). As in a case of the rats, survival level was about 25-30%. The survived free-moving animals were subjected to continuous 24-hr (12/12 LD) digital paper-less polygraphic recording on the 5th, 9th, 18th and 24th day since the operation as well as before it (0 day). Preliminary results confirmed an important gradual increase in PS following occlusion: from 1.6% of RT on the control day 0 (0.8% during the dark and 2.4% during the light period) up to 4.5% on the 18th day (3.1 and 5.9%, consequently). SWS also increased from 31.5% of RT on day 0 to 48% on the 18th day. On the 24th day both parameters begun to decrease (46% of SWS and 4.3% of PS). The results strongly support a hypothesis of the paramount role of PS in cerebral tissue restoration. Minor discrepancies between two experimental series could be attributed to species and age differences as well as experimental conditions. It is known from both clinical and experimental studies that cerebral stroke is followed by a general shift of the brain activity down to a predominance of phylogenetically old mechanisms, that is in a good concordance to an idea of evolutionary ancient origin of PS mechanisms . Thus, the present results could be used in clinical studies for estimation of a stroke itself and time-course of its restoration . References 1. Gasanov R.I. et al. The dynamics of sleep structure in patients with stroke. In: Modern problems of somnology. Ìoscow, 1998. P. 29. 2. Kositsyn N.S. et al. Dokl.Biol.Sci. 1988. 298. 247-250. 3. Kovalzon V.M. Origin of sleep.Neurobiology of Sleep-Wakefulness Cycle. 2002. 2. 33-36 (http://www.sleepcycle.org). 4. Loginov V.V., Dorokhov V.B. JVND. 2003. 53, 677-680. 5. Oswald I. In: Self-regulation of sleep process. Leningrad: Nauka, 1977. P.12-14. (15 min)
The control of the Slow-Wave sleep development changes anxiety manifestations. E.V.Verbitsky, I.A.Topchiy, Kogan Research Institute for Neurocybernetics of Rostov State University, Rostov-on-Don, Russia, Washington State University, Pullman, WA, USA Anxiety manifestations and disturbances are closely connected with the "sleep-wakefulness" cycle development . Rhythmic interactions between loci of the cerebral cortex and sites of the medial thalamus are of great importance for the development of slow-wave sleep, being reflected in the sleep spindle generation . Later the method for changing these interactions by the rhythmic sub-threshold stimulation of several sites in the medial thalamus and modeling some of sleep disturbances, has been developed . The possibilities of the on-line computer control of slow-wave sleep according this method and influences of resulting effects on anxiety manifestations in rabbits have been investigated in this paper. METODS. States of wakefulness, cycles, phases and stages of sleep were investigated in 18 rabbits with recording and stimulating electrodes chronically implanted in 8 areas of sensorymotor, parietal and visual cortex and sites of n. Centralis medialis (NCM) of the thalamus. Bipolar tungsten electrodes in glass isolation were placed in thalamic sites with low thresholds of initiation of recruiting response in the cortex (10 Hz, 81-124 µA, 0.08 msec; distance between electrodes tips: 80µ). Animals were placed in the box, elastic tether of recording electrodes did not restrict their locomotions. The character of wakefulness and depth of sleep have been analyzed off-line by the polygraph SAGURA-2000 ("Sagura Medizintechnik GmbH", Germany) and on-line by the computers . RESULTS AND DISCUSSION. During the development of sleep stages 2-3 microstimulations of 2 NCM loci with low thresholds of recruiting responses by 2 successions of weak current pulses (10 Hz, 2-43 µA, 0.08 ms) did not evoke spindles of recruiting response in the cortex. The time shift of 0.2 ms between sequences of current pulses allowed us to increase the synchronism in the appearance of