Inhibitory and facilitatory effect of two related peptides on extinction of avoidance behavior.

The polypeptide chain which constitutes the first ten amino acids of the ACTH molecule inhibits extinction of a shuttle-box avoidance response. If the phenylalanine molecule in the 7th position of this peptide is replaced by its dextrorotatory form, extinction is facilitated.

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.

Behavioural physiology of serotonergic and steroid-like anxiolytics as antistress drugs.

Pharmacological studies are useful tools to understand the neurobiological basis of behavioural and physiological stress mechanisms. Ipsapirone, a 5-HT1A autoreceptor agonist is a representative of novel anxiolytics without the disadvantages of benzodiazepam-like drugs. Behavioural, physiological and neuroendocrine studies in the rat are reviewed which were aimed to investigate the antistress properties of ipsapirone during reexposure to various conditioned emotional stress situations. It is demonstrated that in certain situations, probably due to a stress-induced sensitisation of postsynaptic 5-HT1A receptors, anxiolytic doses of the drug may show prostress (anxiogenic) behavioural and neuroendocrine effects. Furthermore, brain corticosteroid receptors, probably interacting with the serotonergic transmission, are involved in anxiogenic/prostress processes. In this respect antagonists of the brain mineralocorticoid-like (type I) receptors may be important antistress drugs of the future.

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.

Sites of behavioral and neurochemical action of ACTH-like peptides and neurohypophyseal hormones.

In order to localize the site of action of neuropeptides in relation to their effects on behavior and memory various approaches have been used. As a result of studies using rats bearing lesions in different areas of the limbic system as well as of studies in which neuropeptides were locally applied into various areas of the brain it appeared that the limbic system (amygdala, hippocampus, septum and some thalamic areas) plays an essential role in the effect of vasopressin and ACTH and their derivatives on behavior and memory. Neurochemical studies generally indicate that changes occur in catecholamine utilization in these various limbic regions upon administration of these neuropeptides. It can be concluded that the effects of vasopressin in the terminal regions of the coeruleo-telencephalic noradrenalin system correlate with its effects on consolidation of memory. It is likely that the effects of vasopressin on other transmitter systems (e.g. dopamine in the amygdala and serotonin in the hippocampus) correspond with the effect of this neuropeptide on retrieval processes. In addition, regional differences in biotransformation of the neurohypophyseal hormones suggest that different patterns of behaviorally active fragments of these peptides may be present locally in the brain.

Effect of low amphetamine doses on cardiac responses to emotional stress in aged rats.

In young Wistar rats conditioned emotional stress can be characterized by a learned bradycardiac response to an inescapable footshock. In aged rats this bradycardiac response is attenuated and accompanied by suppressed behavioral arousal in response to novelty. In the present study, cardiac responses to emotional stress and behavioral reactivity to a novel experience in an open field were tested in aged and young rats under the influence of a low dose of d-amphetamine (AMPH, 0.5 mg/kg IP). AMPH administration in 27-month-old rats reinstated the bradycardiac response to emotional stress, while it failed to influence the resting heart rate in the home cage. Age-associated differences in open-field ambulation, present in drug-free conditions, were antagonized by low doses of AMPH (0.25-1.0 mg/kg). It is concluded that enhanced arousal by aminergic stimulation with AMPH in the aged rat invoked cardiac and behavioral response patterns resembling those at younger ages.

Inhibition of centrally-evoked pressor responses by neurohypophyseal peptides and their fragments.

Intracerebroventricular administration of fragments of [arginine8]-vasopressin (AVP) such as AVP1-6 and AVP7-9 attenuated the pressor response evoked by electrical stimulation of the mesencephalic reticular formation in urethane-anaesthetized rats. Oxytocin (OXT) and the fragment OXT7-9 were also active, although OXT1-6 did not affect the pressor response. These peptides did not influence the bradycardia accompanying the rise in blood pressure, nor the basal blood pressure. The inhibition of the pressor response was shown for OXT7-9 to be dose-dependent up to 25 ng. These data suggest that oxytocin, vasopressin and some neuropeptide fragments have an inhibitory role in the regulation of blood pressure. Both the covalent pressinoic ring structure and the C-terminal linear portion of vasopressin contain active sites, while the activity of oxytocin appears to be present in the C-terminal tripeptide Pro-Leu-Gly.

Comparative effects of the ACTH 4-9 analogue (ORG 2766), ACTH 4-10 and [D-Phe7] ACTH 4-10 on medial septal self-stimulation behaviour in rats.

