Effect of Stress on the Expression of Galanin Receptors in Rat Heart.

Neuropeptide galanin, galanin-like peptide and galanin receptors 1, 2 and 3 are a crucial part of the so-called galaninergic system. Our previous studies have shown the possible role of this system in mood modulation, especially regarding stress. So far, the galanin receptors have been found in different tissues including brain and heart. Our study deals with changes in expression of galanin receptor subtypes in the heart of Wistar rats exposed to three different types of stress. Galanin receptor subtypes were determined in fluorescently labelled samples using specific primary antibodies, and all sections were analysed in an immunofluorescent microscope and microphotographs. Image analyses were subsequently performed by software ImageJ, using the threshold method with calculation of the DAPI/galanin receptor signal ratio. We found all three types of receptors in the right and left atria and left and right ventricles. Changes in the density of galanin receptors after application of the stressor depended on the observed heart compartment. We found a significant decrease of galanin receptor 1 in all compartments after all types of stress. For GalR2 and GalR3, the increase/decrease of density was dependent on the tested compartment. These results show that galanin receptors could be involved in the function of heart during the cardiac cycle.

The Effect of Stress on the Galaninergic System in the Rat Adenohypophysis: mRNA Expression and Immunohistochemistry of Galanin Receptors.

The neuropeptide galanin is a widely distributed neurotransmitter/neuromodulator that regulates a variety of physiological processes and also participates in the regulation of stress responses. The effect of stress is dependent on the activity of the hypothalamic-adenohypophyseal-adrenal axis. Although the adenohypophysis is a crucial part of this axis, galanin peptides and their receptors have not yet been identified in this part of the pituitary after activation of the stress response. Since there are many controversies about the occurrence of individual galanin receptor subtypes in the adenohypophysis under basal conditions, we decided to verify their presence immunohistochemically, and we clearly demonstrated that the adenohypophysis expresses neuropeptides galanin, galanin-like peptide, and subtypes of galanin receptors GalR1, GalR2 and GalR3. The specificity of the reactions was confirmed by Western blots for galanin receptors. Using real-time qPCR we also demonstrated the presence of three GalR subtypes, with the highest expression of GalR2. In addition, we tested the effect of stress. We found that acute stress did not induce any changes in the GalR2 expression, but increased expression of GalR1 and decreased that of GalR3. We confirmed the involvement of the galanin system in the stress regulation in the adenohypophysis.

Modulary effects of oxytocin and carbetocin on stress-induced changes in rat behavior in the open-field.

Oxytocin (OXY) has been shown to attenuate some of the physiological and behavioral alterations appearing in stressed rats. Carbetocin (CBT), an oxytocin analog [deamino-1-monocarba-(2-O-methyltyrosine)-oxytocin], was designed to exert prolonged action. In the present study we investigated the impact of these peptides on the behavioral changes in rats exposed repeatedly to restraint stressors. Wistar male rats were exposed to restraint for 1 hour; saline or drugs were administered intraperitoneally immediately after stress termination. Recording of the exploratory activity in the open-field started 60 min later. To explore the possibility of persisting effects of stress and/or drugs, the procedure was repeated for three consecutive days. Restraint moderately suppressed locomotion and rearing, and increased grooming. OXY in 0.3 mg/kg dose showed a tendency to restore the suppressed exploratory activity. In contrast, 1 mg/kg dose potentiated the stress-induced behavioral deficit. Both OXY doses slightly increased grooming. CBT in the same two doses restored the stress-induced deficits in locomotion and rearing but did not influence grooming. The locomotor depression after 1 mg dose of OXY was found also in non-stressed rats in contrast to the increased activity after CBT. The data support the view that post-stress administered CBT exerts a significant effect on the stress-altered spontaneous behavior.

Effects of two types of restraint stress on spontaneous behavior of Sprague-Dawley and Lewis rats.

