Peter Salchner

Induction of c-Fos expression in specific areas of the fear circuitry in rat forebrain by anxiogenic drugs.

BACKGROUND The fact that induction of anxiety- and panic-related symptoms is a property common to a range of drugs suggests that common neural substrates underlie their behavioral effects. METHODS We used Fos immunocytochemistry to test the effects of four anxiogenic drugs (FG-7142, yohimbine, m-chlorophenylpiperazine [mCPP], and caffeine) on anxiety-related circuitry in rat forebrain. RESULTS All four drugs commonly increased Fos-like immunoreactivity in 7 of 41 brain areas investigated, namely, central nucleus of the amygdala, bed nucleus of the stria terminalis, lateral septum, paraventricular nucleus of the hypothalamus, lateral hypothalamus, infralimbic and prelimbic cortex. All drugs but one (mCPP) also increased Fos expression in the basolateral and medial amygdala, the dorsomedial hypothalamus, cingulate cortex, and parts of the motor cortex. CONCLUSIONS The results suggest that the anxiogenic drugs selected activate a restricted set of forebrain areas. Most of these areas have previously been shown to be activated by environmentally evoked anxiety and to have anatomic connections with hindbrain regions that are activated by the same drugs and by environmentally evoked anxiety. Together, these data are consistent with the theory of an integrated forebrain and hindbrain neuronal system that is important for anxiety states evoked by both drug and environmental manipulations.

Reduced anxiety and improved stress coping ability in mice lacking NPY-Y2 receptors.

Neuropeptide Y (NPY) has been implicated in the pathophysiology of certain mood disorders, including depression and anxiety. It is, however, not known which of the five cloned NPY receptors mediate these functions. We investigated the effect of Y2 receptor deletion on anxiety and stress‐related behaviours. In the elevated plus maze, Y2 knock out (Y2−/−) mice showed a 2.7‐fold higher frequency of entering into, and spent 3.8 times more time within, the open arms compared to controls, while entries into the closed arms did not differ. Similarly Y2−/− mice entered the central area of the open field 1.7 times more frequently and also spent 1.8 times more time there. In the light/dark test Y2−/− mice had a 4.8‐fold lower latency to enter the lit area but stayed there 2.6 times longer than control mice. Y2−/− mice displayed 3.2‐fold less immobility in the forced swim test, indicating improved stress coping ability. Y2 receptors are predominantly located presynaptically where they mediate feedback inhibition of neurotransmitter release. Deletion of these receptors may result in enhanced release of NPY, GABA and/or glutamate in brain areas linked to the manifestation of anxiety, and stress‐related behaviour such as the amygdala. Taken together, deletion of the Y2 receptor has revealed an important role of Y2 receptors in the generation of anxiety‐related and stress‐related behaviours in mice.

Neurobiological correlates of high (HAB) versus low anxiety-related behavior (LAB): differential Fos expression in HAB and LAB rats

BACKGROUND Two Wistar rat lines selectively bred for either high (HAB) or low (LAB) anxiety-related behavior were used to identify neurobiological correlates of trait anxiety. METHODS We used Fos expression for mapping of neuronal activation patterns in response to mild anxiety-provoking challenges. RESULTS In both lines, exposure to an open field (OF) or the open arm (OA) of an elevated plus-maze induced Fos expression in several brain areas of the anxiety/fear circuitry. Rats of the HAB type, which showed signs of a hyperanxious phenotype and a hyperreactive hypothalamic-pituitary-adrenal axis compared with LAB rats, exhibited a higher number of Fos-positive cells in the paraventricular nucleus of the hypothalamus, the lateral and anterior hypothalamic area, and the medial preoptic area in response to both OA and OF. Less Fos expression was induced in the cingulate cortex in HAB than in LAB rats. Differential Fos expression in response to either OA or OF was observed in few brain regions, including the thalamus and hippocampus. CONCLUSIONS The present data indicate that the divergent anxiety-related behavioral response of HAB versus LAB rats to OF and OA exposures is associated with differential neuronal activation in restricted parts of the anxiety/fear circuitry. Distinct hypothalamic regions displayed hyperexcitability, and the cingulate cortex showed hypoexcitability, which suggests that they are main candidate mediators of dysfunctional brain activation in pathologic anxiety.