sleep spindles over the cortex (as being in the 3rd sleep stage) and the shift of 20 ms decreased it (as being in the 2nd sleep stage). Correlation between amplitude and time indices of the spindles in the cortex and in the thalamus (the amplitude, the duration, phases of increase and decrease) revealed the increase or decrease of thalamo-cortical interactions (during the shift of 0.2 or 20 ms between sequences of microstimulations, respectively). On-line control of these interactions by the computer switch between regimes of NCM microstimulation allowed us to initiate prolongation or deprivation of the 2 or 3 sleep stages during 12-36 min. It has been revealed, that high anxious rabbits as compared with low anxious ones were characterized by more fragmented sleep with the significant variability of 2-3 stages of its slow-wave phase. Representation of deep sleep stages in them was decreased and the latency of their development increased. A lot of activations of different nature were registered in their NREM sleep. Computer-aided prolongation of 2-3 stages of slow-wave sleep promoted the decrease of the stages segmentation and initiated manifestations of low anxiety during subsequent wakefulness (by the character of locomotions, grooming, etc.) as well as by development of drowsiness and 2-3 stages of sleep, characterized by the decrease of the quantity of activations and movements. As opposed to the prolongation computer-aided deprivation of 2-3 stages initiated both the increase of segmentation of sleep stages during the slow-wave sleep and high anxiety manifestations during subsequent wakefulness. It also evoked an increased quantity of activations and episodes of motor activity in subsequent sleep. The data obtained are regarded in the mutual connection with processes of synchronization of slow thalamo-cortical oscillations in wakefulness and slow-wave sleep. CONCLUSION. The development of slow-wave sleep is of great importance for the anxiety manifestations. Thus, the prolongation of 2-3 stages of sleep produces subsequent reduction of anxiety, while their deprivation initiates high anxiety, reflected in changes of behavior and development of sleep. Interactions between cortical loci and sites of medial thalamus could not only play an important role in the development of slow-wave sleep, but also influence electrophysiological and behavioral manifestations of high or low anxiety of the organism. REFERENCES: 1. Vein A.M., Heht K. Human sleep. Physiology and pathology.- M.:Medicina, 1989. 2. Verbitsky E.V. Investigation of the organization of the thalamo-cortical system by indices of spindle activity during the development of slow-wave sleep. Ph. D. diss. Rostov-on-Don, 1980. 3. Feldman G.L., Verbitsky E.V. Method of simulation of sleep disturbances. Patent 1168266 USSR, sub. 3488716, 4. Steriade M. J. Neurophysiol. 86 (1):1-39, 2001. Authors are grateful to Dr. V.B.Dorokhov for the possibility of working on the somnograph SAGURA-2000. (15 min for the two)
Slow-Wave Sleep, Inter-System Interactions in the Brain and Anxiety. E.V.Verbitsky, Institute for Neurocybernetics, Rostov State University, Rostov-on-Don, Russia. Anxiety as the stable peculiarity of the organism to connect the increase uncertainty of the current situation with the potential threat attracts the growing attention of specialists, searching adequate approaches to investigation of its certain manifestations in animal models. However, strong stimuli and aversive situations are rather more adequate to study stress, than anxiety [1,2]. Additionally, the gradual decrease of uncertainty during the reduction of wakefulness, transition to drowsiness and development of sleep is of undoubted interest for the search of the united methodological approach both in somnology and in the analysis of anxiety manifestations. Accordingly, the approach, based on the investigation of anxiety manifestations in animals under gradual decrease of both environmental uncertainty and suppression of orienting response has been developed. METHODS. 