The purpose of this study was to evaluate the action of two types of stressors in Sprague-Dawley (S-D) and Lewis (LEW) rats differing in their hypothalamic-pituitary-adrenal axis activity on locomotion and rearing in an open space. Exposure to restraint immobilization alone (IMO) or this immobilization combined with cold water (22 degrees C) immersion (IMO+C) lasted for 1 h and started 2 or 5 h before the test. To evaluate the acute and persisting effects of both stressors, four trials were performed in one-week intervals; rats were exposed to the stressors in trial 1 and 3. While in LEW rats both acute stressors produced reduction of locomotion and rearing in all stressed groups, S-D rats responded with a decrease of both parameters only after IMO+C presented 2 h before testing. Neither strain displayed a changed performance one week after the first stress exposure. One week after the second stress exposure rats of both strains exhibited a tendency to an increase of both parameters reaching the significance in several experimental groups. The findings indicate: a) the IMO+C produced stronger behavioral alteration than IMO alone; b) the behavioral responses to stressors were more pronounced in LEW compared to S-D strain; c) change from the reduction of activity to its increase may be interpreted as bi-directional manifestation of long-term effects of immobilization stress.

Effects of stress and of amphetamine on passive avoidance conditioning in rats.

This study examined the effects of immobilization stress combined with water immersion (ICS) and/or amphetamine (AM) on different memory phases in the passive avoidance task in rats. The performance of rats was evaluated in the retention tests 24 and 48 h after a single acquisition trial. ICS exposure lasting 1 h impaired retention of the learned avoidance response if applied 2 to 4 h before or immediately after training. The stressor did not affect retrieval if presented 5 or 2 h before the retention test. AM was used i.p. at the dose of 8 or 1 mg/kg. Neither 8 mg AM administered 4 h before nor 8 or 1 mg doses given after training did not impair the retention performance in unstressed rats. The 1 mg AM prevented the impairment of retention in animals exposed to the stressor 3 or 4 h before training but had no effect when the stronger impairment was induced by ICS 2 h before training. However, when given 1 h before retention testing, 1 mg AM attenuated even the severe impairment induced by the pre-training stressor exposure. Our results suggest that ICS impairs primarily the early phase of memory consolidation and a low dose of AM can prevent this effect.

In situ detection of cyclic AMP-phosphodiesterase activity in the heart of Lewis and Sprague-Dawley rats: the effect of restraint stress or amphetamine application.

Cyclic AMP plays an important role in heart functions under normal as well as pathological conditions. Since phosphodiesterase (PDE), responsible for the hydrolysis of cAMP, is equally important as synthesizing adenylyl cyclase, we decided to determine its activity by cytochemical procedure after exposure of rats to restraint stress or an acute dose of amphetamine. Sprague-Dawley (S-D) and Lewis (LE) rats, the latter known to have a deficient hypothalamo-pituitary-adrenal axis activity, were used in order to disclose the possible significance of rat strain on PDE activity. Animals were divided into 3 groups: controls, rats treated with an acute dose of amphetamine (8 mg/kg, i.p., for 60 min) and rats under restraint stress for 60 min. Control hearts of both strains revealed PDE activity on sarcolemma of cardiomyocytes and plasmalemma of endothelial cells of microvessels. In LE rats we observed an additional enzyme reaction in junctional sarcoplasmic reticulum. In addition, cardiomyocytes of LE rats revealed a higher PDE activity when compared to S-D rats. Restraint stress decreased PDE activity in cardiomyocytes of LE rats while amphetamine markedly inhibited enzyme activity in cardiomyocytes of S-D rats. Endothelial PDE was more resistant to stress. Our results indicate differences in PDE localization and variations in sensitivity of myocardial cAMP-PDE of LE and S-D rat strains to restraint stress and amphetamine application.

Impaired passive avoidance acquisition in Wistar rats after restraint/cold stress and/or stresscopin administration.

