EARLY POSTNATAL DYSFUNCTIONING OF THE BRAIN MUSCARINIC CHOLINERGIC SYSTEM AND THE DISORDERS OF FEAR-MOTIVATED LEARNING AND MEMORY.

The aim of the study was to investigate the early postnatal dysfunctioning of the brain muscarinic cholinergic system as a major factor in the development of cognitive disorders similar with somewhat is noted in animal models of depression and in patients with Major Depressive Disease. The present study examined the processes of learning and long-term retention of information obtained in the two important tasks of non-declarative memory - active avoidance, motivated by foot shock-induced fear, and the elevated plus-maze, based on the natural fear.Experiments were carried out on male white wild rats (n=60). Four groups of animals (15 rats in each) were used with special procedures for each one. Early postnatal dysfunctioning of the brain muscarinic cholinergic system was produced by the new method worked out by Nachkebia and co-authors. Two-way shuttle-box and elevated plus maze devices were used for the investigation of fear-motivated non-declarative memory and anxiety.The lasting effects of early postnatal dysfunctioning of the brain muscarinic cholinergic system on non-declarative learning and memory and anxiety were studied for the first time in the present work. Following new facts were obtained: 1) This procedure produces in adult age initial impairment of learning in a non-declarative memory test that is based on the enhancement of the level of anxiety in both active avoidance and elevated plus maze tests; 2) Dysfunctioning of the brain muscarinic cholinergic system, in the period of early ontogenesis, does not change in adult animals the consolidation and long-term retention of information obtained in the two important tasks of non-declarative memory, active avoidance, motivated by foot shock-induced fear, and the elevated plus-maze, based on the natural fear; 3) Intraperitoneal injection of Scope in adult animals, subjected during early ontogenesis to the dysfunctioning of the muscarinic cholinergic system, reduce the level of anxiety and facilitates by this way learning and memory of natural fear-motivated non-declarative test. Early postnatal dysfunctioning of the brain muscarinic cholinergic system enhances the level of anxiety in adult age and complicates acquisition of information in fear-motivated non-declarative learning and memory tests, but does not change its consolidation and long-term retention.

CHARACTER OF THE CHANGES IN FEAR MOTIVATED DECLARATIVE MEMORY IN THE HIGH IMMOBILIZATION "DEPRESSIVE" RATS.

Present study investigated possible differences in the learning and memory of declarative memory task in rats selected according to the differences in immobilization response that is in high immobilization "depressive" and low immobilization "non-depressive" rats. Understanding the character of learning and memory disturbances in basal conditions of animal models of depression is still very topical for more intimate definition of the pathophysiology of major depressive disorder and appropriate searching the ways of its correction. Experiments were carried out on the adult white wild rats (with the weight 200-250 g, n=20). Selection of rats according to the level of immobilization was made by means of forced swim test. Learning and memory disturbances were studied using passive avoidance test that is fear motivated one trial declarative memory task. It was shown by us that 100% of low immobilization "non-depressive" rats remember painful stimulation and therefore they are not enter in the dark compartment during whole period of observation during testing session. Behavior of high immobilization "depressive" rats is not similar in passive avoidance camera; 50% of "depressive" rats, with long escape latency during training session (92±10 sec), remember painful stimulation during testing session and therefore they are not enter in the dark compartment during whole observation period. The remaining 50%, that are not differ significantly from the low immobility "non-depressive" rats by the latency of escape (5±1 sec) during training session, are not able to remember painful stimulation during testing session and therefore they enter in the dark compartment with shortest escape latency (6±1 sec). In conclusion, high immobility "depressive" rats perform passive avoidance declarative memory task at the chance level that is a direct indicator for the serious disturbances of declarative memory mechanisms in "depressive" rats selected in forced swim test according to the level of immobility.

ACQUISITION AND CONSOLIDATION OF TWO-WAY ACTIVE AVOIDANCE IN THE HIGH IMMOBILIZATION "DEPRESSIVE" RATS.

Present study investigated possible changes in acquisition and consolidation of associative memory in high immobilization "depressive" and low immobilization "non-depressive" rats. Question is very topical because understanding the character of learning and memory disturbances, one of symptoms of major depressive disease, is very significant for more intimate definition of the pathophysiology of major depressive disorder and appropriate searching the ways of its correction. Selection of rats according to the level of immobilization was made by means of forced swim test. Learning and memory disturbances were studied using two-way active avoidance test that is fear motivated multi trial associative memory task. It was shown that ability to avoid/escape an aversive event by learning to perform a specific behavior, in response to a stimulus cue, is retained at the high level in high immobility "depressive" rats, selected by forced swim test. Acquisition of new information about an aversive stimulus is significantly facilitated and processes of consolidation are realized without any impairment. Thus, acquisition and consolidation of associative learning and memory is not impaired in high immobility "depressive" rats in two-way active avoidance task.

