Effects of LPS on the behavioural stress response of genetically selected aggressive and nonaggressive wild house mice.

The bacterial endotoxin lipopolysaccharide (LPS) exerts strong effects on the immune-neuroendocrine network. On behaviour, LPS induces the symptoms of sickness behaviour. Otherwise, LPS challenge shares with psychological stress some common physiological adaptations. The proposal of this study was to analyse the effects of the LPS injection on the behavioural response in the shock-probe defensive burying test of two wild house mouse lines genetically selected for short (SAL) and long (LAL) latency to attack a conspecific. It is known that with previous exposure to stress, each of these lines exhibits behaviour in the burying test that is closely related to their different neuroendocrine patterns of response, with higher expression of burying in the SAL and immobility in the LAL mice. LPS (0.5 ml, 375 microg/Kg) or sterile saline (0.9%) was i.p. injected 3.5h before the beginning of the test. Non-injected mice were used as a general control of stress of handling and drug effect. The following behaviours were analyzed: defensive burying, immobility, rearing, grooming, exploration and jumping. The procedure of injection was found to be a stimulus that induced behavioural alterations in the SAL and LAL mice. Some behavioural changes induced by saline injection resembled that induced by LPS injection; in both lines an increase in immobility as well as a decrease in burying behaviour was observed. It is noteworthy that the LAL mice increased more their immobility than the SAL mice after saline or LPS injection, and the decrease in burying in the saline and LPS-injected mice was lower in the SAL than in the LAL mice. These results and others discussed in the text suggest that the active coping strategy of SAL mice and the passive coping strategy of the LAL mice, the hallmark of each line in the shock-probe burying test is present after psychological as well as LPS challenge exposure.

The angiotensin converting enzyme I/D polymorphism in long distance runners.

AIM: There is an assumption that ACE I/D polymorphism represents one of the possible genetic factors that might be associated with sports excellence. Recent studies have identified an increased frequency of I allele in elite endurance athletes, long distance runners, rowers and mountaineers. The aim of this study was to test the hypothesis that the ACE I/D polymorphism is associated with enhanced endurance performance. METHODS: We examined this hypothesis by determining ACE I/D allele frequency in 215 marathon runners, 222 half-marathon runners and 18 inline skaters classified by performance (marathon competition results). ACE genotype and allele frequencies were compared with 252 healthy controls. RESULTS: ACE genotype frequency in the whole cohort did not differ from that in the sedentary controls (P < 0.56). However, there was an increase of the I/I genotype incidence amongst successful marathon runners scoring on places from 1st to 150th (P < 0.01). These findings were confirmed in the group of inline skaters, similarly demonstrating an increase of the I/I genotype (P < 0.01). There was no association found between half marathon runners and the ACE genotype (P < 0.59). CONCLUSIONS: An excess of the I allele in long distance runners confirms the association between the ACE I/D polymorphism and endurance sports performance.

Temporal and spatial dynamics of corticosteroid receptor down-regulation in rat brain following social defeat.

The experiments explored the nature and time course of changes in glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) binding in homogenates of various brain regions and pituitary of male Wistar rats following social defeat stress. One week after defeat, the binding capacity of GRs was decreased in the hippocampus and the hypothalamus while no changes were observed in the parietal cortex and the pituitary. The number of MRs remained at the same level as in undefeated rats. Three weeks postdefeat, the initially down-regulated GR returned to baseline level in the hippocampus and the hypothalamus. However, GR binding was now decreased in the parietal cortex. Severe down-regulation of MRs was detected in the hippocampal and septal tissue. The results show that brief but intense stress like social defeat induces a long-lasting down-regulation of corticosteroid receptors and that the temporal dynamics of these changes are not only differential for GRs and MRs but also for brain sites.

Postnatal treatment with ACTH-(4-9) analog ORG 2766 attenuates N-methyl-D-aspartate-induced excitotoxicity in rat nucleus basalis in adulthood.