Experiments were performed to examine the effects of various analogues of ACTH on electrical self-stimulation behaviour elicited from the medial septal area using an ascending or descending sequence of stimulus intensities within a session. When an ascending sequence of threshold multiples was used ACTH 4-10 and the ACTH 4-9 analogue (ORG 2766) enhanced level pressing for low intensity stimulation but attenuated self-stimulation at greater current intensities. The analogue ORG 2766 appeared to be a thousand times more potent than ACTH 4-10; [D-Phe7] ACTH 4-10 inhibited the response rate at threshold level but was inactive at greater current intensities. The same effect was found following administration of ORG 2766 in a dose which was 20 times greater (1 microgram/rat) than that used in the first experiments. Lever pressing was not affected by treatment with ACTH 4-10 or ORG 2766 when a descending sequence of stimulus intensities was used within a session. Thus, ACTH-related peptides may affect motivational processes involved in self-stimulation rather than the reward of the stimulation per se. It is suggested that although ORG 2766 mimicked the action of ACTH 4-10 this synthetic peptide may have additional behavioural properties.

Long-term increase in protein kinase C-gamma and muscarinic acetylcholine receptor expression in the cerebral cortex of amygdala-kindled rats--a quantitative immunocytochemical study.

Kindling is an animal model for epilepsy in which repeated application of an electrical stimulus to brain pathways results in an epileptic focus. The animal holds a permanent state of hyperexcitability to the stimulus for the rest of its life. Understanding the cellular and molecular processes underlying hyperexcitability could provide insight into epileptogenesis. Furthermore, it could elucidate cellular and molecular bases of synaptic plasticity in the central nervous system. In the present study the long-term effect of a kindled focus in the amygdala on the gamma-isoform of protein kinase C and the muscarinic cholinergic receptor as cellular messengers was evaluated in the cerebral cortex of rats. Following an average of 10 bilaterally generalized seizures kindling stimulation was terminated and rats were left undisturbed for approximately three months. Brains were processed by immunocytochemistry using monoclonal antibodies against protein kinase C-gamma and muscarinic cholinergic receptor protein. Digital image analysis of sections through the entire forebrain revealed an increase in optical density of both protein kinase C-gamma and the muscarinic cholinergic receptor in the piriform and entorhinal cortex of the hemisphere contralateral to the stimulation site in kindled rats. However, on the ipsilateral side no change was observed in comparison with electrode implanted nonkindled control rats. The observed increase in expression of muscarinic cholinergic receptor protein and a component of the phosphoinositide second messenger system (protein kinase C-gamma) located in specific areas of the cerebral cortex in kindled rats could serve as a basis for the permanent state of hyperexcitability in these rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Dose-dependent suppression of adrenocortical activity with metyrapone: effects on emotion and memory.

Different levels of circulating corticosterone are considered to produce different emotional states and effects on learning and memory. The purpose of the present study was to use different doses of the 11-beta-hydroxylase inhibitor metyrapone to produce dose-dependent inhibition of the synthesis of corticosterone and examine the consequences of that on several cognitive and emotional parameters. Systemic (SC) injections of metyrapone (25 or 50 mg/kg) dose-dependently suppressed increases in plasma concentrations of corticosterone induced by spatial training in a water maze, but did not affect plasma corticosterone levels in non-stressed rats. Treatment with the higher and lower dose of metyrapone also differentially affected behavioral measures of emotion and memory. Administration of 50 mg/kg, but not 25 mg/kg, of metyrapone impaired acquisition performance in the spatial water maze task. Both doses of metyrapone impaired retention. The impairment in retention was attenuated by dexamethasone (0.3 mg/kg) given systemically immediately after training, but not by corticosterone (0.3 mg/kg). During the exposure to a conditioned stressor of inescapable footshock, the higher, but not the lower dose of metyrapone attenuated fear-induced immobility. In contrast, the lower, but not the higher dose attenuated the anxiety state in an elevated plus-maze in a novel environment immediately after exposure to the conditioned stressor. It is suggested that emotion, learning, and memory are differentially affected by the different doses of metyrapone due to interference with different types of adrenal steroid receptors and consequent induction of various corticosterone receptor states.

Effects of beta-endorphin and its fragments on inhibitory avoidance behavior in rats.