12 cats were selected to high and low anxiety groups using behavioral tests. EEG was recorded from visual and acoustic cortices, parietal associative cortex, hypothalamus, caudate nucleus by platinum, chronically implanted electrodes. Fragments of EEG after stimuli (flashes of white light: 1 ms; 5J; .017Hz) were analyzed by the correlation method with calculation of the association coefficient, reflected isolability of investigating brain areas as the main condition of their interaction . Analysis of the involvement of cortico-cortical and cortico-subcortical levels of the brain to anxiety manifestations was carried out before the orienting response suppression, in the middle of this process and after its total suppression during the following transition to the drowsiness and sleep Cycles, phases and stages of sleep were determined by the computer-based polygraph SAGURA-2000 (Sagura Medizintechnik GmbH, Germany). RESULTS AND DISCUSSION. It was found, that in animals with low anxiety as compared with high anxious ones connections between visual, parietal and somatosensory cortices decreased after the total suppression of the orienting reflex (from 28.6 to 3.3%), that could explain the total absence of reactions on acoustic stimuli. High anxious animals as compared with low anxious ones were characterized by close mutual connections between parietal cortex and caudate nucleus (89.3%). Connections between somatosensory cortex and caudate nucleus were slightly less strong in them (48.3%). After the total suppression of the reflex connections between caudate nucleus and parietal cortex decreased to 16.1% on average and connections between caudate nucleus and somatosensory cortex sharply increased up to 81.3%, that specifies the information from other authors . Such changes of interactions on the background of more close connections between somatosensory, visual and parietal cortices in high anxiety cats as compared with low anxiety ones (14.3 vs. 6.8%) demonstrate the priority of the frontal cortex influences and point out the conditions for the spread of excitation, that could explain the preserved ability of these animals to react to acoustic stimuli even after the total suppression of the orienting response. According the somnological analysis, animals with high anxiety as compared with low anxious ones had more segmented sleep with the significant variability in 2-3 stages of slow-wave sleep. These animals had decreased representation of deep sleep stages with longer latency of their development. During NREM sleep they also showed activation (EEG, EOG, EMG etc.), resembling some sleep disturbances in anxious and neurotic states . CONCLUSION. High anxiety could be explained from the standpoint of the preservation of conditions for the spread of excitation not only from the frontal cortex to another cortical areas, but also both to the caudate nucleus "the center of the inhibitory control of the motor activity" and to the other subcortical structures, probably, such as the medial thalamus, inhibition of which results in the decrease of thalamo-cortical synchronizing influences. The latter can be the reason of abnormal switches between slow-wave sleep stages, reflected in both increased latency and segmentation of these stages. REFERENCES. 1. Kalueff A.V. Stress, anxiety and behavior. Kiev, 1998. 2. Krupina N.A. et al. ZhVND, 49(5):864-75, 1999. 3. Verbitsky E.V. Psychophysiology of anxiety. Rostov-on-Don: RSU, 2003. 4. Merzhanova G. Kh. et al. ZhVND, 43(3):487-92, 1993. 5. Iznak A.F. Proc. 2 All-Russ. Conf. "Sleep - the window to the world of wakefulness". Moscow, 2003: 43-44. Authors are grateful to Dr. V.B.Dorokhov for the possibility of working on the somnograph SAGURA-2000.