Genetic predisposition to anxiety-related behavior determines coping style, neuroendocrine responses, and neuronal activation during social defeat

Genetic background may influence an individuals susceptibility to, and subsequent coping strategy for, an acute stressor. When exposed to social defeat (SD), rats bred for high (HAB) or low (LAB) trait anxiety, which also differ in depression-like behavior, showed highly divergent passive and active coping behaviors, respectively. HABs spent more time freezing and emitted more ultrasound vocalization calls during SD than LABs, which spent more time rearing and grooming. Although the behavioral data confirmed the prediction that heightened trait anxiety would make rats more prone to experience stress, adrenocorticotropin and corticosterone were secreted to a higher extent in LABs than in HABs. In the latter, Fos expression upon SD was enhanced in the amygdala and hypothalamic areas compared with LABs, whereas it was diminished in prefrontal and brainstem areas.

Differential Stress-Induced Neuronal Activation Patterns in Mouse Lines Selectively Bred for High, Normal or Low Anxiety

There is evidence for a disturbed perception and processing of emotional information in pathological anxiety. Using a rat model of trait anxiety generated by selective breeding, we previously revealed differences in challenge-induced neuronal activation in fear/anxiety-related brain areas between high (HAB) and low (LAB) anxiety rats. To confirm whether findings generalize to other species, we used the corresponding HAB/LAB mouse model and investigated c-Fos responses to elevated open arm exposure. Moreover, for the first time we included normal anxiety mice (NAB) for comparison. The results confirm that HAB mice show hyperanxious behavior compared to their LAB counterparts, with NAB mice displaying an intermediate anxiety phenotype. Open arm challenge revealed altered c-Fos response in prefrontal-cortical, limbic and hypothalamic areas in HAB mice as compared to LAB mice, and this was similar to the differences observed previously in the HAB/LAB rat lines. In mice, however, additional differential c-Fos response was observed in subregions of the amygdala, hypothalamus, nucleus accumbens, midbrain and pons. Most of these differences were also seen between HAB and NAB mice, indicating that it is predominately the HAB line showing altered neuronal processing. Hypothalamic hypoactivation detected in LAB versus NAB mice may be associated with their low-anxiety/high-novelty-seeking phenotype. The detection of similarly disturbed activation patterns in a key set of anxiety-related brain areas in two independent models reflecting psychopathological states of trait anxiety confirms the notion that the altered brain activation in HAB animals is indeed characteristic of enhanced (pathological) anxiety, providing information for potential targets of therapeutic intervention.

Neuroanatomical substrates involved in the anxiogenic-like effect of acute fluoxetine treatment.

An initial exacerbation of anxiety can be observed in animals and humans treated with selective serotonin reuptake inhibitors (SSRIs). The neurobiological substrates and mechanism(s) underlying this effect are not clear. We used Fos expression as a marker of neuronal activation to investigate effects of acute fluoxetine treatment in rats submitted to two different models of emotional stress, airjet and immobilization. Exposure to both stressors induced Fos expression in various brain regions implicated in fear/anxiety mechanisms. Acute treatment with 5 mg/kg fluoxetine facilitated airjet-induced escape responses and enhanced the airjet-, as well as immobilization-induced Fos expression exclusively in the locus coeruleus (LC), but not in other areas including the amygdala, hypothalamus or septum. Fluoxetine also facilitated airjet-induced noradrenaline efflux in the medial prefrontal cortex, a projection area of LC noradrenergic neurons. A higher dose of fluoxetine (10 mg/kg) did not change escape responses and had no effect on stress-induced Fos expression in the LC, but decreased airjet-induced Fos expression in the medial amygdala. The results indicate that anxiogenic effects of acute fluoxetine treatment occur in a specific dose range and can be mimicked by exacerbation of escape responses in the airjet model. Furthermore, facilitation of escape responses by fluoxetine is linked to enhanced activity in the LC/noradrenaline system.

Decreased social interaction in aged rats may not reflect changes in anxiety-related behaviour.

There is evidence that ageing both in humans and animals is accompanied by changes in emotional behaviour. Behavioural studies in rats point to an increase in emotional reactivity and/or anxiety-related behaviour with age. Here we studied social interaction in young adult (3 months) and aged (30 months old) rats using an established test system for anxiety-related behaviour. Using Fos expression as a marker of neuronal activation, we aimed to investigate whether age-related differences in anxiety would be reflected by changes in neuronal activity in brain regions known to be sensitive to fear- and anxiety-related stimuli. Aged rats spent significantly less time (75%) in active social interaction than young rats, without concomitant changes in general locomotor activity. Social interaction enhanced Fos expression both in young and aged rats in several anxiety-related brain areas. Lower Fos response in aged versus young rats was noted in the dorsomedial, dorsolateral and ventrolateral part of the periaqueductal grey, the medial and basolateral amygdala and parvocellular region of the paraventricular hypothalamic nucleus, while no differences in Fos expression were observed in the other regions examined, including the hippocampus, septum or locus coeruleus. These results demonstrate age-related reduction in social interaction, indicative of enhanced anxiety-related behaviour in aged rats. However, since the supposedly increased anxiety level was not accompanied by augmented Fos expression in any of the key brain areas of the fear/anxiety circuitry known to be activated by anxiogenic stimuli, it is suggested that reduced social interaction does not reflect enhanced anxiety in aged rats.

