The Effect of Acute and Repeated Stress on CRH-R1 and CRH-R2 mRNA Expression in Pituitaries of Wild Type and CRH Knock-Out Mice.

The activation of the HPA axis is the endocrine measure of stress responsiveness that is initiated by corticotropin-releasing hormone (CRH). CRH exerts its effects via CRHR1 and CRH-R2 receptors coupled to the cAMP signaling system and this process involves transcription factor cAMP-responsive element-binding protein (CREB).This study investigated the role of CRH and the possible involvement of CREB in gene regulation of CRH receptor, under basal conditions and after stress application in the pituitary. We used wild type (wt +/+) controls and CRH knock-out (CRH-KO -/-) male mice. Using CRH-deficient mice, we were able to investigate the consequences of the lack of the CRH on the expression of CRH receptors and transcriptional regulation mediated by CREB. We estimated the effect of acute (IMO 1×) and repeated (IMO 7×) restraint stressors lasting 30 and 120 min on the expression of mRNA CREB, CRH-R1, and CRH-R2 by qPCR. We found very significant difference in the expression of these peptides under the effect of single and repeated stress in control and CRH-KO mice. Our results indicate that both CRH receptors and CREB might be involved in the regulation of stress response in the pituitary of mice. We propose that regulation of the stress response may be better understood if more were known about the mechanisms of CRH receptor signal transduction and involvement of CREB system.

Effects of immobilizations stress with or without water immersion on the expression of atrial natriuretic peptide in the hearts of two rat strains.

Atrial natriuretic peptide (ANP) is produced and released by mammalian cardiomyocytes and induces natriuresis, diuresis, and lowering of blood pressure. The present study examined localization of ANP and a possible role of the hypothalamic-pituitary-adrenal axis (HPA) activity on the expression of proANP gene in the heart. The Sprague Dawley (SD) and Lewis (LE) rat strains were used. The animals were exposed to the two types of stress: immobilization and immobilization combined with water immersion for 1 hour. Localization of ANP was detected by immunohistochemistry and expression of the proANP mRNA by real-time qPCR in all heart compartments of control and stressed animals after 1 and 3 hours after stress termination (IS1, IS3, ICS1, and ICS3). Relatively high density of ANP-immunoreactivity was observed in both atria of both rat strains. In control rats of both strains, the expression of the proANP mRNA was higher in the atria than in ventricles. In SD rats with the intact HPA axis, an upregulation of ANP gene expression was observed in the right atrium after IS1, in both atria and the left ventricle after IS3 and in the left atrium and the left ventricle after ICS3. In LE rats with a blunted reactivity of the HPA axis, no increase or even a downregulation of the gene expression was observed. Thus, acute stress-induced increase in the expression of the proANP gene is related to the activity of the HPA axis. It may have relevance to ANP-induced protection of the heart.

Electronic and vibrational optical activity of several peptides related to neurohypophyseal hormones: disulfide group conformation.

Electronic and vibrational optical activity of the set of neurohypophyseal hormones and their analogs was investigated to clarify the S-S bond solution conformation. The selected compounds include oxytocin (I), lysine vasopressin (II), arginine vasopressin (III), and their analogs (IV-IX), differing widely in their pharmacological properties. We have extended the already known electronic circular dichroism data by new information provided by vibrational circular dichroism (VCD) and Raman optical activity (ROA). The use of VCD brought additional details on three-dimensional structure of the chain reversal in the ring moiety and on its left handedness. Furthermore, Raman scattering and ROA allowed us to deduce the sense of the disulfide bond torsion.

Effects of acute stressors on the expression of oxytocin receptor mRNA in hearts of rats with different activity of HPA axis.