It has been reported that the ACTH-(4-9) analog H-Met(O(2))-Glu-His-Phe-D-Lys-Phe-OH (ORG 2766) administered in adulthood has trophic effects on neuronal tissue and when given postnatally, it can induce long-lasting changes in brain development. In the present study, we investigated whether early postnatal treatment with ORG 2766 affects adult neuronal vulnerability, i.e. the sensitivity of cholinergic neurons against excitotoxic damage. Wistar rat pups received injections of ORG 2766 or saline on postnatal days 1, 3 and 5 and were then left undisturbed until adulthood. At the age of 6 months, the animals were subjected to unilateral lesion of magnocellular basal nucleus by infusion of high dose of N-methyl-D-aspartate (NMDA). The effects of the excitotoxic insult were studied 28 hours and 12 days after the lesion by measuring both the acute cholinergic and glial responses, and the final outcome of the degeneration process. Twenty eight hours after NMDA infusion, postnatally ACTH-(4-9)-treated animals showed stronger suppression of choline-acetyltransferase immunoreactivity and increased reaction of glial fibrillary acidic protein -immunopositive astrocytes in the lesioned nucleus compared to control animals. However, 12 days post-surgery, the NMDA-induced loss of cholinergic neurons, as well as the decrease of their acetylcholinesterase -positive fibre projections in the cortex, were less in ACTH-(4-9) animals. Our data indicate that the early developmental effects of ACTH-(4-9) influence intrinsic neuroprotective mechanisms and reactivity of neuronal and glial cells, thereby resulting in a facilitated rescuing mechanism following excitotoxic injury.

The influence of postnatal handling on adult neuroendocrine and behavioural stress reactivity.

Environmental stimuli during early stages of life can influence the development of an organism and may result in permanent changes in adult behaviour and physiology. In the present study we investigated the influence of early postnatal handling on adult neuroendocrine and behavioural stress reactivity in Wistar rats. Pups were subjected to handling from postnatal day 1-21. The young were taken from the nest every day for 15 min and each of the pups was handled separately. Control nests were left undisturbed. When the animals had reached an adult age of 3-4 months they were individually housed and subjected to a series of tests to measure their stress reactivity. In the first experiment we established adult behavioural coping with stressors and anxiety in the following series of tests: open field test, shock prod defensive burying test, elevated plus maze and conditioned fear test. Collectively, the data clearly indicate that handled animals are characterized by a lower stress-induced anxiety. Yet, handled and control animals do not differ in their general way of coping with stressors. Although the lower anxiety in handled animals is often reflected in a higher activity, they are not more active per se. In a second experiment, animals were provided with a permanent jugular vein canula for repeated blood sampling to determine stress hormones: noradrenaline, adrenaline, prolactin and corticosterone. Animals were subjected to a novelty test and a conditioned fear test. The neuroendocrine response profile is consistent with the conclusion that handled animals are less anxious than controls but are not different in their general strategy of coping with stressors. The handled animals showed an attenuated adrenaline, prolactin and corticosterone response. Yet, in neither of the two tests there was a difference in noradrenaline response, a typical marker for an active coping strategy. Interestingly, the differences in neuroendocrine reactivity already appeared in response to a mild novelty challenge when there were no clear behavioural differences yet. The neuroendocrine measures are in line with the behavioural data but more sensitively reflect the differences between handled and control animals.

Long-lasting deficient dexamethasone suppression of hypothalamic-pituitary-adrenocortical activation following peripheral CRF challenge in socially defeated rats.