Airjet and FG-7142-induced Fos expression differs in rats selectively bred for high and low anxiety-related behavior

We reported recently that two rat lines bred for either high (HAB) or low (LAB) anxiety-related behavior display differential Fos expression in restricted parts of the fear/anxiety circuitry when exposed to mild anxiety evoked in exploratory anxiety tests. Since different forms of anxiety are thought to activate different parts of the anxiety circuitry, we investigated now whether (1) an aversive stimulus which elicits escape behavior (airjet) and (2) the anxiogenic/panicogenic drug FG-7142 would reveal further differences in Fos expression as a marker of neuronal activation between HAB and LAB rats. Both airjet exposure and FG-7142 induced Fos expression in both lines in various anxiety-related brain areas. HAB rats, which displayed exaggerated escape responses during airjet exposure, exhibited increased Fos expression in brain areas including the hypothalamus, periaqueductal gray and locus coeruleus, as well as blunted Fos activation in the cingulate cortex in response to airjet and/or FG-7142. The results corroborate previous findings showing that trait anxiety affects neuronal excitability in hypothalamic and medial prefrontal areas. Furthermore, by using airjet as well as FG-7142, we now reveal that enhanced trait anxiety is also associated with neuronal hyperexcitability in the locus coeruleus and the periaqueductal gray, suggesting that investigation of an array of different anxiogenic stimuli is important for the detection of altered neuronal processing in trait anxiety.

5-HT receptor subtypes involved in the anxiogenic-like action and associated Fos response of acute fluoxetine treatment in rats

RationaleWe have recently reported that acute treatment with the selective serotonin reuptake inhibitor fluoxetine exacerbates escape responses to airjet and facilitates airjet-induced activation of locus coeruleus (LC) neurons.ObjectiveHere we aimed to identify the 5-HT receptor subtype(s) mediating the anxiogenic-like effects of acute fluoxetine in this paradigm and to study whether chronic fluoxetine treatment would alter these responses.MethodsThe expression of the immediate early gene c-fos was used as a marker of neuronal activation.ResultsAcute fluoxetine increased the airjet-induced escape behaviour and Fos expression in the LC of saline-pretreated rats. Pretreatment with the 5-HT2C/2B antagonist SB 206553, but not with the 5-HT1A antagonist WAY 100635, the 5-HT1B antagonist SB 224289 or the 5-HT3 antagonist Y-25130 inhibited the fluoxetine-induced increase in escape behaviour and the associated elevated LC Fos response. The selective 5-HT2C agonist MK-212 mimicked the anxiogenic response of fluoxetine. Chronic treatment with fluoxetine abolished the anxiogenic-like effect and led to a normalization of the enhanced fluoxetine-induced Fos response to airjet.ConclusionsTaken together, the results indicate that the anxiogenic-like effect as well as the facilitated neuronal reactivity induced by acute fluoxetine in the airjet model is mediated primarily by activation of 5-HT2C receptors.

Genetic functional inactivation of neuronal nitric oxide synthase affects stress-related Fos expression in specific brain regions.

To identify neuronal substrates involved in NO/stress interactions we used Fos expression as a marker and examined the pattern of neuronal activation in response to swim stress in nNOS knock-out (nNOS–/–) and wild-type (WT) mice. Forced swimming enhanced Fos expression in WT and nNOS–/– mice in several brain regions, including cortical, limbic and hypothalamic regions. Differences in the Fos response between the two groups were observed in a limited set (6 out of 42) of these brain areas only: nNOS–/– mice displayed increased stressor-induced Fos expression in the medial amygdala, periventricular hypothalamic nucleus, supraoptic nucleus, CA1 field of the hippocampus, dentate gyrus and infralimbic cortex. No differences were observed in regions including the septum, central amygdala, periaqueductal grey and locus coeruleus. During forced swimming, nNOS–/– mice displayed reduced immobility duration, while no differences in general locomotor activity were observed between the groups in the home cage and during the open field test. The findings indicate that deletion of nNOS alters stress-coping ability during forced swimming and leads to an altered pattern of neuronal activation in response to this stressor in specific parts of the limbic system, hypothalamus and the medial prefrontal cortex.