OBJECTIVES: Cardiovascular system is regulated by a diverse array of hormones, neurotransmitters and neuropeptides. Oxytocin and its receptors (OTR) were also shown to regulate cardiovascular functions and this hormone was even called cardiovascular hormone. In recent publication, we demonstrated the expression of mRNA of OTR by real-time quantitative PCR (RT qPCR) in all rat heart compartments. The aim of this study was to investigate the effects of acute restraint stress on OTR mRNA expression in two rat strains with different activity of HPA axis. METHODS: Adult male Sprague-Dawley and Lewis rats, the latter strain reported to have lower HPA activity, were used in RT qPCR studies and Wistar rats in immunofluorescent ones. Both acute restraint (IS) and this stress combined with the immersion of rats in water (ICS) lasted 60 min. Gene expression of OTR mRNA was estimated in all heart compartments after 1 or 3 hours after stress termination (IS1, IS3, ICS1, ICS3). The relative expression was calculated using 2(-ΔΔC)T method. In immunofluorescent studies we used commercial specific OTR antibodies. RESULTS: In RT qPCR studies we found higher expression of OTR mRNA in atria than in ventricles and no statistical differences between Sprague-Dawley and Lewis rats under basal conditions. Relative expression of OTR mRNA after 60 min lasting stress exposure differed in dependence on the stress type and partly on the time interval after the stress termination. When compared to controls, in rat left atria both stressors caused inhibition of OTR mRNA expression in both rat strains. In rat ventricles, which have very low OTR mRNA expression, there was a significant difference in the effect of two stressors. In most groups ICS displayed the increase of OTR mRNA expression if compared to IS groups. Immunofluorescent studies revealed changes induced by acute restraint stress in all heart compartments. The immunofluorescent studies suggested that acute stress induces higher colocalization of OTR with the nuclei than it was observed in the controls. CONCLUSIONS: The expression of OTR mRNA in all heart compartments of controls as well as after stress exposure in Sprague-Dawley and Lewis rats support the notion that OTR plays a regulatory role in the cardiovascular system and is also involved in the regulations in the heart after stress. The immunofluorescent observation that OTRs coexpress in areas of cell nuclei in certain heart compartments and after acute stress, compared to controls, requires further studies.

Galanin modulating effect on restraint stress-induced short- and long-term behavioral changes in Wistar rats.

The neuropeptide galanin has been recognized as a possible neurotransmitter/neuromodulator, and in addition has been implicated in anxiety- and depression-related behaviors. The present study demonstrates increased locomotion and rearing after galanin (0.3mg/kg) that was given intraperitoneally (i.p.) to intact Wistar rats which were tested 1h later in the open field (OF). These effects, which suggest an anxiolytic-like action, were blocked by i.p. administered peptidic galanin antagonist M40. Further, the locomotion increase caused by galanin and the inhibitory effect of M40 persisted for 48h without additional treatment. Rats exposed to restraint stress (lasting 60min) for three consecutive days and tested 1h after stress termination exhibited reduced locomotion and exploration in the OF. Galanin (0.3 and 1.0mg/kg) given immediately after each stress exposure prevented the decrease of locomotion and exploration induced by stress in all trials. When the test was repeated 6 days later without stress and galanin treatment the reduction of locomotion produced by stress persisted; the anti-stress behavioral effects of both galanin doses were also present. Testing performed on the 12th day after the last stress and galanin treatment with 0.3mg/kg revealed an increased locomotion compared with unstressed and stress-exposed rats. Our results demonstrate that behavioral effects of the peptide galanin are evident even after i.p. administration. These results also suggest that galanin elicits stress-modulatory action, and support the notion that the galaninergic system may serve as a drug target in stress-related conditions.

Expression of heart oxytocin receptor and its mRNA in two rat strains with different activity of HPA axis.