The present study focuses on the long-term changes in the regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis following two short-lasting episodes of intensive stress in the rat stress model of social defeat and the possible similarities with HPA functioning in human affective disorders. Male Wistar rats experienced social defeats on 2 consecutive days by an aggressive male conspecific. The long-term effect of these defeats on resting and ovine corticotropin-releasing factor (oCRF; intravenous (i.v.) 0. 5 microg/kg) induced levels of plasma ACTH and corticosterone (CORT) were measured 1 and 3 weeks later. In a second experiment the glucocorticoid feedback regulation of HPA function was tested in a combined dexamethasone (DEX)/CRF test (DEX; 25 microg/kg s.c., 90 min before oCRF injection, 0.5 microg/kg). The oCRF challenges were performed between 11.00 and 13.00 h (about three hours after start of the light phase). One week after defeat the ACTH response to CRF was significantly enhanced in defeated rats as compared to controls. Three weeks after defeat the ACTH response was back to control levels. The increased ACTH response 1 week after the stressor was not reflected in higher CORT levels. Neither were baseline ACTH and CORT levels affected by the prior stress exposure. DEX pretreatment inhibited pituitary adrenocortical activity, reflected both in reduced baseline and response values of ACTH and CORT. The ACTH response to CRF following DEX administration was significantly higher in defeated rats as compared to controls both at one and three weeks after defeat. A reduced DEX suppression of baseline secretion of ACTH appeared 3 weeks after defeat. The same tendency was apparent in response and baseline values of CORT. The differences in CORT between socially stressed and control treated rats, however, did not reach significance. The possible role of changes in glucocorticoid-(GR) and mineralocorticoid receptor (MR) binding in the altered regulation of HPA activity following defeat were studied in brain and pituitary of male Wistar rats 1 and 3 weeks after defeat. One week after defeat GR-binding decreased in hippocampus and hypothalamus. No changes were observed in GR-binding in the pituitary nor in MR-binding in any of the regions analysed. Three weeks after defeat GR-binding recovered in hippocampus and hypothalamus but at this time MR-binding in hippocampal tissue was seriously decreased. In a fourth experiment vasopressin (AVP) and CRF stores in the external zone of the median eminence (ZEME) were measured by quantitative immunocytochemistry one and three weeks after defeat and compared with controls. Social defeat failed to induce a change in the immunocytochemical stores of AVP or CRF. The present findings show that in rats short-lasting stressors like defeat induce long-lasting, temporal dynamic changes in the regulation of the HPA axis. Since these changes in time are reflected in GRs and MRs in different brain areas an altered corticosteroid receptor binding might play an important role in the affected HPA activity following defeat.

Fear-potentiation in the elevated plus-maze test depends on stressor controllability and fear conditioning.

The purpose of the study was to determine which stressor qualities (escapable vs. inescapable stress and unconditioned vs. conditioned stress) can potentiate fear in the elevated plus-maze. While inescapable stress potentiated fear, escapable stress did not, but escapable stress increased the locomotor activity (closed arm entries). Inescapable stress only potentiated fear when re-exposure to the former shock compartment, 24 h after footshock and without further footshock, took place just before to 90 min before testing in the elevated plus-maze. We conclude that fear-potentiation in the plus-maze depends on stressor controllability and contextual conditioning. Fear-potentiation was reduced by the anxiolytic diazepam (0.5, 1.0 and 2.0 mg/kg, s.c.) and was further enhanced by the anxiogenic DMCM (1.0 mg/kg, s.c). The fear-potentiated plus-maze test may be a valuable tool in the search for novel anxiolytics and in the study of the neurobiology of fear-potentiation, fear conditioning and generalization of fear.

Differential effects of CRH infusion into the central nucleus of the amygdala in the Roman high-avoidance and low-avoidance rats.

