Věra Klenerová

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.

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.

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.

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.

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.

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.

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.

Exposure to intermittent high altitude induces different changes in adenylyl cyclase activity in hearts of young and adult Wistar rats

Abstract This study investigates changes of adenylyl cyclase activity in the heart of young and adult Wistar rats exposed to experimental conditions simulating high altitude hypoxia as a model for interpretation of some adaptive changes of adenylyl cyclase observed in human. The exposure of rats to intermittent high altitude (IHA) hypoxia (5000 m) showed significant adaptive changes. The right ventricular weight and the ratio of right/left ventricular weights of adult rats exposed to IHA were significantly increased when compared to appropriate controls; adaptive changes of cardiac adenylyl cyclase being dependent on the age of the animals. The isoprenaline‐stimulated activity was higher in the left than in the right ventricle, and in both ventricles it was higher in young rats than in adult rats. When compared to controls, isoprenaline stimulation was decreased in the right ventricles of adapted young rats and, by contrast, it was increased in the left ventricles of adapted adult rats. This decrease and increase of adenylyl cyclase activity evoked by isoprenaline was paralleled by forskolin‐induced adenylyl cyclase activity in these experimental groups. It seems therefore that the changes in the pattern of total adenylyl cyclase activity observed under IHA hypoxia may at least be partially explained by the changes of beta‐adrenergic receptor susceptibility following IHA hypoxia.