OBJECTIVES: Oxytocin (OT) is a neuropeptide acting both as a peripheral hormone and in the brain as neurotransmitter and neuromodulator. In addition to its well-known effects on milk-ejection and uterine contraction, OT was shown to exert neuroendocrine regulation of heart functions. The aim of this study was to investigate the expression of mRNA of OT receptors (OTR) in rat hearts by real-time quantitative PCR (qPCR). The study was performed in Sprague-Dawley (SD) and Lewis (LE) rat strains, the latter having lower activity of HPA axis. METHODS: We used adult male SD and LE rats. OTR mRNA expression was detected in all heart chambers by comparing their threshold cycle values (CT) to CT of reference gene ß-actin. The relative expression ratios were calculated using the 2-ΔΔCT method. The specificity of reaction of primary antibody with OTRs was tested by Western Blot and localization of OTR in the heart compartments was performed by immunofluorescence with commercial OTR specific antibodies. RESULTS: We found expression of OTR mRNA in all heart compartments. The expression of OTR mRNA in both atria (LA, RA) was much higher than in the ventricles (RV, LV). By using two-way ANOVA we found no statistical differences between corresponding compartments of SD and LE rats. Immunohistochemical studies showed that OTR staining is not related to neuronal tissue and findings from left atrium indicate that prevalent localization of OTR is on cell membranes of cardiomyocytes. CONCLUSIONS: The finding of expression of OTR mRNA by real-time qPCR and proof of OTR staining by immunohistochemistry in all heart compartments indicate that OT and its receptors may have function as a cardiovascular hormone. The differences in the HPA axis activity, as is exemplified in Sprague-Dawley and Lewis rat strain, do not project in the expression of OTR mRNA under basal condition. The effect of activity of HPA on OTR expression should be studied under stimulated conditions as it was performed in the behavioral studies.

Oxytocin and carbetocin ameliorating effects on restraint stress-induced short- and long-term behavioral changes in rats.

OBJECTIVES: Carbetocin (CBT), an oxytocin (OXY) analog, was designed to exert prolonged peripheral actions. It has also been proposed as potential therapeutic mean in certain psychiatric disorders where OXY role has been implicated. This study examined the effects of both peptides on behavior of naive and restraint stress exposed rats in the open field (OF) and elevated plus maze (EPM) tests. METHODS: Spontaneous behavior in the OF and EPM was measured in Wistar rats after intraperitoneal (i.p.) application of OXY or CBT and/or repeated restraint stress. Behavioral parameters were recorded and subsequently elaborated by an automated activity monitoring system (AnyMaze, Stoelting, U.S.A.). Changes in the total movement distance (TMD) and movement in the center area (CMD) were postulated as indicators of the anxiety level. RESULTS: OXY (0.05 mg/kg) and CBT (0.3 mg/kg) increased TMD but not CMD 60 min after the i.p. treatment; the increased locomotion/exploration indicate participation of arousal/vigilance. Daily stress exposures for three consecutive days, followed by behavioral tests, reduced locomotion of rats in OF and EPM tests; OXY and CBT partly prevented these effects. Five days after the last stress, rats exhibited an increase of both TMD and CMD in the OF. CBT but not OXY prevented these long-term post-stress changes. In the EPM the stressed rats exhibited an increase in time spent in open arms; CBT accelerated this time development. Similar prevention of stress behavioral sequel in OF were obtained in study when stress and peptides were applied for three consecutive days but behavioral testing was postponed for several days to determine the long-lasting effects. CBT reduced the developed locomotor enhancement (6-11 days post-stress) irrespectively whether injected before or after stress. CONCLUSIONS: Repeated restraint stress exposure produced acute and persisting effects on Wistar rat behavior in the OF and EPM tests. CBT either injected before or after stress practically abolished the developed changes in the mobility parameters. The CBT effectiveness to ameliorate the late post-stress behavioral alteration supports the notion of its therapeutic potential in psychiatric disorders in which the role of OXY has been implicated.

Oxytocin and carbetocin effects on spontaneous behavior of male rats: modulation by oxytocin receptor antagonists.

OBJECTIVES: Oxytocin (OXY) in addition to peripheral actions has many central regulatory functions which can be studied on animal models. In the present study we examined in rats, which behavioral actions of OXY and long-acting carba-analog of OXY carbetocin (CBT) in the open-field can be inhibited by OXY-receptor antagonists. Our interest focused on the behavioral patterns considered indicative of anxiety-related behavior. To determine what is the participation of OXY receptor on OXY and CBT induced behavioral changes, we used two peptide and one nonpeptide OXY antagonists differing in selectivity for OXY receptor. METHODS: OXY, CBT as well as OXY antagonists were injected intraperitoneally, and spontaneous behavior (horizontal and vertical activity, grooming) of Wistar rats was observed in the circular open-field arena 60 min after application of drugs; in some experiments testing was performed without treatment few days after drug administration. RESULTS: OXY at the dose 0.05 mg/kg increased locomotion indicating anxiety attenuation, but 1.0 mg/kg reduced both locomotion and rearing. CBT in the dose range 0.1-3.0 mg/kg either did not change or increased horizontal activity. The increase in exploration after both peptides persisted for several days. A marked difference in the behavioral effects of the two peptides was grooming enhancement induced by OXY compared with the absence of this effect after CBT. The increase of the activity induced by OXY and CBT indicating anxiolytic-like action was blocked by OXY antagonists. However, the reduction of exploration induced by 1.0 mg dose of OXY was only partially reversed. The OXY induced enhanced grooming was completely antagonized by all used antagonists. CONCLUSIONS: Behavioral effects of OXY and its antagonists after their i.p. application indicate that they penetrate blood brain barrier. The diversity in potency of OXY antagonists to inhibit grooming and other behaviors induced by OXY suggests that receptors participating in these behaviors may differ in brain localization, receptor conformation and/or in the utilized signaling pathways.