Roman-high (RHA/Verh) and low (RLA/Verh) avoidance rats are selected and bred for rapid learning versus non-acquisition of two-way, active avoidance behavior in a shuttle box. RHA/Verh rats generally show a more active coping style than do their RLA/Verh counterparts when exposed to various environmental challenges. The central nucleus of the amygdala (CeA) is known to be involved in the regulation of autonomic, neuroendocrine and behavioural responses to stress and stress-free conditions, and it is considered in relation to coping strategies. Corticotropin-releasing hormone (CRH) seems to be a key factor in the control of the CeA output. Neuroanatomical studies have revealed that the majority of CRH fibers from the CeA have direct connections with autonomic regulatory nuclei in the brainstem, e.g. lateral parabrachial nucleus (lPB), ventrolateral periaquaductal gray (vlPAG). The modulating effects of CRH (30 ng) on CeA activity were studied by infusion of CRH into the CeA in freely moving male RHA/Verh and RLA/Verh rats under stress-free conditions. Heart-rate and behavioural activities were repeatedly measured before, during and after local administration of CRH or vehicle, after which early gene product FOS immunocytochemistry and CRH-mRNA in situ hybridisation were carried out in selected brain areas. CRH infusion into the CeA caused a long lasting increase in heart-rate and behavioural activation in the RHA/Verh rats, leaving the RLA/Verh rats unaffected. As a result of CRH infusion, the number of FOS positive cells in the CeA and lPB of RLA/Verh rats was increased whereas an opposite response was found in the RHA/Verh rats. However, CRH into the CeA of the Roman rat lines induced no pronounced effects on FOS staining in the vlPAG and CRH mRNA levels in the CeA. These results indicate that the CRH system of the CeA, connected with the output brainstem areas, is differentially involved in cardiovascular and behavioural responses.

Vulnerability to arrhythmias during social stress in rats with different sympathovagal balance.

An increased activity of the sympathetic nervous system is an important factor in the genesis of ventricular arrhythmias. Changes in average R-R interval, R-R interval variability (indirect measure of sympathovagal balance), occurrence of arrhythmias, and plasma norepinephrine concentrations were measured during a social stress episode (defeat) in two strains of rats, Wistar and wild type, which were supposed to differ in their autonomic stress responsiveness. Electrocardiograms were telemetrically recorded, and blood samples were withdrawn through jugular vein catheters from healthy, freely moving animals. R-R interval variability was estimated by the following time-domain parameters: the standard deviation of the mean R-R interval, the coefficient of variance, and the root mean square of successive differences in R-R interval. Average R-R interval and R-R interval variability measures, as well as plasma norepinephrine concentrations, indicated a higher sympathetic tone, a larger sympathetic responsiveness, and a lower parasympathetic antagonism after sympathetic activation in wild-type animals, which also showed a much higher incidence of arrhythmias (ventricular premature beats), compared with Wistar rats. These two strains might represent a valuable experimental model for studying the mechanisms (cellular/electrophysiological) responsible for the susceptibility to arrhythmias in healthy individuals exposed to stressful situations.

Repeated blockade of mineralocorticoid receptors, but not of glucocorticoid receptors impairs food rewarded spatial learning.

Corticosteroids from the adrenal cortex influence a variety of behaviours including cognition, learning and memory. These hormones act via two intracellular receptors, the mineralo-corticoid receptor (MR) and the glucocorticoid receptor (GR). These two receptor types display a high concentration and distinct distribution in the hippocampus, a brain region which is directly involved in the regulation of spatial orientation and learning. In this study, repeated subcutaneous administration of the mineralocorticoid receptor antagonist RU28318 (1.0 mg/100 g body weight), the glucocorticoid receptor blocker RU38486 (2.5 mg/100 g body weight), or a combination of both antagonists were investigated for their effects on working--and reference memory in morning and afternoon trials during 8 subsequent days in food rewarded spatial learning in a hole board task. Each rat received one dose of either vehicle (2% ethanol in PEG 400), RU28318, RU38486 or the combination of both antagonists directly after the first trial on training days 1, 3, 5, and 7. The experiments demonstrated that repeated blockade of mineralocorticoid receptors impairs reference memory reflected in the morning--as well as in the afternoon trial, whereas blockade of glucocorticoid receptors has little effect on this type of cognitive behaviour. Furthermore, combined blockade of MRs and GRs resulted in a decrease, in both daily trials, in reference memory as well as working memory performance. These findings suggest that in this spatial learning paradigm, the impairment of working memory required blockade of both receptor types, while reference memory performance involves predominantly the mineralocorticoid receptors.