STRESS-INDUCED ALTERATIONS IN NEUROPHYSIOLOGICAL AND NEUROENDOCRINE CHARACTERISTICS OF SLEEP-WAKE CYCLE IN YOUNG RATS. Aristakesyan E.A., Kuzik V.V., Makina D.M., Oganesian G.A., Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS, St-Petersburg, Russia. Stress is known to be one of the causes of sleep disturbances and the formation of different pathologic conditions. The adolescent period is the stressful stage of ontogenesis . Therefore the analysis of sleep-wake cycle (SWC) and the changes in neuroendocrine systems during a stress in young adolescent organisms is actual problem of aging physiology. The aim of the study was to investigate how photostimulating (PhS) and sleep deprivating (SD) stresses influent the behavioral reactions of young and the dynamics of SWC, of the morphofunctional characteristics in hypothalamo-hypophysial neuroendocrine system. Method. The study was taken in the male of Wistar rats (30-45 day age). The animals was divided to three groups: A (n=30), B (n=30) and C (n=14). The locomotor and exploratory activity in "open field" test after 6-h SD and after 1, 3, and 5 PhS was analyzed in group A only. The morphofunctional parameters of the hypothalamo-hypophysial neuroendocrine cells and triiodthyronin (T3), thyroxin (T4), thyrostimulating (TSH) and adrenocorticotopic hormones (ACTH) levels in blood serum was studied in the animals of B group under the same experimental conditions. The SWC was investigate (C group rats) with electrodes implanted into somatosensor and visual cortex, n.caudatus, hippocampus and preoptic hypothalamus. The SD procedure was performed by the putting rats into the automatically rolling wheel (4 rotation/min). The PhS (10s light:10s dark for 1/2 h. daily) was made for 5 days. This procedure promoted the photogenic catalepsy in amphibians, reptilians, birds and some mammals . This condition was also found in SWC only during pubertant period their life (20-45 days) . The cataleptic state is considered as an ancient form of sleep behavior in vertebrates . It is presented in the SWC of all poikylotherm vertebrates and birds and disappeared in mammals. Results and discussion. The quantitative analysis of locomotor and exploratory reactions of the young rats in "open field" was practically the same that in adult animals. After 6-h. SD the locomotor activity was decreased and exploratory activity was increased. After PhS led both reactions inhibited (p<.05). The SWC in intact young rats was presented as a rule by wake (47.2+5.6 %), slow-wave or NREM sleep (32.3+5.8%) and paradoxical or REM sleep (12.0+3.8%) and cataleptic stage (8.8+2.4%). Cataleptic stage was twice increased to the end of SD and during the first 30-40min of postdeprivated sleep. Real sleep rebound was development during the 2-th h. of postdeprivation period. The duration of NREM and REM sleep increased. A tendency to the restoring of SWC parameters during the 3-d h. of postdeprivation period was found. Increasing of wake and decreasing of NREM and REM sleep durations accompanied the first PhS. The quantity characteristics of SWC came back to the basic level to the 4-th PhS procedure only. The duration of cataleptic stage increased from 6.9% to 16.3% to the 3-d PhS procedure. Then it decresed gradually to background levels. The episodes of NREM sleep were become shorter during posttimulation sleep and were often interrupted by short EEG microactivations, quality of which was increased twice to the 3-d PhS. The deep stages of NREM sleep were registered more seldom compare to intact rats. The duration of REM sleep episodes increased two-three times to the 4-th PhS only. The EEG power spectra of brain investigated structures did not differ from the initial level. The remarkable quantity of neurosecretory material was found (PAH-reaction) in neurosecretory cells (NSC) of paraventricular (PVN) and supraoptical nuclei (SON) and in the middle eminence of rats after SD. This neurosecretory material contained mainly vasopressin. The nucleolus volume in NSC of PVN became 160% and in SON 121% (both p<.05) vs. controls, meaning the activation of vasopressin releasing processes and stimulation of its synthesis in hypothalamic NSC. The processes were more active in PVN. PhS activated the processes releasing of neurosecretory materials. Its quantity was increased gradually in the middle eminence. Balance of releasing and synthesis in NSC of PVN and SON was observed after the 4-5th PhS. This can be due to adaptation of rat's organism to the