Novel and simple behavioral paradigm for assessing anxiety in rats: effect of diazepam.

OBJECTIVES: Anxiety is an emotional state experienced by people, and is not readily modeled in animals. In order to extend till now ethologically derived paradigms used in the evaluation of anxiety and fear in rodents, a modified open-field was designed. METHODS: Spontaneous behavior of male rats was investigated in the elevated arena; the bottom was divided by inter-space of different width (2, 4, 6, 8 and 10 cm) into two identical parts. Anxiolytic effects of diazepam (DZP) at doses 0.5, 1.0, 2.5 and 5.0 mg/kg were investigated in the newly designed device and compared with the effects of similar doses in a large circular open-field arena. RESULTS: In Experiment 1 the progressive extension of the inter-space prolonged the first passing, decreased the total number of passing, and increased the inter-space sniffing in intact animals. In Experiment 2 DZP at doses 1.0, 2.5 and 5.0 mg/kg significantly enhanced the readiness to cross, the frequency of passing the inter-space and decreased inter-space sniffing as compared to controls. In Experiment 3 we found that DZP at doses 0.1 and 0.3 mg/kg increased behavioral activity both along the perimeter and in the center of the arena, thus indicating lower level of anxiety. CONCLUSION: The presented modified open-field test is a useful paradigm to investigate risk assessment behavior in rats, and may provide a sensitive novel model of anxiety and fear level.

Dipeptide "alaptide" prevented impairments in spontaneous behavior produced with trimethyltin in male rats.

OBJECTIVES: On the animal model of trimethyltin (TMT) induced behavioral deficits the effect of chronic treatment with spirocyclic dipeptide cyclo/alanyl-l-amino-l-cyclopentane-carbonyl (alaptide, AL) was evaluated in adult male rats. METHODS: Changes in the spontaneous behavioral repertoire were investigated in the open-field test on Day 21 (Session 1) and Day 28 (Session 2) after a single oral TMT administration. RESULTS: In Experiment 1, rats given the highest TMT dose (7.5 mg/kg) exhibited significantly increased total number of behavioral patterns, the floor sniffing being the most frequent pattern. While the medium TMT dose (5 mg/kg) had a similar effect only in Session 1, the lowest TMT dose (2.5 mg/kg) was entirely ineffective. In Experiment 2, an explicit beneficial influence of both AL doses (5 and 10 mg/kg) given for 10 days before and 10 days after TMT (7.5 mg/kg) on the spontaneous behavior repertoire was observed in both Session 1 and Session 2. The total number of patterns and the time spent in individual patterns of AL+TMT treated animals did not differ from the controls and those given AL alone. CONCLUSION: We conclude that sufficiently long AL treatment interfered with deleterious effects of TMT and forestalled changes in the structure and timing of spontaneous behavioral patterns. Thus, AL can be designated as a substance having "neuroprotective" effects.

Effects of melanotan II, a melanocortin agonist, on grooming and exploration in rats after repeated restraint/immobilization.