Autonomic brainstem projections to the pancreas: a retrograde transneuronal viral tracing study in the rat.

The present study describes brainstem nuclei that participate in the autonomic innervation of the pancreas, using a retrograde viral transneuronal tracing technique. It aimed at identifying the neuronal architecture of the parasympathetic, gustatory-induced insulin release by the endocrine pancreas (preabsorptive insulin response, PIR). Autonomic pathways organized for reflex adjustments of the end organ, as it happens in the PIR, involve relatively simple circuits. This implies a short brainstem circuit from the rostral gustatory nucleus of the solitary tract to the dorsal motor nucleus of the vagus. The present findings confirm projections to the pancreas, originating from preganglionic neurons in the dorsal motor nucleus of the vagus. Transneuronal labeling was detected in the medial, and to a lesser extent in the lateral nucleus of the solitary tract mainly at caudal and intermediate levels. Furthermore, infected neurons were seen in the brainstem in the dorsal and ventral part of the medullary reticular formation, in the area postrema and in the raphe nuclei. Sparse labeling was found in the gustatory zone of the nucleus tractus solitarius. These results indicate that a direct connection between the rostral nucleus tractus solitarius and the medial dorsal motor nucleus of the vagus is very unlikely, so that one or more intermediate stations may be involved. Candidates to complete this pathway are the intermediate or caudal nucleus tractus solitarius, the medullary reticular formation or the parabrachial nucleus.

Coping with stress in rats and mice: differential peptidergic modulation of the amygdala-lateral septum complex.

This chapter focuses on the parvicellular vasopressin (VP) system originating from the medial nucleus of the amygdala (MeA) and bed nucleus of the stria terminalis (BNST). The vasopressinergic fibers of these nuclei innervate a number of limbic brain areas including the septum-hippocampal complex. Interestingly, this VP system is sexually dimorphic and the VP synthesis in this system depends on circulating gonadal steroids. Studies in rats and mice show that the variation in the lateral septal VP network within the male gender is as large as the variation between the sexes as reported in the literature. Non-aggressive males are characterized by a far more extensive VP network and a higher VP content in the lateral septal area than aggressive males. A review of the literature on the function of lateral septal VP in the organization of behavior reveals not only a modulatory role of behavior in a social context, but also of fear- and anxiety-related behaviors. It is argued that these seemingly diverse functions might be explained by the concept of coping style. Extensive behavioral and physiological analyses in a variety of animal species show that males may be characterized by the way in which they cope with environmental challenges in general. Aggressive males tend to cope actively with their environment whereas non-aggressive males seem to accept the situation as it is more easily. In several tests, we determined the effects of chronic infusion of the V1 receptor antagonist locally into the lateral septal area in male rats. The main conclusion from these experiments is that LS VP does not modulate coping style in general. However, the experiments confirm the idea that LS VP has a certain degree of functional specificity in social behavior and social learning tasks. Together with the observation that the size and distribution of the vasopressinergic system may be highly variable between individual males in relation to their coping style, this suggests that the lateral septal vasopressinergic system is involved in the differential capacity of individuals to cope behaviorally with challenges of a social nature.

The vasopressin deficient Brattleboro rats: a natural knockout model used in the search for CNS effects of vasopressin.