HYPOTHALAMIC OREXINE SYSTEM ACCELERATES REGULATION OF SLEEP HOMEOSTASIS AND SLEEP-WAKEFULNESS CYCLE RECOVERY FROM BARBITURATE ANESTHESIA-INDUCED ARTIFICIAL SLEEP.

The work was aimed for the ascertainment of following question - whether Orexin-containing neurons of dorsal and lateral hypothalamus and brain Orexinergic system in general are those cellular targets which can accelerate recovery of disturbed sleep homeostasis and restoration of sleep-wakefulness cycle behavioral states from barbiturate anesthesia-induced artificial sleep. Investigation was carried out on 18 wild type white rats (weight 200-250gr). Different doses of Nembutal Sodium were used for the initiation of deep anesthesia. 30 min after barbiturate anesthesia induced artificial sleep serial electrical stimulations of dorsal or lateral hypothalamus were started. Stimulation period lasted for 1 hour with the 5 min intervals between subsequent stimulations applied by turn to the left and right side hypothalamic parts. EEG registration of cortical and hippocampal electrical activity was started 10 min after intra-peritoneal administration of Nembutal Sodium and continued continuously during 72 hour. According to obtained new evidences, serial electrical stimulations of dorsal and lateral hypothalamic Orexin-containing neurons significantly accelerate recovery of wakefulness, sleep homeostasis, disturbed because of barbiturate anesthesia induced artificial sleep and different behavioral states of sleep-wakefulness cycle. Hypothalamic Orexin-containing neurons can be considered as the cellular targets for regulating of sleep homeostasis through the acceleration of recovery of wakefulness, and SWC in general, from barbiturate anesthesia-induced deep sleep.

Influence of serial electrical stimulations of perifornical and posterior hypothalamic orexin-containing neurons on regulation of sleep homeostasis and sleep-wakefulness cycle recovery from experimental comatose state and anesthesia-induced deep sleep.

The work was aimed for the ascertainment of following question - whether Orexin-containing neurons of dorsal and lateral hypothalamic, and brain Orexinergic system in general, are those cellular targets which can speed up recovery of disturbed sleep homeostasis and accelerate restoration of sleep-wakefulness cycle phases during some pathological conditions - experimental comatose state and/or deep anesthesia-induced sleep. Study was carried out on white rats. Modeling of experimental comatose state was made by midbrain cytotoxic lesions at intra-collicular level.Animals were under artificial respiration and special care. Different doses of Sodium Ethaminal were used for deep anesthesia. 30 min after comatose state and/or deep anesthesia induced sleep serial electrical stimulations of posterior and/or perifornical hypothalamus were started. Stimulation period lasted for 1 hour with the 5 min intervals between subsequent stimulations applied by turn to the left and right side hypothalamic parts.EEG registration of cortical and hippocampal electrical activity was started immediately after experimental comatose state and deep anesthesia induced sleep and continued continuously during 72 hour. According to obtained new evidences, serial electrical stimulations of posterior and perifornical hypothalamic Orexin-containing neurons significantly accelerate recovery of sleep homeostasis, disturbed because of comatose state and/or deep anesthesia induced sleep. Speed up recovery of sleep homeostasis was manifested in acceleration of coming out from comatose state and deep anesthesia induced sleep and significant early restoration of sleep-wakefulness cycle behavioral states.

Changes of open field behavior in animal model of depression.

Animal model of depression was developed by means of chronic exposure of rat pups to anticholinergic drugs (Atropine, Scopolamine) during the early life period from postnatal day 7 (P7) and/or 14 (P14) to P21 and/or P28, respectively. Such procedure resulted in lasting behavioral changes that were evident long after drug discontinuation and persisted at mature age (2-3 month period). Behavioral changes included most indices of open field behavior. Modeled animals exhibited significant depression of locomotor activity certified by sharp reduction of the number of crossed squares, rising of a head and vertical standings. Grooming behavior was also significantly decreased. Frequency of center entrance and the time of staying in the center of open field were sharply shortened. Modeled animals exhibited complete loss of exploratory motivation which wasn't related to the enhancement of fear emotion so far as values of incidence of urination and defecation remained unchangeable. These findings indicate that postnatal exposure of rat pups to Atropine and/or Scopolamine induces lasting behavioral "despai"' or "refractory loss of interest" at mature age. In sum animal model of depression which are characterized by super sensitivity of brain muscarinic cholinergic system exhibit more depressed behavioral items in open field than other types of animal models of depression. These data imply the preference of muscarinic cholinergic super sensitivity for the development of depressive state and therefore they are very significant for both basic science and clinical research issues.

