C-fos mRNA In Response Research Articles

Age-related decline in social interaction is associated with decreased c-Fos induction in select brain regions independent of oxytocin receptor expression profiles

Social behavior decreases with aging, and we have previously found a substantial decline in social investigative behavior of old female rats. In this study we examined the neural activation pattern (c-Fos mRNA) of young (3 month) and old (18 month) female rats after brief 10 min exposure to a novel female rat in order to identify forebrain regions that show selective age-related alterations in their neural response to social investigation. We also measured relative oxytocin receptor expression (Oxtr mRNA) as a possible factor in age-related declines in c-Fos induction after social interaction. Young rats exposed to a social partner had a greater c-Fos mRNA response than those exposed to novel context alone in the lateral septum and septohypothalamic area, with blunted increases evident in old rats. In addition, c-Fos mRNA levels in the lateral septum were positively correlated with social investigative behavior. Interestingly, age-related differences in c-Fos gene induction were unrelated to the local amount of Oxtr expression within specific brain regions, although we found an age-related decline in Oxtr expression in the ventromedial hypothalamus. This functional neuroanatomical characterization may point to certain brain regions that are especially sensitive to age-related declines associated with social interaction behavior.

Repeated exposure to cat urine induces complex behavioral, hormonal, and c-fos mRNA responses in Norway rats (Rattus norvegicus).

Prey species show specific adaptations that allow recognition, avoidance, and defense against predators. This study was undertaken to investigate the processing of a chronic, life-threatening stimulus to Norway rats (Rattus norvegicus). One hundred forty-four Norway rats were tested by repeated presentation of cat urine for 1h at different days in a defensive withdrawal apparatus. Rats exposed to urine for short periods showed significantly larger defensive behavioral and medial hypothalamic c-fos messenger RNA (mRNA) responses than other groups. These defensive responses habituated shortly after the presentation of cat urine. Serum levels of adrenocorticotropic hormone and corticosterone increased significantly when animals were repeatedly exposed to cat urine. However, the hormonal responses took longer to habituate than the behavioral and molecular responses did. We conclude that the behavioral and c-fos mRNA responses are "primed" for habituation to repeated exposures to cat urine, while the hormonal responses show "resistance." The results support our hypothesis that the strongest anti-predator responses at three levels would occur during short-term exposure to cat urine and that these responses would subsequently disappear on prolonged exposure. This study assists understanding the way in which the different levels of defensive responses are integrated and react during chronic stress.

Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus.

Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus—derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A—positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability.

An unexpected increase in restraint duration alters the expression of stress response habituation

While habituation develops to a repeated psychological stressor, manipulating certain parameters of the stress challenge experience may lead to dishabituation of the stress response. In this experiment, we investigated whether the behavioral, endocrine, and neural responses (c-fos mRNA immediate early gene expression) to a psychological stressor (restraint) differ when the duration of the stressor given on the test day violates expectations based on prior stress experience. Rats experienced 10min of daily restraint on Days 1–4 followed by a challenge with either the same duration (10min) or a longer duration (30min) of restraint on Day 5. Rats' behavior was video recorded during the Day 5 restraint episode, and trunk blood and brain tissue were collected 30min following restraint onset. Struggling behavior was manually scored as active attempts to escape the restraint device. Rats who experienced the same duration of repeated restraint showed a significant decrease of plasma corticosterone (CORT) compared to the 10min acute restraint group (habituation). In addition, these rats showed decreased active struggling over repeated restraint trials. Conversely, the rats showed an increased CORT response (dishabituation) when they experienced a longer duration of restraint on Day 5 than they had previously. These rats showed a habituated behavioral response during the first 10min of restraint, however struggling behavior increased once the duration of restraint exceeded the expected duration (with a peak at 12min). This peak in struggling behavior did not occur during 30min acute restraint, indicating that the effect was related to the memory of previous restraint experience and not due to a longer duration of restraint. In contrast, these animals showed habituated c-fos mRNA expression in the paraventricular nucleus (PVN), lateral septum (LS), and medial prefrontal cortex (mPFC) in response to the increased stressor duration. Thus, there was a dissociation between c-fos mRNA expression in key stress responsive brain regions and the behavioral and endocrine response to increased stressor duration. This dissociation may have been due to a greater lag time for c-fos mRNA responses to reflect the impact of a dishabituation response. In conclusion, habituation of the endocrine and behavioral stress response occurred when the duration of the stressor matches the previous experience, while dishabituation of the stress response was triggered (with remarkable temporal precision) by an unexpected increase in stress duration.