Behavioral neuroscience is using more and more gene knockout techniques to produce animals with a specific deletion. These studies have their precedent in nature. A mutation may result in a limited genetic defect, as seen in the vasopressin (VP) deficiency in the Brattleboro rat. The mutation is in a single pair of autosomal loci, and the sequences of VP gene from wild-type and homozygous Brattleboro rats are identical except for a single nucleotide deletion in the second exon. The deletion results in the synthesis of an altered VP precursor that is unable to enter the secretory pathway. The genetic disturbance results in a central diabetes insipidus comparable to that found in humans. Starting with our work during the early 1970s we found that the genetic defect in the availability of VP causes deficits in central nervous system (CNS) functions. Behavioral processes from cognition to drug tolerance appeared to be disturbed by the absence of VP, but not all behaviors are affected. The specificity of the absence of VP in causing behavioral deficits is shown in many cases. However, certain deficits are due to genetic factors other than the deletion of the VP gene. The picture is further complicated by differences in testing conditions, the absence of proper controls, i.e. heterozygous and wild-type Brattleboro rats, sex, compensation phenomena, and the absence of neuropeptides co-localized with VP. Interestingly, an age dependent spontaneous shunt to a heterozygous phenotype in vasopressinergic neurons might also compensate for the disturbance. Accordingly, findings in knockout animals should be interpreted with caution. One should realize that brain functions are modulated by multiple neuropeptides and that neuropeptides possess multiple CNS effects.

Effect of corticosterone and adrenalectomy on NMDA-induced cholinergic cell death in rat magnocellular nucleus basalis.

The present study demonstrates the effects of adrenalectomy and subcutaneously administered corticosterone on N-methyl-D-aspartate-induced neurodegeneration in the cholinergic magnocellular basal nucleus of the rat. NMDA was unilaterally injected into the nucleus basalis at different plasma corticosterone concentrations in adrenalectomized rats, in adrenalectomized animals with subcutaneously implanted cholesterol-corticosterone pellets containing 25% or 100% corticosterone, and in sham-adrenalectomized controls. The neurotoxic impact of the NMDA injection in the various experimental groups was assessed by the loss of cholinergic fibers stained with acetylcholinesterase histochemistry in the parietal neocortex. Reactive cortical astrocytes as a result of the treatments were detected by glial fibrillary acidic protein immunohistochemistry. Measurements of the densities of astrocytes and cholinergic fibers at the injected side of the brain were carried out by image analysis. Adrenalectomy significantly potentiated the NMDA-induced neurodegeneration by 50%, while chronic administration of corticosterone significantly attenuated the NMDA-neurotoxicity in a dose-dependent manner. Compared to the ADX group, 25% corticosterone application reduced the NMDA damage by 37%, whereas the 100% corticosterone pellet diminished NMDA neurotoxicity by 75%. Both ADX and ADX + corticosterone implantation enhanced the NMDA-induced GFAP immunoreactivity. The increase of GFAP immunoreactivity was most pronounced in the adrenalectomized rats supplied with the 100% corticosterone pellets. The results demonstrate that corticosterone exerts a potent neuroprotective effect on NMDA-induced neurotoxicity in the magnocellular nucleus basalis. The activated astroglia suggest that astrocytes may contribute to the beneficial effect of corticosterone in the neuroprotective mechanisms against excitotoxic neuronal injury.

Permanent upregulation of hippocampal mineralocorticoid receptors after neonatal administration of ACTH-(4-9) analog ORG 2766 in rats.

The development of brain corticosteroid receptors may be permanently modified by perinatal hormone treatments, in particular by hormones of the hypothalamic-pituitary-adrenal axis. Changes in binding characteristics of corticosteroid receptors were investigated in rats treated subcutaneously with 1 microg/g body wt of the ACTH-(4-9) analog peptide ORG 2766 once daily at postnatal days 1, 3 and 5. [3H]Corticosterone (CORT) binding capacity (Bmax) and affinity (Kd) were determined at 1-, 2- and 3-weeks old and adult ages in the hippocampal cytosol by using saturation analysis. Mineralocorticoid type receptor (MR) and glucocorticoid receptor (GR) sites were measured separately with single-point analysis applying a selective glucocorticoid ligand RU 28362 saturating GR. An increase in [3H]CORT binding capacity was found during postnatal development which remained permanently high up to adult age. Separate analysis of MR and GR expression indicated that the increment in the number of corticoid receptor sites was due to an increase in number of MRs in both the young and adult rats. It was concluded that neonatal injections of ACTH-(4-9) peptide resulted in a permanent and selective upregulation of hippocampal MRs, which may underlie the previously observed increased vigilance and novelty-induced behavioral reactivity of the peptide-treated adult rats (Felszeghy, K., Sasvari, M. and Nyakas, C., Horm. Behav., 27 (1993) 380-396).