Changes of locomotor, exploratory and emotional behavior in animal model of depression induced by deficiency of brain monoamine content.

The character of changes of open field behavior was not studied extensively in animal model of depression with deficiency of brain monoamine/serotonin content and obtained results are controversial. Both, enhancement and invariability of locomotor activity has been obtained. Additional investigation of this question is motivated also by insufficient study of exploratory and emotional behaviors in animal model of depression of this type. Animal model of depression was developed by chronic administration of Clomipramine and/or Melipramine in rat pups from postnatal day 7 (P7) and/or 14 (P14) to P21 and/or P28, respectively. Studies of open field behavior were started in adult age rats i.e. 8-12 weeks after the end of treatment. Control animals were the same age old. Two-week period of postnatal development starting at the P7 and/or P14 appeared equally sensitive to early antidepressant treatment. Modeled animals exhibited significant increase of horizontal locomotor activity. Frequency of center entrance and the time of staying in the center of open field were increased significantly indicating that animal models of depression can not percept really the level of stressfulness of novel surroundings. All of these changes indicate also to the significant level of exploratory behavior in modeled animals. Postnatal exposure of rat pups to Clomipramine or Melipramine produces significant increase of locomotor activity but dos not induces behavioral 'despair' or "refractory loss of interest" at mature age.

Effects of trihexyphenydil, the structural analog of phencyclidine, on neocortical and hippocampal electrical activity in sleep-waking cycle.

Finding about structural and functional relation between NMDA receptors specific binding and phencyclidine sites was very important for a possible modulation of NMDA receptors' function. We have therefore got interested what would happen with EEG and vegetative patterns of PS in the case when NMDA receptors function is modulated by blocking of phencyclidines' site. Consequently, we studied the effects of Trihexyphenydil, the structural analog of phencyclidine, on neocortical and hippocampal electrical activity in SWC. On cats (n=5) metallic electrodes were implanted under Nembutal anesthesia. EEG registration lasting 12 hr daily started after animals' recovery. Trihexyphenydil was administered intraperitoneally (0.5 mg/kg - 1 mg/kg). Statistical processing was made by Students' t-test. Trihexyphenydil resulted in dissociated triggering of PS. Rapid eye movements and PGO waves appeared on the face of active waking state. Therefore on the background of behavioral active waking according to electrical activity of the visual cortex and rapid eye movements, electrographic patterns of paradoxical sleep were recorded. Thus in our experiments it was shown firstly that the mechanism of hallucinogenic action of Trihexyphenydil is closely related to the disturbance of paradoxical sleep integrity. Blocking of NMDA receptors phencyclidines site and therefore functional modulation of these receptors produce the splitting of PS patterns and their intrusion in waking state. Such an effect never takes place in normal conditions since the waking system has the powerful inhibitory influence on the PS triggering system. Suggestion is make that NMDA glutamate receptors must be involved in mechanisms providing structural and functional integrity of PS and that fulfillment of such function is possible in the case when the NMDA receptors phencyclidine site isn't in blocked state. Normal functioning of NMDA receptors phencyclidine site represents the mechanism which inhibits and/or hampers appearance of hallucination. NMDA glutamate receptors, possessing phencyclidine site, are implicated in the mechanisms providing structural and functional integrity of PS.

Influence of diazepam on different behavioral states of sleep-waking cycle.