stimulation and the compensatory increase in NREM sleep. The concentration TSH and T3 in rat's blood serum increased after the 1-3-d PhS. THS level returned to the normal level only after the 5th PhS, but T3 concentration remained rather high. The T4 concentration decreased immideately after the 1st PhS, but then it increased and came to normal level to the 5-th PhS. The ACTH concentration increased gradually to the 3-d PhS. The SD procedure did not influence TSH and T3 concentrations. Only the concentration of T4 was decreased. Conclusion. The changes of SWC, neurosecretory systems in rats of pubertant age life under small stress procedures are deeper than in adult animals. They cause distortion of SWC structure, appearance and increasing in it evolutionary ancient form of sleep - cataleptic form of passive-defensive behavior. This appearance makes it possible to include the compensatory mechanisms NREN sleep, which is evolutionary more progressive form of sleep and has reparative and antistressor functions (2). The work was performed according to the state contract 4/2003. 1. Aristakesyan E.A. J Evol. Biochem. Physiol. 1997. 33. 622-629. 2. Borbelly A.A. 1982. Hum. Neurobiol. 1. 195-204. 3. Karmanova I.G. Evolution of sleep. 1982. 4. Spear L.P. Neurosci. Biobehav. Revs. 2000. 4. 417-463. (10 min)
THALAMOCORTICAL AND INTRACORTICAL RELATIONS IN STRESS AND SLEEP STATES. R.V. Yasenkov, A.A. Burikov, A.G. Sukhov, L.A. Konyahina. Rostov State Pedagogical University, A.B. Kogan Research Institute for Neurocybernetics, Rostov-on-Don, Russia. The comparative studies of functional organization of the brain at different levels of activation, in particular, during stress and sleep, are important in today biological psychiatry. Novel experimental animal models of stress are necessary, representing the only way to investigate organization and functioning of neural networks at the different dynamic states of the brain, as well as to study regularities of various CNS disorders. METHODS: The study was performed on 20 non-narcotized, partly immobilized (d-tubocurarine) adult rats. The assessment of the current functional state of animals during stress and initial stages of sleep was performed by evoked activities, EEG, ECG, and recording of vibrissal movements. Registration and stimulation of the brain were performed by glass microelectrodes (tip resistances 1-2 MÎm), placed stereotaxically in ventroposterior (VP) and intralaminar thalamic nuclei as well as within the supragranular (I-III) and infragranular (IV-VI) layers of the somatic cortex. We used 2 functional states: 1) stress, observed in post-operating period, caused by short hypoxia or weak electrotactile stimulation; and 2) different depths of sleep induced by phased perfusion of nembutal (3-5-10-20 mg/kg). Statistical analysis included: spectra of powers, coherency and phase cross-correlative spectra, calculated for different brain structures. RESULTS AND DISCUSSION: The state of stress was characterized by the high level of the brain activations with domination of theta-rhythm (stress-rhythm, Guselnikov, Supin, 1968) in sensorimotor cortex, thalamus, as well as by activated vibrissal movements. The prevalence of the frequency range of theta-rhythm in lower, infragranular layer of the cortical module (Sukhov, 1995) was confirmed by our data. In stress we observed the suppression of evoked rhythmic alpha-frequency activities, the oppression of background alpha-activities in (EEG) and heart arhythmia. The typical particularity of motor activity in stress was that reinforcement of the theta-rhythm preceded it, and is often developed synchronously in sensor and motor cortex, as well as in thalamic nuclei and reticular formation. During the transitions to early stages of sleep induced by nembutal, the suppression of the theta-rhythm in cortex and thalamus and also a typical spindle activity were found (Naricashvili et al., 1965; Burikov, 1975, 1985). Rare spontaneous motions of vibrissae on early stages of sleep appeared after the spindles, spontaneous or caused by tactile stimulation, which more often existed in upper associative layers of the somatic cortex. The state of sleep was characterized by the phase shifts of spindle oscillations in thalamus, comparatively with the upper and lower cortical layers. Herewith, the coherency of rhythmic activity of the thalamic nuclei with lower layers of the cortex was higher, than coherency with upper layers, indicating the prevalence of corticofugal