Exposure to chronic psychosocial stress and corticosterone in the rat: effects on spatial discrimination learning and hippocampal protein kinase Cgamma immunoreactivity.

Previous reports have demonstrated a striking increase of the immunoreactivity of the gamma-isoform of protein kinase C (PKCgamma-ir) in Ammon's horn and dentate gyrus (DG) of rodent hippocampus after training in a spatial orientation task. In the present study, we investigated how 8 days of psychosocial stress affects spatial discrimination learning in a hole board and influences PKCgamma-ir in the hippocampal formation. The acquisition of both reference memory and working memory was significantly delayed in the stressed animals during the entire training period. With respect to cellular plasticity, the training experience in both nonstressed and stressed groups yielded enhanced PKCgamma-ir in the CA1 and CA3 regions of the posterior hippocampus but not in subfields of the anterior hippocampus. Stress enhanced PKCgamma-ir in the DG and CA3 pyramidal cells of the anterior hippocampus. In stressed animals that were subsequently trained, the PKCgamma-ir was increased in the posterior CA1 region to the same level as that found in nonstressed trained animals. Stress apparently abrogated the PKCgamma-ir training response in the CA3 region. In a second experiment, the elevation of plasma corticosterone levels to values that are found during stress did not significantly influence reference memory scores but slightly and temporarily affected working memory. The training-induced enhancement of PKCgamma-ir in the CA1 region was similar in trained and corticosterone-treated trained animals, but the learning-induced PKCgamma-ir response in the posterior CA3 area was absent after corticosterone pretreatment. These results reveal that prolonged psychosocial stress causes spatial learning deficits, whereas artificial elevation of corticosterone levels to the levels that occur during stress only mildly affects spatial memory performance. The spatial learning deficits following stress are reflected only in part in the redistribution of hippocampal PKCgamma-ir following training.

Muscarinic acetylcholine receptor immunoreactivity in the amygdala--I. Cellular distribution correlated with fear-induced behavior.

This study examined the distribution of muscarinic acetylcholine receptor-immunoreactive neurons in the amygdaloid complex of the rat, with emphasis on the central nucleus. The monoclonal antibody M35 raised against purified muscarinic acetylcholine receptor protein was used to visualize muscarinic acetylcholine receptor-immunoreactive cells. Muscarinic acetylcholine receptor immuno-reactivity was high in the central nucleus and low to moderate in all other regions of the amygdaloid complex. Within the central nucleus, the muscarinic acetylcholine receptor-immunoreactive neurons were found predominantly in the lateral subdivision. This region contained medium-sized neurons (largest diameter ranging from 10 to 15 microns), with a round or slightly ovoid cell shape. At the subcellular level, however, the labeled neurons revealed relatively few muscarinic acetylcholine receptor-immunoreactive postsynaptic densities. Immunofluorescent double-labeling demonstrated that nearly all of the muscarinic acetylcholine receptor-immunoreactive neurons (98.6%) in the central nucleus expressed abundant amounts of nicotinic acetylcholine receptors, further substantiating the cholinoceptive character of these cells. In addition, the vast majority of these muscarinic acetylcholine receptor-immunoreactive neurons (94.3%) were GABAergic neurons. The muscarinic acetylcholine receptor-immunoreactive neurons expressed moderate levels of protein kinase gamma, one of the likely intracellular mediators between muscarinic acetylcholine receptors and their elicited physiological response. The number and staining intensity of muscarinic acetylcholine receptor-immunoreactive neurons in the central nucleus varied dramatically among rats. This individual variation correlated positively with the rat's expression of conditioned immobility and correlated negatively with active shock avoidance performance. These results suggest that the GABAergic/cholinoceptive neuronal elements in the central nucleus are involved in the expression of fear-induced behaviors. This interpretation is further elaborated in a forthcoming paper.

