Neuropeptide Y, calcitonin gene-related peptide, and neurokinin A in brain regions of HAB rats correlate with anxiety-like behaviours.

Anxiety disorders are pervasive psychiatric disorders causing great suffering. The high (HAB) and low (LAB) anxiety-related behaviour rats were selectively bred to investigate neurobiological correlates of anxiety. We compared the level of neuropeptides relevant for anxiety- and depression-related behaviours in selected brain regions of HAB and LAB rats. Increased anxiety and depression-like behaviours of male and female HAB rats in the elevated plus-maze and forced swim tests were accompanied by elevated levels of neuropeptide Y (NPY) in the prefrontal (PFC), frontal (FC) and cingulate cortex (CCx), the striatum, and periaqueductal grey (PAG). Moreover, HAB rats displayed sex-dependent, elevated levels of calcitonin gene-related peptide (CGRP) in PFC, FC, CCx, hippocampus, and PAG. Higher neurokinin A (NKA) levels were detected in CCx, striatum, and PAG in HAB males and in CCx and hypothalamus in HAB females. Increased neurotensin was detected in CCx and PAG in HAB males and in hypothalamus in HAB females. Elevated corticotropin-releasing hormone (CRH) levels appeared in female HAB hypothalamus. Significant correlations were found between anxiety-like behaviour and NPY, CGRP, NKA, and neurotensin, particularly with NPY in CCx and striatum, CGRP in FC and hippocampus, and NKA in entorhinal cortex. This is the first report of NPY, CGRP, NKA, Neurotensin, and CRH measurements in brain regions of HAB and LAB rats, which showed widespread NPY and CGRP alterations in cortical regions, with NKA and neurotensin changes localised in sub-cortical areas. The results may contribute to elucidate pathophysiological mechanisms underlying anxiety and depression and should facilitate identifying novel therapeutic targets.

Anti-aggressive effects of neuropeptide S independent of anxiolysis in male rats.

Neuropeptide S (NPS) exerts robust anxiolytic and memory enhancing effects, but only in a non-social context. In order to study whether NPS affects aggressive behavior we used Wistar rats bred for low (LAB) and high (HAB) levels of innate anxiety-related behavior, respectively, which were both described to display increased levels of aggression compared with Wistar rats not selectively bred for anxiety (NAB). Male LAB, HAB, and NAB rats were tested for aggressive behavior toward a male intruder rat within their home cage (10 min, resident-intruder [RI] test). Intracerebroventricular (icv) infusion of NPS (1 nmol) significantly reduced inter-male aggression in LAB rats, and tended to reduce aggression in HAB and NAB males. However, local infusion of NPS (0.2 or 0.1 nmol NPS) into either the nucleus accumbens or the lateral hypothalamus did not influence aggressive behavior. Social investigation in the RI test and general social motivation assessed in the social preference paradigm were not altered by icv NPS (1 nmol). The anti-aggressive effect of NPS is most likely not causally linked to its anxiolytic properties, as intraperitoneal administration of the anxiogenic drug pentylenetetrazole decreased aggression in LAB rats whereas the anxiolytic drug diazepam did not affect aggression in HAB rats. Thus, although NPS has so far only been shown to exert effects on non-social behaviors, our results are the first demonstration of anti-aggressive effects of NPS in male rats.

Measuring virgin female aggression in the female intruder test (FIT): effects of oxytocin, estrous cycle, and anxiety.

