Sixtus Hynie

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.

Influence of some new beta-sympathotropic agents on lipid mobilizationin vitro

The influence of some beta-sympatholytic agents on the lipid mobilization from rat epididymal adipose tissuein vitro was followed. DCI, Nethalide (Net), 1-(2,4-dichlorophenyl)-1-hydroxy-2-(t-butylamino)- ethane (I), 1-(3,4-dichlorophenyl)-1-hydroxy-2-(methylethyl)-ethane (II), and M2,4-dibromphenyl)-l-hydroxy-2-(t-butylamino)-ethane (III) were shown to act as dualists of mimetic amines. The mimetic effect of these drugs decreases in the line DCI > Net > II = III = I. The intensity of the lytic action of these drugs in antagonizing the norepinephrine lipid mobilization increases in the line DCI = Net = II < III < I. The lytic effectiveness calculated as the difference of the lytic and untoward mimetic effect, increases as follows: DCI < Net = II < III = l. Therefore, substance I seems to be the most effective lytic when used againstin vitro norepinephrine-induced lipid mobilization. Some aspects of interactions between the drugs used are discussed.

SOME ASPECTS OF THE ACTION OF BETA ADRENERGIC BLOCKING DRUGS ON ADRENERGIC LIPID MOBILIZATION

Although only beta adrenergic blocking drugs have been shown to specifically antagonize adrenergic lipid mobilization,”2 very unexpected results were obtained when the degree of this specific antagonistic action was examined.3’4 In six of a series of nine blocking drugs very different pA2 values were obtained for the same agent when comparing pA2 parameters obtained from different concentrations of the antagonists. As far as the pAz value represents the negative logarithm of the dissociation constant of the complex “receptor” + “antagonist” it should remain constant. The usual method for determining the pA2 value assumes quantitative relationships characterizing a simple bimolecular reaction (drug + receptor). However, in the lipid mobilizing actions of some sympathomimetic drugs “quadratic” dose-response relations, very similar t o trimolecular-reaction laws (drug + two receptor sites), were also d e ~ c r i b e d . ~ ’ ~ In this paper an attempt is made to examine the actions of beta adrenergic antagonists in terms of one of the possible hypothetical models concerning a “two-receptor’’ reaction.

Effect of directly acting sympathomimetic drugs on lipid metabolismin vitro

Abstract When following up the adrenergic lipid mobilization from the rat depot adipose tissue in vitro it was found that the “intrinsic activity” of isoproterenol, norepinephrine, and epinephrine with regard to this function is identical. The “intrinsic affinity” investigated both by the reaction of mimetics per se and with the aid of an interaction with the antagonist phentolamine, is graded in the sequence ISO/pD 2 = 5·8/>NOR/pD 2 = 5·5/>EPI/pD 2 = 5·3 . The “pure” alpha-mimetic phenylephrine had only a minimal lipid mobilizing effect. The betalytic DCI acts as a competitive dualist with an affinity close to that of ISO and with a relatively high activity. In the alpha-lytic phentolamine no appreciable mimetic changes have been observed. The maximum of the mimetic effect of active drugs lay in the vicinity of the concentration 2–5 × 10 −5 M. Higher concentrations of NOR and DCI led to an autoinhibition. The pD 2 ′ values of the autoinhibitory effect were determined approximately in DCI (3.9) and in NOR (3.5). Certain specific characteristics of the adrenergic lipid mobilization in contrast to other adrenergic functions have been pointed out.

Rat Strain Differences in Responses of Plasma Prolactin and PRL mRNA Expression After Acute Amphetamine Treatment or Restraint Stress

Abstract1. 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 theCORT 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 mediatingthe 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.

Inhibitory effects of neuropeptide Y on adenylate cyclase of rabbit ciliary processes

The inhibitory effect of neuropeptide Y (NPY) was studied on the adenylate cyclase (AC) activity in homogenates of rabbit ciliary processes and compared with that of the alpha 2-adrenergic agonist clonidine (CLN). NPY inhibited basal AC activity as well as AC activity stimulated by isoproterenol (ISO), vasoactive intestinal polypeptide (VIP) or forskolin (FSK). The extent of this inhibition corresponded well to the inhibition elicited by CLN. The inhibitory effects of NPY and CLN appeared to be nonadditive. AC activity stimulated by ISO was considerably more sensitive to the effects of either NPY or CLN than basal, VIP- or FSK-stimulated AC activity. It was inferred that NPY inhibitory effects were mediated by the activation of NPY receptors coupled negatively to the catalytic unit of AC via the inhibitory Gi protein. Moreover, involvement of NPY in physiological modulation of AC activity in ciliary processes and in the regulation of aqueous humor formation and intraocular pressure is suggested.

Inhibitory effects of clonidine and dopamine on adenylate cyclase of rabbit ciliary processes.

The inhibitory effects of the alpha 2-adrenergic agonist clonidine and that of dopamine were studied on the adenylate cyclase activity in homogenates of ciliary processes. Clonidine inhibited in a dose-dependent manner basal adenylate cyclase activity as well as that stimulated by isoproterenol or forskolin. However, the adenylate cyclase activity stimulated by isoproterenol was sensitive to at least one order lower inhibitory concentrations of clonidine than basal or forskolin-stimulated adenylate cyclase. Dopamine inhibited adenylate cyclase stimulated by isoproterenol considerably less potently than clonidine. The slope of the dopamine dose-response curve was, however, similar to that of the dose-response curve of clonidine. The inhibitory effects of clonidine and dopamine were antagonized by an alpha 2-adrenergic antagonist, yohimbine, in a manner suggesting a competitive nature of this interaction. On the contrary, the inhibitory effects of neither clonidine nor dopamine were prevented by an alpha 1-adrenergic antagonist, prazosin. In addition, the effect of dopamine was not antagonized by the D2-antagonist, tiapride. Taken together, these results strongly indicate that both clonidine and dopamine exert their inhibitory effects by the stimulation of alpha 2-adrenergic receptors. Accordingly, they provide experimental evidence that both basal and drug-stimulated adenylate cyclase activity of ciliary processes can be inhibited via stimulation of alpha 2-adrenergic receptors. The substantially higher sensitivity of isoproterenol-stimulated than basal or forskolin stimulated adenylate cyclase to alpha 2-adrenergic inhibition seems to be a unique feature of this enzyme of ciliary processes. It is suggested that this may reflect an involvement of alpha 2-adrenergic receptors in the physiological feedback mechanism preventing the over-stimulation of adenylate cyclase of ciliary processes during excessive adrenergic drive.