Milena Girotti

Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain

Rats repeatedly exposed to restraint show a reduced hypothalamic-pituitary-adrenal axis response upon restraint re-exposure. This hypothalamic-pituitary-adrenal axis response habituation to restraint does not generalize to other novel stressors and is associated with a decrease in stress-induced c-fos expression in a number of stress-reactive brain regions. We examined whether habituation to repeated restraint is also associated with adaptation of immediate early gene expression in brain regions that process and relay primary sensory information. These brain regions may not be expected to show gene expression adaptation to repeated restraint because of their necessary role in experience discrimination. Rats were divided into a repeated restraint group (five 1-hour daily restraint sessions) and an unstressed group (restraint naïve). On the sixth day rats from each group were either killed with no additional stress experience or at 15, 30 or 60 min during restraint. Immediate early gene expression (corticotrophin-releasing hormone heteronuclear RNA, c-fos mRNA, zif268 mRNA) was determined by in situ hybridization. A reduction in stress-induced hypothalamic-pituitary-adrenal axis hormone secretion (plasma corticosterone and adrenocorticotropic hormone) and immediate early gene expression levels in the paraventricular nucleus of the hypothalamus, the lateral septum and the orbital cortex was observed in repeated restraint as compared with restraint naïve animals. This reduction was already evident at 15 min of restraint. Unexpectedly, we also found in repeated restraint rats a reduction in restraint-induced c-fos expression in primary sensory-processing brain areas (primary somatosensory cortex, and ventroposteriomedial and dorsolateral geniculate nuclei of thalamus). The overall levels of hippocampal mineralocorticoid receptor heteronuclear RNA or glucocorticoid receptor mRNA were not decreased by repeated restraint, as may occur in response to severe chronic stress. We propose that repeated restraint leads to a systems-level adaptation whereby re-exposure to restraint elicits a rapid inhibitory modulation of primary sensory processing (i.e. sensory gating), thereby producing a widespread attenuation of the neural response to restraint.

Diurnal expression of functional and clock-related genes throughout the rat HPA axis: system-wide shifts in response to a restricted feeding schedule

The diurnal rhythm of glucocorticoid secretion depends on the suprachiasmatic (SCN) and dorsomedial (putative food-entrainable oscillator; FEO) nuclei of the hypothalamus, two brain regions critical for coordination of physiological responses to photoperiod and feeding cues, respectively. In both cases, time keeping relies upon diurnal oscillations in clock gene (per1, per2, and bmal) expression. Glucocorticoids may play a key role in synchronization of the rest of the body to photoperiod and food availability. Thus glucocorticoid secretion may be both a target and an important effector of SCN and FEO output. Remarkably little, however, is known about the functional diurnal rhythms of the individual components of the hypothalamic-pituitary-adrenal (HPA) axis. We examined the 24-h pattern of hormonal secretion (ACTH and corticosterone), functional gene expression (c-fos, crh, pomc, star), and clock gene expression (per1, per2 and bmal) in each compartment of the HPA axis under a 12:12-h light-dark cycle and compared with relevant SCN gene expression. We found that each anatomic component of the HPA axis has a unique circadian signature of functional and clock gene expression. We then tested the susceptibility of these measures to nonphotic entrainment cues by restricting food availability to only a portion of the light phase of a 12:12-h light-dark cycle. Restricted feeding is a strong zeitgeber that can dramatically alter functional and clock gene expression at all levels of the HPA axis, despite ongoing photoperiod cues and only minor changes in SCN clock gene expression. Thus the HPA axis may be an important mediator of the body entrainment to the FEO.

Immediate–early gene induction in hippocampus and cortex as a result of novel experience is not directly related to the stressfulness of that experience

The stressful quality of an experience, as perceived by rats, is believed to be largely represented by the magnitude of a hypothalamic–pituitary–adrenal (HPA) axis response. The hippocampus may be especially important for assessing the stressfulness of psychological stressors such as novel experience. If such is the case then experience‐dependent immediate–early gene expression levels within the hippocampus may parallel relative levels of HPA axis activity. We examined this prospect in rats that were placed in four different novel environments (empty housing tub, circular arena, elevated pedestal or restraint tube). Restraint and pedestal produced the largest magnitude of increased ACTH and corticosterone secretion, arena an intermediate level (Experiment 2) and tub the least magnitude of increase. We saw a very similar experience‐dependent pattern of relative Fos protein, c‐fos mRNA and zif268 mRNA expression in the paraventricular nucleus of the hypothalamus. However, in hippocampus (and select regions of cortex), immediate–early gene expression was associated with the exploratory potential of the novel experience rather than level of HPA axis activity; pedestal and arena elicited the greatest immediate–early gene expression, tub an intermediate level and restraint the least amount of expression. We conclude that the stressfulness of psychological stressors is not represented by the amount of immediate–early gene induction elicited in hippocampus and cortex, nor does there appear to be a general enhancing or depressive influence of acute stress on immediate–early gene induction in those brain regions.

Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2α in regulating interfacial kinetics and binding and cellular function

Group IVA cytosolic phospholipase A2 (cPLA2α) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA2α (IMLF-/-) were reconstituted with wild type and cPLA2α mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP2) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA2α translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA2α to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA2α to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF-/- expressing phosphorylation site (S505A) and PIP2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA2α, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA2α reduced activation by PIP2 but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA2α hydrolytic activity.

Repeated ferret odor exposure induces different temporal patterns of same-stressor habituation and novel-stressor sensitization in both hypothalamic-pituitary-adrenal axis activity and forebrain c-fos expression in the rat

Repeated exposure to a moderately intense stressor typically produces attenuation of the hypothalamic-pituitary-adrenal (HPA) axis response (habituation) on re-presentation of the same stressor; however, if a novel stressor is presented to the same animals, the HPA axis response may be augmented (sensitization). The extent to which this adaptation is also evident within neural activity patterns is unknown. This study tested whether repeated ferret odor (FO) exposure, a moderately intense psychological stressor for rats, leads to both same-stressor habituation and novel-stressor sensitization of the HPA axis response and neuronal activity as determined by immediate early gene induction (c-fos mRNA). Rats were presented with FO in their home cages for 30 min a day for up to 2 wk and subsequently challenged with FO or restraint. Rats displayed HPA axis activity habituation and widespread habituation of c-fos mRNA expression (in situ hybridization) throughout the brain in as few as three repeated presentations of FO. However, repeated FO exposure led to a more gradual development of sensitized HPA-axis and c-fos mRNA responses to restraint that were not fully evident until after 14 d of prior FO exposure. The sensitized response was evident in many of the same brain regions that displayed habituation, including primary sensory cortices and the prefrontal cortex. The shared spatial expression but distinct temporal development of habituation and sensitization neural response patterns suggests two independent processes with opposing influences across overlapping brain systems.

Chronic Intermittent Cold Stress Sensitizes Neuro-Immune Reactivity in the Rat Brain

Chronic stress contributes to many neuropsychiatric disorders in which the HPA axis, cognition and neuro-immune activity are dysregulated. Patients with major depression, or healthy individuals subjected to acute stress, present elevated levels of circulating pro-inflammatory markers. Acute stress also activates pro-inflammatory signals in the periphery and in the brain of rodents. However, despite the clear relevance of chronic stress to human psychopathology, the effects of prolonged stress exposure on central immune activity and reactivity have not been well characterized. Our laboratory has previously shown that, in rats, chronic intermittent cold stress (CIC stress, 4°C, 6h/day, 14 days) sensitizes the HPA response to a subsequent novel stressor, and produces deficits in a test of cognitive flexibility that is dependent upon prefrontal cortical function. We have hypothesized that CIC stress could potentially exert some of these effects by altering the neuro-immune status of the brain, leading to neuronal dysfunction. In this study, we have begun to address this question by determining whether previous exposure to CIC stress could alter the subsequent neuro-immune response to an acute immunological challenge (lipopolysaccharide, LPS) or an acute heterologous stressor (footshock). We examined the response of the pro-inflammatory cytokines, IL1β and IL6, the enzyme cyclooxygenase 2, and the chemokines, CXCL1 and MCP-1 in plasma, hypothalamus and prefrontal cortex. There was no effect of CIC stress on basal expression of these markers 24h after the termination of stress. However, CIC stress enhanced the acute induction of the pro-inflammatory cytokines, IL1β and particularly IL6, and the chemokines, CXCL1 and MCP-1, in plasma, hypothalamus and prefrontal cortex in response to LPS, and also sensitized the hypothalamic IL1β response to acute footshock. Thus, sensitization of acute pro-inflammatory responses in the brain could potentially mediate some of the CIC-dependent changes in HPA and cognitive function.

Restraint-induced fra-2 and c-fos expression in the rat forebrain: relationship to stress duration

The protein product of the fra-2 gene (Fra-2), a fos-family member, can compete with Fos protein for participation in activating protein-1 (AP-1) transcription factor complexes and each protein can contribute different transactivational consequences to an AP-1 complex. To date, there is limited characterization of fra-2 mRNA expression in the rat forebrain. We examined basal and restraint-induced mRNA expression (in situ hybridization) of fra-2 in the rat forebrain and compared its temporal-spatial pattern to c-fos. In contrast to the very low basal expression of c-fos, fra-2 basal expression was moderately high throughout cortex and some subcortical structures, including prominent basal expression in the hypothalamic paraventricular nucleus (PVN). Restraint-induced fra-2 expression was quantified in the prefrontal cortex (PFC), lateral septum (LS) and PVN. Maximal fra-2 gene induction in the PFC and LS was delayed (60 min) after restraint onset with respect to c-fos (15 min), whereas in the PVN, fra-2 mRNA increased within 15 min of restraint. Additionally we compared c-fos and fra-2 gene expression in rats given shorter or longer restraint durations, but equal total time from stress onset to sample collection, to determine the extent to which the kinetics of gene induction matched that of a hypothalamic-pituitary-adrenal axis hormone response. Rats given 45 min recovery after 15 min restraint showed less c-fos expression in the PVN, less fra-2 expression in the prelimbic and infralimbic PFC, and no difference in the LS compared with rats restrained for 60 min. Thus, the expression of both genes was sensitive to stressor duration, but this sensitivity varied with brain region. Differential basal and stress-induced expression patterns of the fra-2 and c-fos genes are likely to have important functional consequences for AP-1 transcription factor dependent regulation of neural plasticity.