The costs of violence and aggression in our society have stimulated the scientific search for the predictors and causes of aggression. The majority of studies have focused on males, which are considered to be more aggressive than females in most species. However, rates of offensive behavior in girls and young women are considerable and are currently rising in Western society. The extrapolation of scientific results from males to young, non-maternal females is a priori limited, based on the profound sex differences in brain areas and functioning of neurotransmitters involved in aggression. Therefore, we established a paradigm to assess aggressive behavior in young virgin female rats, i.e. the female intruder test (FIT). We found that approximately 40% of un-manipulated adult (10-11 weeks old) female Wistar rats attack an intruder female during the FIT, independent of their estrous phase or that of their intruder. In addition, adolescent (7-8 weeks old) female rats selected for high anxiety behavior (HABs) displayed significantly more aggression than non-selected (NAB) or low-anxiety (LAB) rats. Intracerebroventricular infusion of oxytocin (OXT, 0.1 µg/5 µl) inhibited aggressive behavior in adult NAB and LAB, but not HAB females. Adolescent NAB rats that had been aggressive towards their intruder showed increased pERK immunoreactivity (IR) in the hypothalamic attack area and reduced pERK-IR in OXT neurons in the paraventricular hypothalamic nucleus compared to non-aggressive NAB rats. Taken together, aggressive behavior in young virgin female rats is partly dependent on trait anxiety, and appears to be under considerable OXT control.

Local oxytocin expression and oxytocin receptor binding in the male rat brain is associated with aggressiveness.

We recently demonstrated in male wild-type Groningen rats that enhancing brain oxytocin (OXT) levels acutely produces marked pro-social explorative and anti-aggressive effects. Moreover, these pharmacologically-induced changes are moderated by the individual's aggressive phenotype, suggesting an inverse relationship between aggressiveness and tonic endogenous OXT signaling properties. Aim of the present study was to verify the hypothesis that variations in OXT expression and/or OXT receptor (OXTR) binding in selected brain regions are associated with different levels or forms of aggression. To this end, male resident wild-type Groningen rats that repeatedly contested and dominated intruder conspecifics were categorized as being low aggressive, highly aggressive or excessively aggressive. Their brains were subsequently collected and quantified for OXT mRNA expression and OXTR binding levels. Our results showed that OXT mRNA expression in the hypothalamic paraventricular nucleus (PVN), but not in the supraoptic nucleus (SON), negatively correlates with the level of offensiveness. In particular, the excessively aggressive group showed a significantly lower OXT mRNA expression in the PVN as compared to both low and highly aggressive groups. Further, the excessively aggressive animals showed the highest OXTR binding in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST). These findings demonstrate that male rats with excessively high levels and abnormal forms of aggressive behavior have diminished OXT transcription and enhanced OXTR binding capacities in specific nodes of the social behavioral brain circuitry.

Increased brain and plasma oxytocin after nasal and peripheral administration in rats and mice.

The possibility to improve socio-emotional behaviors in humans by intranasal administration of synthetic oxytocin (OXT) attracts increasing attention, but its uptake into the brain has never been demonstrated so far. Here we used simultaneous microdialysis in both the dorsal hippocampus and amygdala of rats and mice in combination with concomitant blood sampling from the jugular vein to study the dynamics of the neuropeptide in brain extracellular fluid and plasma after its nasal administration. OXT was found to be increased in microdialysates from both the hippocampus and amygdala with peak levels occurring 30-60min after nasal administration. Despite a similar temporal profile of OXT concentrations in plasma, peripheral OXT is unlikely to contribute to dialysate OXT as calculated from in vitro recovery data, indicating a central route of transport. Moreover, intraperitoneal administration of synthetic OXT in identical amounts caused rapid peak levels in brain dialysates and plasma during the first 30min after treatment and a subsequent return toward baseline. While the precise route(s) of central transport remain to be elucidated, our data provide the first evidence that nasally applied OXT indeed reaches behaviorally relevant brain areas, and this uptake is paralleled by changes in plasma OXT.

High and abnormal forms of aggression in rats with extremes in trait anxiety--involvement of the dopamine system in the nucleus accumbens.