2mg/kg melanotan II (MTII, administered i.p.), a cyclic peptide analog of alpha-melanocyte stimulating hormone, at a single dose increased grooming in naive rats placed in an unfamiliar open-field device without changing locomotion or rearing. Male rats exposed to restraint/immobilization stress (IS) for 1h on three consecutive days displayed increased grooming after the second stressor exposure, compared to pre-stress levels. MTII, administered to the rats after IS, enhanced the grooming response compared both to the pre- and post-stress values. The increase was greatest after the first dose and declined over the following two applications. As to the locomotion of rats in the entire experimental space, IS reduced the distance moved only after the first two stressor exposures; MTII did not influence these alterations. Locomotion in the central part of arena was not reduced by the stressor or by MTII, on the contrary, there was an increase in both groups after the third intervention. The only observed change in rearing was an increase in the MTII group after the third restraint exposure. Thus, MTII selectively increased grooming without markedly affecting the spatio-temporal structure of locomotor behavior in the open-field. The decline of MTII enhanced grooming over the three test days may be interpreted in terms of adaptation to the stressor and of the developing tolerance to the peptide.

Corticotropin-releasing hormone receptor subtypes in the rat anterior pituitary after two types of restraint stress.

The stress response in anterior pituitary (AP) is mediated by corticotropin-releasing hormone (CRH) acting through CRH-R1 and -R2, however, the function of CRH-R2 in AP is still not fully elucidated. We used 1-h long restraint (IMO) as well as restraint combined with water immersion (IMO+C). Using real-time PCR we quantified mRNA expression of CRH-R1, CRH-R2alpha-soluble and -insoluble, and cAMP response element binding (CREB), with reference gene GAPDH. In control AP, CRH-R1 mRNA was up to 20-fold higher than levels of CRH-R2alpha-soluble or CRH-R2alpha-insoluble mRNA. IMO reduced CRH-R1 mRNA to 47% and 63% of control levels 1 and 2 h after the onset of stressor, respectively, while IMO+C did not produce significant changes. Our data demonstrated that these stressors did not change CRH-R2alpha mRNA, unlike the very significant response of CRH-R1 to IMO.

Dynamics of immediate early gene and neuropeptide gene response to prolonged immobilization stress: evidence against a critical role of the termination of exposure to the stressor.

Stress-induced expression of immediate early genes (IEGs) appears to be transient even if the exposure to the stressor persists. However, there are some exceptions which suggest that particular characteristics of stressors can affect the dynamics of IEG expression. We studied in selected telencephalic, diencephalic and brainstem regions the mRNA levels of two clearly distinct IEGs (c-fos and arc) during prolonged exposure to a severe stressor such as immobilization (IMO) and after releasing the rats from the situation. Although regional differences were observed with the two IEGs, overall, c-fos mRNA levels progressively declined over the course of 4 h of continuous exposure to IMO, whereas arc mRNA levels were maintained at high levels in the brain regions that express this gene under stress (telencephalon). Levels of CRF hnRNA in the hypothalamus paraventricular nucleus only slightly declined during prolonged exposure to IMO. Surprisingly, termination of exposure to IMO did not modify CRF gene expression in the paraventricular nucleus or the pattern of IEGs expression, with the exception of c-fos in the lateral septum. Thus, putative signals associated to the termination of exposure to IMO were unable to modify either IEG expression in most brain areas or CRF gene expression in the paraventricular nucleus.

Timing of stress and testing influence the long-lasting behavioral performance in rats.

Three exposures (Days 1, 2 and 3) of rats to immobilization or immobilization combined with cold induced an alteration of exploratory behavior in an open space arena. When tested 1h after both stressors exposure, rats displayed a decrease in locomotor and rearing score. The deficit disappeared when rats were tested five days later and the performance remained unchanged in trials performed on days 9, 10, 15, 22 and 29 of the study. When testing was postponed five days after the third stressor exposure, a gradual reduction of the performance developed and the deficit persisted until the last trial on Day 29. Amphetamine, in a dose of 0.3 mg/kg revealed a sensitized response to the drug in the stressed animals. The results showed short- and long-lasting behavioral consequences of the used stressors, the long-term manifestation of the sequelae being dependent on the sequence and timing of stressor exposure and open space testing.

The association of monoamine oxidase B functional polymorphism with postoperative pain intensity.