Postnatal Aromatase Blockade Increases c-fos mRNA Responses to Acute Restraint Stress in Adult Male Rats

Recent evidence suggests that the aromatization of testosterone to estrogen is important for the organizing effects of neonatal testosterone on neuroendocrine responses to acute challenges. However, the extent to which neonatal inhibition of aromatase alters the stress-induced activation of neural pathways has not been examined. Here we assessed central patterns of c-fos mRNA induced by 30 min of restraint in 65-d-old adult male rats that were implanted with sc capsules of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12 h of birth and removed on d 21 of weaning. Neonatal ATD decreased the expression of arginine vasopressin within extrahypothalamic regions in adults, confirming reduced estrogen exposure during development. As adults, ATD-treated animals showed higher corticosterone responses at 30 min of restraint exposure compared with control animals as well as higher c-fos expression levels in the paraventricular nucleus of the hypothalamus. ATD treatment also increased stress-induced c-fos within several limbic regions of the forebrain, in addition to areas involved in somatosensory processing. Based on these results, we propose that the conversion of testosterone to estrogen during the neonatal period exerts marked, system-wide effects to organize adult neuroendocrine responses to homeostatic threat.

Cannabinoid receptor type 1 antagonism significantly modulates basal and loud noise induced neural and hypothalamic-pituitary-adrenal axis responses in male Sprague–Dawley rats

Altered regulation of the hypothalamic-pituitary-adrenal (HPA) axis is associated with stress-induced changes in cognitive, emotional, and physical health. Recent evidence indicates that the endogenous cannabinoid (eCB) system may modulate HPA-axis function both directly and more centrally, via regulation of limbic brain systems that control HPA-axis activity. The current study examines the contribution of cannabinoid type 1 (CB1) receptor modulation throughout the neuraxis on control and stress-induced HPA-axis activity. Adult male Sprague–Dawley rats were given intraperitoneal injections of either CB1 receptor antagonist (AM251, 2 mg/kg) or vehicle 30 min prior to a session of loud white noise stress (95 dBA for 30 min) or placement in a familiar sound-proof chamber. Immediately following stress and control treatments, rats were killed, the brains and pituitary glands were excised for subsequent immediate early gene (c-fos mRNA) measurement, and trunk blood was collected for subsequent determination of corticosterone (CORT) and adrenocorticotropic (ACTH) hormone levels. AM251 treatment resulted in a potentiated plasma ACTH response to loud noise stress. AM251 treatment also increased stress-induced plasma CORT levels, but that increase may be due to an increase in basal plasma CORT levels, as was evident in control rats. AM251 treatment produced three distinctive c-fos mRNA response patterns across the various brain regions examined. In cortical (prelimbic, infralimbic, somatosensory, and auditory) and some subcortical structures (basolateral amygdala and paraventricular nucleus of the hypothalamus), AM251 treatment produced a substantial increase in c-fos mRNA that was comparable with the elevated c-fos mRNA levels present in those brain regions of both vehicle and AM251-treated stressed rats. In some other subcortical structures (bed nucleus of the stria terminalis and medial preoptic area) and the anterior pituitary, AM251 treatment produced a c-fos mRNA response pattern that was similar to the response pattern of ACTH hormone levels, that is, no effect on no noise control levels, but an augmentation of stress-induced levels. Conversely, in the medial geniculate and ventral posterior thalamus, AM251 treatment inhibited stress-induced c-fos mRNA induction. These data indicate that disruption of eCB signaling through CB1 receptors results in potentiated neural and endocrine responses to loud noise stress, but also substantial increases in activity in various brain regions and the adrenal gland.This article is part of a Special Issue entitled: Stress, Emotional Behavior and the Endocannabinoid System.

Medial prefrontal cortex activity can disrupt the expression of stress response habituation

Recent findings suggest that the expression of hypothalamic–pituitary–adrenal (HPA) axis stress response adaptation in rats depends on top–down neural control. We therefore examined whether the medial prefrontal cortex (mPFC) modulates expression of stress response habituation. We transiently suppressed (muscimol microinfusion) or stimulated (picrotoxin microinfusion) mPFC neural activity in rats and studied the consequence on the first time response to psychological stress (restraint) or separately on the development and expression of habituation to repeated restraint. We monitored both the hormonal (corticosterone) and neural (forebrain c-fos mRNA) response to stress. Inactivation of the mPFC had no effect on the HPA-axis response to first time restraint, however increased mPFC activity attenuated stress-induced HPA-axis activity. In a three day repeated restraint stress regimen, inactivation of the mPFC on days 1 and 2, but not day 3, prevented the expression of HPA-axis hormone response habituation. In these same rats, the mPFC activity on day 3 interfered with the expression of c-fos mRNA habituation selectively within the mPFC, lateral septum and hypothalamic paraventricular nucleus. In contrast, inactivation of the mPFC only on day 3, or on all 3 days did not interfere with the expression of habituation. We conclude that the mPFC can permit or disrupt expression of HPA-axis stress response habituation, and this control depends on alteration of neural activity within select brain regions. A possible implication of these findings is that the dysregulation of PFC activity associated with depression and post-traumatic stress disorder may contribute to impaired expression of stress-response adaptation and consequently exacerbation of those disorders.

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.