A better neurobiological understanding of high and abnormal aggression based on adequate animal models is essential for novel therapy and prevention. Selective breeding of rats for extremes in anxiety-related behavior resulted in two behavioral phenotypes with high and abnormal forms of aggression. Rats bred for low anxiety-related behavior (LAB) consistently show highest levels of aggression and little social investigation in the resident-intruder (RI) test, compared with non-selected low-aggressive (NAB) rats. High anxiety-related (HAB) rats also show higher levels of aggression than NAB rats, but to a lesser extent than LAB rats. Accordingly, extremes in inborn anxiety in both directions are linked to an increased aggression level. Further, both LAB and HAB, but not NAB males, display abnormal aggression (attacks towards vulnerable body parts, females or narcotized males), which is particularly prominent in LABs. Also, only in LAB rats, the nucleus accumbens (NAc) was found to be strongly activated in response to the RI test as reflected by increased c-fos and zif268 mRNA expression, and higher local dopamine release compared with NAB males, without differences in local dopamine receptor binding. Consequently, local pharmacological manipulation by infusion of an anesthetic (lidocaine, 20 µg/µl) or a dopamine D2 (haloperidol, 10 ng/µl), but not D1 (SCH-23390 10 ng/µl), receptor antagonist significantly reduced high aggression in LAB rats. Thus, LAB rats are an adequate model to study high and abnormal aggression. In LAB males, this is likely to be linked to hyper-activation of the reward system, as found in psychopathic patients. Specifically, activation of the accumbal dopamine system is likely to underlie the high aggression observed in LAB rats.

Chronic subordinate colony housing (CSC) as a model of chronic psychosocial stress in male rats.

Chronic subordinate colony housing (CSC) is an adequate and reliable mouse model of chronic psychosocial stress, resulting in reduced body weight gain, reduced thymus and increased adrenal weight, long-lasting anxiety-like behaviour, and spontaneous colitis. Furthermore, CSC mice show increased corticotrophin (ACTH) responsiveness to acute heterotypic stressors, suggesting a general mechanism which allows a chronically-stressed organism to adequately respond to a novel threat. Therefore, the aim of the present study was to extend the CSC model to another rodent species, namely male Wistar rats, and to characterize relevant physiological, immunological, and behavioural consequences; placing particular emphasis on changes in hypothalamo-pituitary-adrenal (HPA) axis responsiveness to an acute heterotypic stressor. In line with previous mouse data, exposure of Wistar rats to 19 days of CSC resulted in a decrease in body weight gain and absolute thymus mass, mild colonic barrier defects and intestinal immune activation. Moreover, no changes in stress-coping behaviour or social preference were seen; again in agreement with the mouse paradigm. Most importantly, CSC rats showed an increased plasma corticosterone response to an acute heterotypic stressor (open arm, 5 min) despite displaying similar basal levels and similar basal and stressor-induced plasma ACTH levels. In contrast to CSC mice, anxiety-related behaviour and absolute, as well as relative adrenal weights remained unchanged in CSC rats. In summary, the CSC paradigm could be established as an adequate model of chronic psychosocial stress in male rats. Our data further support the initial hypothesis that adrenal hyper-responsiveness to ACTH during acute heterotypic stressors represents a general adaptation, which enables a chronically-stressed organism to adequately respond to novel challenges.

Aggression and anxiety: social context and neurobiological links.

Psychopathologies such as anxiety- and depression-related disorders are often characterized by impaired social behaviours including excessive aggression and violence. Excessive aggression and violence likely develop as a consequence of generally disturbed emotional regulation, such as abnormally high or low levels of anxiety. This suggests an overlap between brain circuitries and neurochemical systems regulating aggression and anxiety. In this review, we will discuss different forms of male aggression, rodent models of excessive aggression, and neurobiological mechanisms underlying male aggression in the context of anxiety. We will summarize our attempts to establish an animal model of high and abnormal aggression using rats selected for high (HAB) vs. low (LAB) anxiety-related behaviour. Briefly, male LAB rats and, to a lesser extent, male HAB rats show high and abnormal forms of aggression compared with non-selected (NAB) rats, making them a suitable animal model for studying excessive aggression in the context of extremes in innate anxiety. In addition, we will discuss differences in the activity of the hypothalamic-pituitary-adrenal axis, brain arginine vasopressin, and the serotonin systems, among others, which contribute to the distinct behavioural phenotypes related to aggression and anxiety. Further investigation of the neurobiological systems in animals with distinct anxiety phenotypes might provide valuable information about the link between excessive aggression and disturbed emotional regulation, which is essential for understanding the social and emotional deficits that are characteristic of many human psychiatric disorders.