OBJECTIVES: The monoamine oxidase B (MAO-B) is an enzyme involved in metabolism of dopamine, benzylamine, phenylethylamine, tyramine and tryptamine. The A/G polymorphism in intron 13 of the MAO-B gene has been previously found to be associated with variability of the MAO-B enzyme activity. The aim of the present association study has been to examine the relationship between the A/G polymorphism in intron 13 and postoperative pain intensity. METHODS: 284 subjects (105 males and 179 females) undergoing planned tonsillectomy were examined. The intensity of pain was evaluated using 100-mm visual analogue scale (VAS). A PCR method with allele specific primers for detection of A/G polymorphism was used. RESULTS: We found a relationship between the A/G polymorphism in intron 13 of the MAO-B gene and average intensity of postoperative pain in male subjects. Higher average intensity of postoperative pain was detected in males with the G allele (3.96) in comparison with males with the A allele (3.45) and the difference was statistically significant (p<0,03). CONCLUSIONS: Results of this study indicate the relationship between the MAO-B polymorphism and postoperative pain intensity in the Czech male population. A potential role of MAO-B in the perception of pain intensity is discussed.

Differences in the brain expression of c-fos mRNA after restraint stress in Lewis compared to Sprague-Dawley rats.

In order to study the contribution of genetic factors to the pattern of stress-induced brain activation, we studied the expression of c-fos mRNA, a marker of neuronal activity, in male Sprague-Dawley and Lewis strains, the latter being known to have a deficient responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis. Immobilization (IMO) alone or combined with the immersion into water at 21 degrees C was applied for 15 or 60 min. The expression of c-fos mRNA was quantified by in situ hybridization in those brain areas that represent important parts of neuronal circuits activated by stress: medial prefrontal cortex, medial amygdala, lateral septum ventral part, paraventricular nucleus of the hypothalamus and locus coeruleus. While in controls, c-fos mRNA was not detectable in tested brain areas, both types of stressors induced a strong expression of this immediate early gene. There were only small differences in c-fos mRNA expression related to the type of stressor or the length of exposure to them. However, there were remarkable differences in the expression between the two rat strains. When compared to Sprague-Dawley rats, Lewis rats showed a reduced c-fos mRNA expression after both stressors in most brain areas, which may be related to the reduced responsiveness of HPA axis and also with other abnormal responses in this strain. However, this hyporesponsiveness was not observed in all brain areas studied, suggesting that there is not a generalized defective c-fos response to stress in Lewis rats and that some responses to stress may be normal in this strain.

Effects of two types of restraint stress on the spontaneous behaviour in rats.

Our previous findings suggested the existence of stressor-specific behavioural and cognitive responses in rats. In the present study, restraint stressor (immobilization, IMO) and restraint stressor combined with partial immersion of rats into water (IMO+C) were applied for 1 hour to Wistar male rats and their spontaneous behaviour was examined in the open field test. The classic behavioural parameters were recorded: crossing, rearing, and resting. When tested 1 and 4 hours after IMO+C, animals exhibited strong suppression of locomotor and exploratory activity (crossing and rearing); partial inhibition of both behavioural variables was found after IMO. Thus, substantial differences were observed in dependence on the length of period between the end of stressor application and the start of testing. In testing performed one week later, the locomotor and exploratory activity levels of both IMO and IMO+C animals corresponded to the control ones. These data suggest a differential behavioural response to both used stressors that may result from their different proportion of psychical and physical components. In conclusion, our results provide other data for the support of differential effects of two types of restraint stressors on spontaneous behaviour of animals exposed to a novel environment.

Combined restraint and cold stress in rats: effects on memory processing in passive avoidance task and on plasma levels of ACTH and corticosterone.