Stresscopin (SCP) and related peptides are new members of the corticotropin-releasing factor (CRF) peptide family that are selective ligands for CRF type 2 receptor; these ligands are essential for maintaining homeostasis after stress. SCP (i.p. injections) was tested on the passive avoidance learning task in stressed Wistar rats; it impaired the formation of memory trace. The retention performance deficit induced by SCP was comparable with the deficit induced by the stressor of restraint/cold. More profound impairment of avoidance response occurred following combined application of SCP and stressor. More specific actions of SCP can be expected from its studies with targeted intracerebral applications.

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

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.

Differences in the behaviour of Sprague--Dawley and Lewis rats during repeated passive avoidance procedure: effect of amphetamine.

The present paper investigated the differences in passive avoidance learning between Sprague--Dawley and Lewis rats. After initial habituation (experimental Part 1), measured as latencies to enter the dark, preferable compartment, the effect of treatment with amphetamine (8 mg kg(-1)b.w.), the retention performance compared with controls (saline) was tested in both rat strains in Parts 2--4. The intervals between Parts 2--4 were 24 or 49 days. Each experimental part consisted of testing lasting 6 days. On the 7th day the rats received drug treatment 1 h before the application of foot shock. The differences between rat strains were already detectable at the beginning of the study. During the repeated exposures of rats in Part 1, only Lewis rats, in contrast to Sprague--Dawley rats, exhibited the habituation. The repeated testing of rats in Parts 2--4, due to previous experience with an aversive stimulus, was considered as the retention test. In Parts 2--3 we observed only minor differences in the responses of both rat strains tested. Also no significant differences were observed between rat strains after amphetamine treatment that induced an amnesia-like effect in all retention trials. However, data shown in Part 4 revealed the largest differences between both strains. Control Lewis rats exhibited significantly higher retention responses than Sprague--Dawley rats. In the latter strain we observed no differences in avoidance latencies between controls and amphetamine treated rats. In Lewis rats the difference in avoidance performance between controls and amphetamine treated animals was highly significant due to their enhanced retention performance. In conclusion, the results presented in this study extend the known behavioural differences in tested rat strains to the passive avoidance procedure that, in addition, was performed for a total period of 4 months. Due to a known deficiency of hypothalamo-pituitary-adrenal axis activity in Lewis rats it can be hypothesized that the behavioural dissociation of this strain from Sprague--Dawley rats could be related to the different activity of this regulatory axis in the rat strains tested.

Rat strain differences in responses of plasma prolactin and PRL mRNA expression after acute amphetamine treatment or restraint stress.

1. The aim of this study was to compare the effects of acute amphetamine (AMPH) treatment and restraint stress on plasma level of prolactin (PRL) and PRL mRNA expression in the adenohypophysis in Sprague-Dawley and Lewis male rats, the latter known to have a deficient hypothalamo-pituitary-adrenal (HPA) axis. 2. Both restraint stress and AMPH treatment (i.p. in a dose of 8 mg/kg of b.w.) were applied 15 or 30 min before termination of the experiment. Plasma PRL and corticosterone (CORT) were determined by radioimmunoassay. PRL mRNA expression was estimated by a dot-blot hybridization. 3. Restraint stress and AMPH treatment induced a significant increase in the CORT plasma level, as an indicator of stress response. Compared to Sprague-Dawley rats, the magnitude of CORT increase after both stimuli was significantly lower in Lewis rats. 4. Although restraint stress significantly increased the PRL plasma levels in both rat strains, AMPH treatment reduced the PRL levels in both rat strains. However, the changes of PRL plasma levels had another pattern in Lewis rats than in Sprague-Dawley rats. Control plasma PRL levels were significantly higher in Lewis rats, and in this rat strain AMPH treatment for 30 min increased the PRL levels as compared to the values obtained after AMPH treatment for 15 min. 5. Expression of PRL mRNA in adenohypophysis by restraint stress and AMPH treatment had a similar pattern. After a 15-min lasting restraint stress, the expression of PRL mRNA was decreased insignificantly in both rat strains. AMPH treatment induced in Sprague-Dawley rats a significant decrease of PRL mRNA after a 15-min interval while after 30 min there was a significant increase. However, in Lewis rats AMPH failed to significantly change PRL mRNA. 6. The results from the present study indicate that the mechanisms mediating the effects of acute restraint stress and acute AMPH treatment differ in PRL response in Sprague-Dawley and Lewis male rat strains. Differences in the observed responses in Lewis rats could be related to the deficient activity of HPA axis in this rat strain.