The effects on retrieval of a one-trial learning inhibitory avoidance response of beta-endorphin, alpha-endorphin, and gamma-endorphin, given prior to test have been studied in rats. beta-Endorphin (beta-LPH 61-91) in a relatively low dose (1.56 micrograms sc. or 50 ng icv.) facilitated inhibitory avoidance behavior, while a higher dose (10 micrograms sc. or 100 ng icv.) caused bimodal changes (facilitation in 50% of the animals and attenuation in another 40%. Peripheral injection of gamma-endorphin attenuated inhibitory avoidance behaviour in a dose-dependent manner. The C-terminus of beta-endorphin (beta-LPH 78-91) was ineffective. alpha-Endorphin facilitated inhibitory avoidance behavior in a dose dependent manner. Naltrexone pretreatment antagonized the bimodal effect of beta-endorphin: following pretreatment with the opiate antagonist the low latency component disappeared, but the facilitatory effect of the neuropeptide remained the same. It is suggested that beta-endorphin carries more than one bit of behavioral information. Inherent activities either related or unrelated to naltrexone-sensitive opiate as well as biotransformation into alpha- and gamma-endorphin may contribute to the multiple behavioral effects of this neuropeptide.

Behavioural activation produced by CRH but not alpha-helical CRH (CRH-receptor antagonist) when microinfused into the central nucleus of the amygdala under stress-free conditions.

The central nucleus of the amygdala (CeA) is known to be involved in the regulation of autonomic, neuroendocrine, and behavioural responses in stress situations. The CeA contains large numbers of corticotropin-releasing hormone (CRH)-containing cell bodies and terminals. In the present study we examined (by continuous behaviour observations) the effects of a high dose of CRH (150 ng) and two doses of the CRH-receptor antagonist (alpha-hCRH: 1.0 and 0.1 micrograms) after microinfusion into the CeA in freely moving male Wistar rats under stress-free conditions. In comparison with control, alpha-hCRH infusion did not cause any behavioural activation. In contrast CRH-infusion revealed a long-lasting increase in grooming and exploration with a concomitant decrease in behaviours specified as resting. These results indicate that the CRH system in the CeA does not seem to be activated in stress-free conditions, but its activation is of importance for active behavioural responses.

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.

Anxiolytic-like effects of selective mineralocorticoid and glucocorticoid antagonists on fear-enhanced behavior in the elevated plus-maze.

The effects of intracerebroventricular (ICV) administration of the mineralocorticoid receptor (MR) antagonist, RU28318, and the glucocorticoid receptor (GR) antagonist, RU38486, were studied on behavior of rats exposed to a compartment previously associated with a stressor, and placed subsequently in an elevated plus-maze test. Fear-motivated immobility behavior was attenuated by the MR antagonist in a dose of 50 or 100 ng ICV, whereas the GR antagonist alone or simultaneous administration of both antagonists had no significant effect. In the elevated plus-maze, immediately after the exposure to the conditioned stressor, both the GR antagonist (50 ng) and MR antagonist (50 ng) increased the percentage of time the rats spent on open arms, and increased the amount of entries into these open arms. These data are interpretated in terms of the involvement of the GR and MR in fear and anxiety.

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.

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.

Colocalization of muscarinic acetylcholine receptors and protein kinase C gamma in rat parietal cortex.

The present investigation analyzes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) and the gamma isoform of protein kinase C (PKC) in the rat parietal cortex employing the monoclonal antibodies M35 and 36G9, respectively. Muscarinic cholinoceptive neurons were most present in layers 2, 3 and 5, whereas most PKC gamma-positive cells were found in layers 2, 5 and 6. Under normal, non-stimulated conditions, approximately 58% of all muscarinic cholinoceptive neurons were immunoreactive for PKC gamma. Conversely, nearly all PKC gamma-positive neurons were M35-immunoreactive. Although both pyramidal and nonpyramidal neurons express the two types of protein, the pyramidal cell type represents the vast majority. Of all cortical neurons, the large (15-25 microns in diameter) muscarinic cholinoceptive pyramidal neurons in layer 5 express the gamma isoform of PKC most abundantly and most frequently. Approximately 96% of these cells are immunoreactive for PKC gamma. Stimulation of mAChRs by the cholinergic agonist carbachol resulted in a pronounced increase in the intensity of 36G9 immunoreactivity, which may suggest that the mAChRs are functionally linked to the colocalized PKC gamma. No change was found in the number of 36G9-immunoreactive neurons. In contrast, the number of immunocytochemically detectable muscarinic cholinoceptive neurons increased by approximately 38% after carbachol stimulation. The high degree of codistribution in cortical neurons of both transduction proteins suggests a considerable cholinergic impact upon the regulation of PKC gamma, a candidate key enzyme in cortical learning and memory mechanisms.