Distinct correlations of vasopressin release within the lateral septum and the bed nucleus of the stria terminalis with the display of intermale aggression.

Arginine vasopressin (AVP) has been implicated in a wide variety of social behaviors ranging from affiliation to aggression. However, the precise functional involvement of AVP in intermale aggression is still a matter of debate. In fact, very little is known about AVP release patterns within distinct brain regions during the display of intermale aggression and, in turn, the behavioral consequences of such release. We used intracerebral microdialysis to monitor local AVP release within the lateral septum (LS) and the bed nucleus of the stria terminalis (BST) of adult male Wistar rats during the resident-intruder (RI) test. Resident males were cohabitated with a female prior to the RI test to stimulate intermale aggression toward the intruder male. AVP release within the LS correlated positively with intermale aggression. The specific AVP V1a receptor antagonist d(CH(2))(5)Tyr(Me)AVP (10 microg/ml) administered via retrodialysis (3.3 microl/min, 30 min) into the LS of high-aggressive rats prior to the second RI test, prevented an increase in aggression in the second compared with the first RI test as seen in vehicle-treated high-aggressive rats. In contrast to the LS, AVP release within the BST correlated negatively with intermale aggression. Moreover, retrodialysis of synthetic AVP (1 microg/ml) administered into the BST of high-aggressive rats significantly reduced the display of aggression during the second RI test. These data reveal that AVP can both promote and inhibit intermale aggression, depending upon the brain region in which AVP is released. Although challenging the general view that central AVP release enhances intermale aggression in rodents, our data support a model in which AVP coordinates a range of social behaviors by eliciting region-specific effects.

Increased levels of cocaine and amphetamine regulated transcript in two animal models of depression and anxiety.

The neurobiological bases of mood disorders remain elusive but both monoamines and neuropeptides may play important roles. The neuropeptide cocaine and amphetamine regulated transcript (CART) was shown to induce anxiety-like behavior in rodents, and mutations in the human CART gene are associated with depression and anxiety. We measured CART-like immunoreactivity (-LI) in genetic rat models of depression and anxiety, i.e. the Flinders Sensitive Line (FSL) and rats selected for High Anxiety-related Behavior (HAB) using a radioimmunoassay. CART-LI was significantly increased in the periaqueductal grey in FSL rats, whereas in the HAB strain it was increased in the hypothalamus, both compared with their respective controls. No line-dependent changes were found in the hippocampus, striatum or frontal cortex. Our results confirm human genetic studies indicating CART as a neurobiological correlate of depression and anxiety, and suggest that its differential regulation in specific brain regions may play a role for the behavioral phenotypes.

Differences in intermale aggression are accompanied by opposite vasopressin release patterns within the septum in rats bred for low and high anxiety.