The effect of restraint stress combined with water immersion (IMO+C), applied at various intervals before and after the acquisition of a passive avoidance task, was studied in rats. The procedure started with two pre-training trials. On the single training trial the rats received a footshock (0.3 mA, 3s) after they entered the preferred dark compartment. The exposure to IMO+C lasting 1 h terminated 4 or 1 h before application of the footshock or started immediately or 3 h after this aversive stimulus. Retention tests were performed 1 and 2 days after the acquisition trial. In an attempt to relate the behavioural responses to the stressor with plasma levels of two stress hormones we measured ACTH and corticosterone under similar conditions as were used in the behavioural experiments. IMO+C exposure terminating 1 h before the training resulted in very short avoidance latencies during retention testing. A similar impairment of retention test performance was found in animals exposed to the stressor immediately after training. When IMO+C exposure terminated 4 h before training the stressed rats exhibited comparably long avoidance latencies as shown by the controls. IMO+C presented 3 h after acquisition trial also did not influence retention of avoidance learning. The hormones were estimated 1 and 4 h after IMO+C, both in the absence and presence of footshock. Both ACTH and corticosterone were significantly increased 1 h after IMO+C termination, and their plasma levels returned to control values within 4 h. Footshock alone increased plasma corticosterone, however, the hormone levels were significantly lower than those estimated after IMO+C terminating 1 h before blood collection. Footshock substantially increased ACTH levels in rats exposed to IMO+C 1 h before footshock, but not in stressed rats with already high levels of corticosterone. In conclusion, IMO+C represents a strong stress stimulus exerting amnesic effect when applied shortly before or after the acquisition trial. Further, the findings indicate the restraint and cold stressor to interfere with consolidation of passive avoidance response. We suggest that the moderate circulating levels of corticosterone found after footshock may be positively related to the memory consolidation, while the exceedingly high levels have an opposite effect.

Effects of two types of restraint stress on the learned behaviour in rats.

To study the effects of stress on cognitive functions, Wistar and Lewis rats were exposed to restraint (immobilization stressor) (IMO) or restraint combined with partial immersion into water (IMO+C). Learned discriminatory avoidance response in Y-maze, with foot-shock as an unconditioned stimulus, was used as a memory test. The latency to enter the correct arm and number of wrong entries were daily recorded during the training period (20 days) until the criterion was reached, which was set at 90% avoidances (choosing the correct arm). After exposure of rats to one of the stressors for 60 min, the rats were returned to the home cage; the latency to enter the safe arm was recorded in 6 daily trials that started 1 h after application of stressor. Both stressors significantly prolonged the avoidance latencies for 2 or 3 days in Wistar and Lewis rats, respectively; then the latencies returned to the values obtained before the stress exposure. In Lewis rats, the latencies more increased after IMO+C than after IMO stressor, and the maximal increase in latencies was higher in Lewis rats than in Wistar rats. The latency did not reach the time limit for foot-shock delivery, and the number of correct choices remained unchanged in both strains. The results indicate that the used restraint stressors did not affect the long-term memory; rather a transient impairment of retrieval can be considered. Further, differences in response of Lewis and Wistar rats may be interpreted by different activity of hypothalamic-pituitary-adrenal axis activity in used strains.

Impaired passive avoidance acquisition in Sprague-Dawley and Lewis rats after restraint and cold stress.

The study examined the effects of restraint combined with cold water stress (IMO+C) on learning and memory of Sprague-Dawley (S-D) and Lewis (LE) rats in the passive avoidance task. The procedure started with 6 days of adaptation to the apparatus during which the recorded latencies to enter the dark compartment were used to assess the process of habituation. On the training day rats were exposed to IMO+C for 60 min and the stressor exposure terminated 1 h before the acquisition trial. Retention trials started 24 h later. To evaluate the possible long-term consequences of the acute and repeated stress presentation on the performance of the two strains with diverse activity of hypothalamic-pituitary-adrenal axis, this procedure was performed three times including stress application (Parts 1-3). Finally, an identical procedure was performed without stress (Part 4). An immediate behavioural effect of the stressor exposure was observed in an increase of latencies to enter the dark compartment before the shock delivery in rats of both strains; this enhancement approached significance after the second and third exposure to the stressor (Parts 2 and 3). Control animals of both strains acquired passive avoidance response after training in Parts 2-4. IMO+C produced significant impairment of this response irrespective of the strain. The three-time repeated exposure did not influence the ability to learn the task in the final procedure without stress. Differences in behaviour of S-D and LE rats were observed already during the first adaptation period. LE rats exhibited longer latencies upon the first exposure to the novel environment compared to S-D rats. Also only LE rats displayed habituation. In Part 4 marked strain differences in the latencies both before and after training were recorded. The results show that the repeated exposure to the IMO+C stressor proved to be a strong amnestic stimulus but without persisting consequences for the ability of rats to acquire the learning task.