Effects of immobilization stress combined with water immersion and chronic amphetamine treatment on the adenylyl cyclase activity in rat neurohypophysis.

Several papers have indicated the participation of cyclic AMP as a second messenger for the release of neurohypophysial hormones. Since very little is known about the effects of stress and drugs of abuse on this process, we studied the activity of adenylyl cyclase in the neurohypophyses after immobilization stress and chronic amphetamine treatment. Our findings indicate the involvement of cyclic AMP in the regulation of neurohypophysis as well as the increase in total adenylyl cyclase both after application of immobilization stress combined with water immersion and after chronic amphetamine treatment.

Different activity of adenylyl cyclase in prefrontal cortex in three rat strains. The effect of amphetamine.

Since the literature data do not provide enough information on the effects of amphetamine on the prefrontal cortex and since many controversial findings were reported in various rat strains we decided to compare adenylyl cyclase activity in the prefrontal cortex in various rat strains and test the effects of chronic amphetamine treatment (for 14 days) on the activity of this enzyme. Basal adenylyl cyclase activity was lower in Wistar rats than in Sprague-Dawley and Lewis rat strains. Amphetamine treatment produced in Wistar rats a substantial decrease in basal adenylyl cyclase activity. In Sprague-Dawley rats, we observed the highest enzyme activity which was slightly reduced after amphetamine treatment. In Lewis rats which had basal activity close to the activity of Wistar rats, amphetamine produced an increase in enzyme activity. The total adenylyl cyclase activity, estimated in the presence of forskolin, was the lowest in Wistar rats. The highest stimulation was observed in Lewis rats. Amphetamine treatment caused a very significant inhibition of total adenylyl activity in Wistar rats and a smaller inhibition in Sprague-Dawley rats. However, in Lewis rats amphetamine treatment increased the dose-response curve of forskolin stimulation. These results show that Lewis rats, compared to the other two strains, develop not only quantitatively but also qualitatively different responses.

Changes in beta-adrenergic receptors in the neurohypophysis and intermediate lobe of rat hypophysis exposed to stress.

Changes in beta-adrenergic receptors in the neurohypophysis and intermediate lobe of the rat have been characterized under physiological and stress conditions. Classical immobilization stress (IMO) was also combined with the immersion of rats into water (IMO + COLD stress). Both types of stress were applied for 30, 60 or 150 min. The intensity of stress stimuli were controlled by measuring the level of plasma ACTH. Changes in the level of plasma ACTH indicate that both types of experimental protocol induced reliable and reproducible stress conditions. Binding studies dealing with beta-adrenergic receptors in the intermediate lobe and neurohypophysis were performed in saturation binding studies by using of 125I-iodopindolol. Binding parameters, maximal binding capacity (Bmax) and dissociation constant (Kd) were assessed by nonlinear analysis with computer program Viewfit. In the neurohypophysis, no changes of Kd were found in the stressed animals. However, maximal binding capacity was decreased significantly with the increased exposure to the stress. In the intermediate lobe Kd values were slightly decreased and Bmax values decreased gradually with increasing duration of stress exposure. Our findings suggest that the process of receptor desensitization of beta-adrenergic receptors can also be detected under stress conditions in the neurohypophysis and intermediate lobe of the pituitary gland where it could contribute to the mechanisms involved in stress reactions.