influences. These results are in agreement with the last studies, in which there are discussed direct participation of corticothalamic inputs in spatiotemporal organization of the spindle oscillations in the thalamus (Contreras et al., 1997; Timofeev, Steriade, 2004). CONCLUSION: In stress we observed higher cortical and subcortical activation than in somnolent states, with domination of the theta-rhythm activity in the lower layers of the somatic cortex and subcortical structures. The spindles stage develops at the level reduction of activation and it is a connecting process to slow-wave sleep, when is still possible the local activation, limited by the upper associative layers of the cortex. We also found the increase in corticofugal influences in the development of the rhythmic processes in the thalamocortical system. (poster)
Ñomparison of the hearing evoked potentials during disturbances of monotonous activity and spontaneous falling asleep. Dorokhov V.B., Verbitskaya Yu.S. Institute of Higher Nervous Activity and Neurophysiology, RAS, Moscow, Russia. In accordance to present views, an analysis of external stimuli is continuing during sleep at subconscious level as a need to estimate biological meaning of external stimuli which serves a flexible contact of sleeping subject to the surrounding still persists during sleep. The method of evoked potentials permits to investigate internal psychological state during transition from wakefulness to sleep and sleep deepening independently of the presence and character of behavioral responses of human subject. Sound is the most adequate distant stimuli to study responses of cerebral information processes to external stimuli during falling asleep and various sleep stages. Comparison of a time-course of hearing evoked potentials changes related to disturbances of performing activity (psychomotor test) during progressive drowsiness and spontaneous falling asleep was the aim of present work. Psychomotor test. 30 subjects were lying in a darken room with eyes closed performing continuously discrete psychomotor test consists of 2 subsequent alternating parts: 1) click counting from 1 to 10 with simultaneous pressing to joystick bars with both hands; 2) clock counting from 1 to 5 without pressing. Monotonous test character and limitation of sensorial input from one hand accelerate an appearance of activity errors; from another hand a discrete structure of the test facilitates self-control and subjective impression of test errors at early stages of drowsy development. Computer-generated sound stimuli have been sent binaurally through the head-phones using 2.3-2.7 sec intervals. Click intensivety was 60 db, duration 50 ms, filling frequency 1 kHz. The session lasted 40-50 min., the first errors appeared with a latency 5-10 min. Eight EEG channels have been recorded as well as horizontal and vertical eye movements and joystick bars pressures. Spontaneous falling asleep. 1.5-2 h. since psychomotor test was over, the same volunteers have been subjected to hearing evoked potentials registration with the same parameters but interstimuli interval of 30 sec on the background of spontaneous falling asleep. Registration and data analysis have been performed using digital paperless polygraph SAGURA-2000 (Germany). Performance accuracy has been estimated by the following parameters: pressing amount in series, amount of stimuli which did not follow by pressings, omissions and lack of pressings. Important performance disturbances accompanied by the "EEG sleep patterns" appearance in polygraphic recording were considered as errors. Then HEPs of the following 4 groups were selected for average procedure: 1) Ia - correct with joystick pressings (series of 10 stimuli with pressings); 2) Ib - correct without joystick pressings (series of 5 subsequent stimuli without pressings); 3) IIa - incorrect with joystick pressings (series of N stimuli with pressings when N is different from 10 for at least 1 stimulus); 4) IIb - incorrect without joystick pressings (series of N stimuli without pressings when N is different from 10 for at least 1 stimulus). Single HEPs of these groups were compared to each other using non-parametric Wilkoxon test (significance level, p<.05), then averaged. The result of analysis was a finding of time intervals when averaged group HEPs were distinct from each other. Results. Comparison of two groups with correct test performance (Ia and Ib) demonstrated significant increase in positive component P2 amplitude, latency of negative component N2 (N350) and decreasing latency of positive component P3 in series without joystick pressings. These changes were more intensive in series IIa and IIb with incorrect test performance. Comparison of HEPs dynamics during psychomotor test performance and spontaneous falling asleep demonstrated that their shapes during psychomotor test performance disturbances and stage1 ("theta-sleep") are similar in their characteristics. (20 min)