Several studies suggest a role for arginine vasopressin (AVP), particularly in the lateral septum, in the regulation of intermale aggression. We used intracerebral microdialysis to monitor the local in vivo AVP release within the mediolateral septum of adult male Wistar rats bred for low (LAB) or high (HAB) anxiety-related behaviour during exposure to the resident-intruder test. LAB residents showed a significantly higher level of aggression than HAB residents, as reflected by more time spent with lateral threat, offensive upright and total aggressive behaviour as well as by more attacks and a shorter attack latency. Septal AVP release was significantly decreased in high-aggressive LAB males, while septal AVP release tended to increase in HAB males during resident-intruder test exposure. Moreover, LAB residents showed reduced neuronal activation of the lateral septum, as indicated by fewer c-Fos-positive cells, 1 h after the resident-intruder test. Pharmacological manipulation of the septal AVP system by local application of either synthetic AVP to LAB residents or the selective V1a receptor antagonist d(CH(2))(5)Tyr(Me)AVP to HAB residents did not change the level of aggression. However, application of AVP into the septum enhanced anxiety-related behaviour on the elevated plus-maze in LAB males, while local administration of the V1a receptor antagonist reduced social investigation in HAB males during the resident-intruder test. In conclusion, although AVP release patterns within the septum are dependent on the level of aggression, locally released AVP does not seem to be directly involved in the regulation of aggression, but rather modulates non-aggressive social and anxiety-related behaviours.

Low inborn anxiety correlates with high intermale aggression: link to ACTH response and neuronal activation of the hypothalamic paraventricular nucleus.

Aggression constitutes a central problem in several psychopathologies, including anxiety and depression disorders and antisocial behaviors. In particular, the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis has been associated with aggression-related disorders. The present study assessed whether genetically determined levels of anxiety-related behavior influence the level of intermale aggression and whether this is associated with differences in neuroendocrine responsiveness and neuronal activation in the brain. Adult male Wistar rats bred for high (HAB) or low (LAB) anxiety-related behavior were used, as well as non-selected rats (NAB) with an intermediate anxiety level. LAB residents displayed more aggressive behavior than HAB and NAB residents during the resident-intruder (RI) test. Moreover, an inverse correlation was found between the level of anxiety and the level of aggression. The plasma corticotropin (ACTH) response to RI-test exposure was significantly higher in LABs than in HABs and NABs, indicating that a higher level of aggression was linked to an elevated hormonal stress response. Furthermore, LAB residents showed more neuronal activation in the parvocellular part of the hypothalamic paraventricular nucleus (PVN) than HAB residents 1 h after the RI-test. In addition, a tendency toward a higher number of c-Fos-positive cells in LABs compared with HABs was observed in the medial amygdala, hypothalamic attack area and central amygdala, areas relevant for the regulation of aggression. These data demonstrate that low trait anxiety is correlated with high intermale aggression. Furthermore, the increased neuronal activation of the PVN along with the higher ACTH responsiveness might underlie the display of high aggression.

Brain oxytocin correlates with maternal aggression: link to anxiety.

The oxytocinergic system is critically involved in the regulation of maternal behavior, which includes maternal aggression. Because aggression has been linked to anxiety, we investigated the maternal aggression and the role of brain oxytocin in lactating Wistar rats selectively bred for high anxiety-related behavior (HAB) or low anxiety-related behavior (LAB) during the 10 min maternal defense test. HAB dams displayed more maternal aggression against a virgin intruder compared with LAB dams, resulting in more defensive behavior and higher anxiety of HAB-defeated virgins. The different levels of aggression were accompanied by opposite oxytocin release patterns within the paraventricular nucleus (PVN; HAB, increase; LAB, decrease). Furthermore, oxytocin release was higher within the central nucleus of the amygdala (CeA) of HAB dams compared with LABs. A direct correlation between the offensive behavior displayed during the maternal defense test and local oxytocin release was found in both the PVN and CeA. Using retrodialysis, blockade of endogenous oxytocin action by infusion of an oxytocin receptor antagonist (des-Gly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT) into the PVN or CeA reduced maternal aggression of HAB dams, whereas infusion of synthetic oxytocin into the PVN tended to increase aggression toward the intruder in LAB dams. There were no significant differences in oxytocin receptor mRNA expression or oxytocin receptor binding between lactating HAB and LAB dams. Therefore, differences in intracerebral release patterns of oxytocin, rather than differences at the level of oxytocin receptors, are critical for the regulation of maternal aggressive behavior.