Muscarinic acetylcholine receptor immunoreactivity in the amygdala--II. Fear-induced plasticity.

Changes in the distribution of muscarinic acetylcholine receptor-immunoreactive neurons were examined in the amygdaloid complex at different time-intervals following a single training session of active shock avoidance in a two-way shuttle-box. Muscarinic acetylcholine receptors were visualized using M35, a monoclonal antibody raised against purified muscarinic acetylcholine receptor protein. Both in naive animals and 2 h after active shock avoidance training, muscarinic acetylcholine receptor immunoreactivity was high in the central nucleus, and only low to moderate in other amygdaloid regions. Twenty-four hours after training, however, the muscarinic acetylcholine receptor immunoreactivity distribution pattern was reversed, showing a dramatic increase in the corticomedial nucleus, while in contrast, in other amygdaloid regions including the central nucleus, muscarinic acetylcholine receptor immunoreactivity was reduced to only a few scattered neurons. Additional studies with a modified experimental design indicated that fear conditioning mechanisms in association with the severity of the aversive stimuli, and not the learning of the avoidance response, may account for the changes in muscarinic acetylcholine receptor immunoreactivity in the amygdala. These results are consistent with the prominent role of the central nucleus in the conditioning and expression of the fear response. A closer examination revealed that 8 h after training the changes in both the central and corticomedial nuclei became significant. The differences still existed after 25 days, but three months after the training session the receptor distribution was returned to normal. The long-lasting, but reversible nature of these changes indicates that fear conditioning is accompanied by a dynamic plasticity of muscarinic acetylcholine receptor immunoreactivity in the amygdaloid complex.

The role of the central amygdala in stress and adaption.

Recent views on stress emphasise the existence of more than one response pattern to stressful events, and the importance of individual differences in coping with environmental challenges. Therefore, in the evaluation of the specific contribution of the central nucleus of the amygdala (CEA) in stress and adaptation it has to be considered whether the stress is acute or conditioned, and whether it requires active or passive styles of coping. Based on series of our own studies, we propose that the CEA is consistently involved in the organisation of processes of passive coping, reflected in immobile behaviour and parasympathetic activity. Furthermore, a differential regulation of the CEA via its peptidergic neuronal input may underlie the distinct behavioural and physiological stress patterns accompanying the different styles of coping. Additionally, the involvement of the CEA in neuroendocrine control is addressed. The CEA exerts a general, modulatory influence on the neuroendocrine control to acute stressors, whereas this output during conditioned stress seems to be independent of the CEA. The neuroendocrine state as achieved during acute stress is, via feedback to the brain, of importance in learning about the situation, and to consolidate the experience. In this review an attempt is made to provide an integrated model of CEA functioning in relation to stress and adaptation.

The temporal dynamics of the stress response.

This paper summarises the available evidence that failure of defense mechanisms in (semi)-natural social groups of animals may lead to serious forms of stress pathology. Hence the study of social stress may provide animal models with a high face validity. However, most of the animal models of human stress-disorders have concentrated on the consequences of chronic exposure to stressors. The present paper considers recent data, indicating that a single experience with a major stressor in the form of social defeat may have long-term consequences ranging from hours to days and weeks. It seems that the experience of a major stressor sensitizes the animal to subsequent stressors. The consequences of these long-term temporal dynamics of the stress response to the development of stress-related disorders and stress-vulnerability are discussed.