Differential glucocorticoid effects on stress-induced gene expression in the paraventricular nucleus of the hypothalamus and ACTH secretion in the rat

Although previous studies have examined the extent to which adrenocorticotropic hormone (ACTH) secretion depends on endogenous glucocorticoid levels, few have examined the parallel glucocorticoid dependency of gene expression within the corticotropin releasing hormone (CRH) neuron containing subregion of the hypothalamic paraventricular nucleus (PVN). This study examined resting and stress-induced expression of three immediate early genes (c-fos, zif268, and NGFI-B mRNAs) and two phenotypic restricted immediate early genes that code for ACTH secretagogues (CRH and arginine vasopressin [AVP] hnRNAs) in the PVN of adrenalectomized (ADX) rats given either 0.9% saline to drink for 5 days or saline with corticosterone (CORT; 25 μg/ml). CORT-containing saline was replaced with saline 18 h before testing to ensure clearance of CORT at the time of testing. Dependent measures were examined 0, 15, 30, 60, or 120 min after 30 min restraint. Compared to sham surgery, ADX produced a large upregulation of basal ACTH secretion but only a trend for an increase in basal PVN CRH and parvocellular (mp) PVN AVP hnRNA expression, and a marked augmentation of restraint-induced ACTH secretion and the expression of all five genes examined. CORT containing saline partially normalized basal and restraint-induced ACTH secretion and restraint-induced AVP hnRNA, c-fos mRNA, and zif268 mRNA in the PVN in ADX rats. In contrast, expression patterns of restraint-induced PVN CRH hnRNA and NGFI-B mRNA were not different between ADX rats with or without CORT replacement. Given that there was no circulating CORT present at the time of restraint challenge in either group of ADX rats, the differential impact of CORT replacement on restraint-induced PVN gene expression must reflect differential dependency of the expression of these genes in the PVN on the prior presence of CORT.

A novel role for brain interleukin-6: Facilitation of cognitive flexibility in rat orbitofrontal cortex

Cytokines, small proteins released by the immune system to combat infection, are typically studied under inflammatory conditions. However, these molecules are also expressed in the brain in basal, nonpathological states, where they can regulate neuronal processes, such as learning and memory. However, little is known about how cytokine signaling in the brain may influence higher-order cognitive functions. Cognitive flexibility is one such executive process, mediated by the prefrontal cortex, which requires an adaptive modification of learned behaviors in response to environmental change. We explored the role of basal IL-6 signaling in the orbitofrontal cortex (OFC) in reversal learning, a form of cognitive flexibility that can be measured in the rat using the attentional set-shifting test. We found that inhibiting IL-6 or its downstream JAK/STAT signaling pathway in the OFC impaired reversal learning, suggesting that basal IL-6 and JAK/STAT signaling facilitate cognitive flexibility. Further, we demonstrated that elevating IL-6 in the OFC by adeno-associated virus-mediated gene delivery reversed a cognitive deficit induced by chronic stress, thus identifying IL-6 and the downstream JAK/STAT signaling pathway as potentially novel therapeutic targets for the treatment of stress-related psychiatric diseases associated with cognitive dysfunction.

Influence of hypothalamic IL-6/gp130 receptor signaling on the HPA axis response to chronic stress

Abnormal basal activity and stress-evoked reactivity of the hypothalamic-pituitary-adrenal (HPA) axis are often seen in depression, implicating HPA axis dysfunction as a potentially causative or exacerbating factor. Chronic stress is also a factor in depression, but it is not known what may underlie the shift from adaptive to maladaptive HPA activity over the course of chronic stress. Interleukin 6 (IL-6), a stress-inducible cytokine that signals through gp130 and IL-6Rα receptors to activate the JAK/STAT3 signaling cascade, is elevated in some subtypes of depression, and may have a modulatory effect on HPA activation, raising the possibility that IL-6 contributes to depression through effects on the HPA axis. In this study, we examined the effects of three different stress modalities, acute footshock, chronic intermittent cold (CIC) stress and chronic unpredictable stress (CUS) on IL-6 signaling in the hypothalamus. We also investigated whether IL-6 modulates the HPA response to chronic stress, by blocking IL-6 signaling in the brain during CIC stress using either a neutralizing antibody or an inhibitor of STAT3 phosphorylation. We show that IL-6 and STAT3 in the hypothalamus are activated in response to footshock and CUS. We also found that basal IL-6 signaling through the JAK/STAT3 pathway is required for the sustained CORT response to chronic, but not acute, cold stress and therefore is a potential determinant of plasticity in the HPA axis specifically during chronic stress exposure.