ON THE ROLE OF NITRIC OXIDE IN REGULATION OF SLEEP NEED. A.V. Kalinchuk, Dag Stenberg, P.A.Rosenberg, T.Porkka-Heiskanen, Institute of Biomedicine, University of Helsinki, Helsinki, Finland, Children's Hospital and Harvard Medical School, Boston, USA. It is has been found that accumulation of adenosine in the basal forebrain can be a key mechanism mediating effects of prolonged wakefulness (Porkka-Heiskanen et al., 1997). Recently we established that local energy depletion in the basal forebrain, induced by infusion of 2,4-dinitrophenol, mimicked the effects of sleep deprivation leading to site-specific accumulation of extracellular adenosine and increasing subsequent sleep (Kalinchuk et al., 2003). Nitric oxide (NO), an intercellular signaling molecule, has been reported to inhibit the energy production and stimulate extracellular adenosine accumulation in rat forebrain neurons in culture (Rosenberg et al., 2000). We suggest that release of NO in the basal forebrain during prolonged wakefulness could be a possible mechanism underling adenosine accumulation and increase in sleep need. In order to test this hypothesis we: 1) infused NO synthesis inhibitor (L-NAME) into the basal forebrain during sleep deprivation and measured changes in subsequent sleep; 2) infused NO donor (DETA NONOate) into the basal forebrain and compared subsequent changes in sleep to the effects of sleep deprivation. Methods. Under anesthesia male rats were implanted with electrodes for recording of EEG and EMG and guide cannulae. Microdialysis probes were aimed to the basal forebrain or adjacent non-cholinergic areas as a control. Experimental schedule for each rat was as follows: baseline sleep-waking cycle recording; sleep deprivation for 3h preceded by 2h baseline and followed by 1h of recovery sleep; sleep deprivation for 3h combined with L-NAME (0.6mM) infusion; infusion of DETA NONOate (1mM) for 3h during normal sleep-wake cycle preceded by 3h baseline. The EEG was recorded for 24h and scored for wakefulness, non-REM and REM sleep. Period of maximal increase in sleep (15pm-9am) was chosen for final quantitative analysis. After the experiments the locations of the probe tips were verified histologically. Statistical significance was assessed using Mann-Whitney rank sum test or Kruskal-Wallis one way analysis of variance on ranks followed by Dunn's post-hoc test. Results. Sleep deprivation induced an increase in sleep by 31.2±5% (Mann-Whitney Rank Sum Test, P<.001). Infusion of L-NAME into the basal forebrain during sleep deprivation completely suppressed the effect of prolonged wakefulness: sleep was increased only by 1.9 ± 4% as compared to control (Kruskal-Wallis, H(3)=24.573, P<0.001, post-hoc Dunn's test Q=0.658, P>.05). Infusion of L-NAME outside the basal forebrain during prolonged wakefulness was ineffective: sleep was significantly elevated by 31.9 ± 2% (Kruskal-Wallis one way analysis of variance on ranks, H(3)=24.573, P<.001, post-hoc Dunn's test Q=2.886, P<.05). Infusion of DETA NONOate during normal sleep-wake cycle into the basal forebrain mimicked the effects of prolonged wakefulness and increased sleep by 37.1±4% (Kruskal-Wallis one way analysis of variance on ranks, H(2)=12.231, P=.002, post-hoc Dunn's test Q=2.596, P<.05). Changes in sleep after infusion of DETA NONOate into the non-cholinergic area (-3.8±2%) were insignificant (Kruskal-Wallis one way analysis of variance on ranks, H(2)=12.231, P=.002, post-hoc Dunn's test Q=0.649, P>.05). Conclusion. Release of NO in the basal forebrain during prolonged wakefulness may be one of the key steps in regulation of sleep need. References: 1. Porkka-Heiskanen et al., Science 1997; 276:1265-1268. 2. Kalinchuk et al., Eur.J.Neurosci. 2003; 17:863-869. 3. Rosenberg et al., J. Neurosci. 2000; 20:6294-6301. Research was supported by NIH grant P50-HL60292-06, CFDA 93.233; ESRS-Sanofi-Synthelabo Group research grant; Academy of Finland; Finska Lakaresallskapet and Juselius Foundation. (20 min)
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