Diazepam is a widely used benzodiazepine. Their prolonged usage leads to deterioration of cognitive functions and to the reduction of the level of vigilance what is explained by hypnotic effect but not by anxiolytic action of this drug. The novelty of our investigation is the elucidation of whether diazepam produces slow wave sleep reduction likely to the other benzodiazepines and if so what is the bases of positive therapeutic effect of diazepam in insomniac patients. For this aim we decided the study of diazepam effects on the ultradian structure of sleep-waking cycle. On cats (n=5) metallic electrodes were implanted under Nembutal anesthesia. EEG registration lasting 12 hr daily started after animals' recovery. Diazepam was administered intraperitoneally (0.37; 0.75; 1.5; 2 mg/kg). Statistical processing was made by Students' t-test. Diazepam prolonged sleep onset latency and waking overall time. Incidence and overall percentage of deep slow wave sleep decreased significantly but doze dependently. Paradoxical sleep latency was also significantly increased after diazepam action. All dozes of drug used by us significantly decrease the duration of each episode of paradoxical sleep. Diazepam significantly increase incidence of paradoxical sleep episodes with maximal duration till to 5 min. Under the influence of diazepam paradoxical sleep coursed without emotional coloring, without any patterns reflecting the level of emotional tension during this phase. The first episode of paradoxical sleep with partial restoration of patterns of emotional tension developed after 9+/-2 postinjectional hours at the 2 mg/kg doze of diazepam. All parameters reflecting the level of emotional tension during paradoxical sleep wholly recovered after 27+/-2 hour of diazepam administration. Diazepam administration worsens the quality of slow wave sleep. It is suggested that positive therapeutic effect of this drug may be related with the reduction of emotional tension during paradoxical sleep and modulation of behavioral and EEG patterns of this behavioral state.

Septo-hippocampal cholinergic/gabaergic relationship and sleep-waking cycle.

There is controversy in the literature in the results of various septal lesions on the sleep-waking cycle (SWC) ultradian structure. Current investigation was aimed to study the effects of interruption of septo-hippocampal cholinergic/GABAergic afferentation on the ultradian structure of SWC and on PS major indices. Experiments were carried out on 12 adult cats, operated under overall anesthesia (Nembutal, 35-40 mg/kg). Three groups of animals were used: I. Implanted sham lesioned control; II. With isolated lesion of medial septal part; III. With combined lesion of medial and lateral septal parts. Lesion was made by passing of direct current. Continuous EEG registration of SWC was lasted 12 hour. Results were evaluated statistically with Student's t test. Isolated lesion of medial septum doesn't produce significant changes of motivational-emotional behavior, but combined lesion of medial and lateral septal parts lead to enhancement of food and water motivation, development of hyper emotionality and hyperactivity. Interruption of septo-hippocampal cholinergic/GABAergic input, increased sleep onset latency, incidence and percentage of active waking (AW) and passive waking (PW) and PS latency but the last effect was dependent from sleep latency change. In the period from appearance of first PS episode to the end of EEG registration PS incidence and percentage wasn't changed significantly. This surgery completely abolished theta rhythm in waking and PS. Combined lesion of medial and lateral septal parts increased sleep latency still more. Total time of AW and PW increased twice. DSWS was significantly decreased. In this case PS latency was also increased still more. PS incidence and total percentage in whole 12 h registration period were reduced substantially, but for the period calculated after appearance of first PS episode until to the end of EEG registration PS mean value was the same as in sham lesioned animals. It is concluded that: 1.Septo-hippocampal cholinergic/GABAergic relationship doesn't play significant role in the triggering mechanisms of SWC ultradian structure; 2. GABAergic part of this input as well as hippocampo-mesodiecephalic descending pathways through the lateral septum have powerful modulatory influence on basic triggering mechanisms of SWS; 3. Development of hippocampal theta rhythm is the only event of PS affected after medial septal lesion; 4. Septo-hippocampal cholinergic input is not essential in triggering mechanisms of PS.

The influence of electrocoagulation of the septum and section of the entorhinal cortex on general behaviour and memory in cats.

In chronic experiments on cats it was shown that the lesion of the medial part of the septum does not result in the "septal syndrome"; the ratio of the different sleep-wakefulness cycle phases remains within the baseline values; the acquisition, retrieval and extinction of instrumental alimentary conditioned reflexes proceed normally; the delayed conditioned reflexes are impaired. Massive septal lesion, including its lateral part, leads to the development of the "septal syndrome"; there are changes in the structure and percentage of the different sleep-wakefulness cycle phases; the acquisition and extinction of instrumental alimentary reflexes with the sound discrimination are markedly retarded; the performance of delayed conditioned reflexes is completely destroyed. The section of the entorhinal cortex produces an increase in the number of repeated errors and perseverative movements during performance of instrumental alimentary reflexes, the deceleration of the acquisition and extinction of instrumental alimentary reflexes, complete disturbance of the delayed conditioned reflexes and does not affect the sleep-wakefulness cycle. The problems of the role of the hippocampus and its main inputs in the regulation of the short-term operative memory in the "pure form" as well as the significance of the descending influence of the hippocampus on the regulation of general animal behavior were also discussed.