Hypothalamic-Pituitary-Adrenal Axis Activity of Newborn Mice Rapidly Desensitizes to Repeated Maternal Absence but Becomes Highly Responsive to Novelty

In CD1 mice we investigated the hypothalamic-pituitary-adrenal (HPA) axis response to maternal separation for 8 h daily from postnatal d 3 to 5. At d 3 a slow separation-induced corticosterone response developed that peaked after 8 h, and the pups became responsive to stressors. On the second and third day, the response to 8 h separation rapidly attenuated, whereas the response to novelty did not, a pattern reflected by the hypothalamic c-fos mRNA response. If maternal separation and exposure to novelty were combined, then after the third such daily exposure, the sensitivity to the stressor was further enhanced. Meanwhile, basal corticosterone and ACTH levels were persistently suppressed 16 h after pups were reunited with their mothers. To explain the HPA axis desensitization after repeated separation, we found that circulating ghrelin levels increased and glucose levels decreased after all periods of maternal separation, ruling out a role of altered metabolism. Glucocorticoid feedback was not involved either because a glucocorticoid receptor antagonist amplified the corticosterone response after the first but became ineffective after the third separation. In contrast, a mineralocorticoid receptor antagonist decreased and increased corticosterone levels after the first and third period of separation, respectively. In conclusion, the newborn's HPA axis readily desensitizes to repeated daily maternal separation, but continues to respond to novelty in a manner influenced by a central mineralocorticoid receptor- rather than glucocorticoid receptor-mediated mechanism.

The Anteroventral Bed Nucleus of the Stria Terminalis Differentially Regulates Hypothalamic-Pituitary-Adrenocortical Axis Responses to Acute and Chronic Stress

The anteroventral region of the bed nucleus of the stria terminalis (BST) stimulates hypothalamic-pituitary-adrenocortical (HPA) axis responses to acute stress. However, the role of the anterior BST nuclei in chronic drive of the HPA axis has yet to be established. Therefore, this study tests the role of the anteroventral BST in physiological responses to chronic drive, using a chronic variable stress (CVS) model. Male Sprague-Dawley rats received either bilateral ibotenate lesions, targeting the anteroventral BST, or vehicle injection into the same region. Half of the lesion and control rats were exposed to a 14-d CVS paradigm consisting of twice-daily exposure to unpredictable, alternating stressors. The remaining rats were nonhandled control animals that remained in home cages. On the morning after the end of CVS exposure, all rats were exposed to a novel restraint stress challenge. CVS induced attenuated body weight gain, adrenal hypertrophy, thymic involution, and enhanced CRH mRNA in hypophysiotrophic neurons of the hypothalamic paraventricular nucleus, none of which were affected by anteroventral BST lesions. In the absence of CVS, lesions attenuated the plasma corticosterone and paraventricular nucleus c-fos mRNA responses to the acute restraint stress. In contrast, lesions of the anteroventral BST elevated plasma ACTH and corticosterone responses to novel restraint in the rats previously exposed to CVS. These data suggest that the anterior BST plays very different roles in integrating acute stimulation and chronic drive of the HPA axis, perhaps mediated by chronic stress-induced recruitment of distinct BST cell groups or functional reorganization of stress-integrative circuits.

Long‐term effects of a single exposure to immobilization: A C‐fos mRNA study of the response to the homotypic stressor in the rat brain

A single exposure to a severe emotional stressor such as immobilization in wooden boards (IMO) causes long-term (days to weeks) peripheral and central desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the same (homotypic) stressor. However, the brain areas putatively involved in long-term desensitization are unknown. In the present experiment, adult male rats were subjected to 2 h of IMO and, 1 or 4 weeks later, exposed again to 1 h IMO together with stress-naive rats. C-fos mRNA activation just after IMO and 1 h after the termination of IMO (post-IMO) were evaluated by in situ hybridization. Whereas in most brain areas c-fos mRNA induction caused by the last IMO session was similar in stress-naive (controls) and previously immobilized rats, a few brain areas showed a reduced c-fos mRNA response: ventral lateral septum (LSv), medial amygdala (MeA), parvocellular region of the paraventricular hypothalamic nucleus (pPVN), and locus coeruleus (LC). In contrast, an enhanced expression was observed in the medial division of the bed nucleus stria terminalis (BSTMv). The present work demonstrates that a previous experience with a stressor can induce changes in c-fos mRNA expression in different brain areas in response to the homotypic stressor and suggests that LSv, MeA, and BSTMv may be important for providing signals to lower diencephalic (pPVN) and brainstem (LC) nuclei, which results in a lower physiological response to the homotypic stressor.

Low Doses of Dexamethasone Can Produce a Hypocorticosteroid State in the Brain

The synthetic glucocorticoid dexamethasone (dex) blocks stress-induced hypothalamic-pituitary-adrenal (HPA) activation primarily at the level of the anterior pituitary because multidrug resistance P-glycoprotein hampers its penetration in the brain. Here, we tested the hypothesis that central components of the HPA axis would escape dex suppression under conditions of potent peripheral glucocorticoid action. We subchronically treated rats with low or high doses of dex. The animals were subjected on the last day of treatment for 30 min to a restraint stressor after which central and peripheral markers of HPA axis activity were measured. Basal and stress-induced corticosterone secretion, body weight gain, adrenal and thymus weight, as well as proopiomelanocortin mRNA in the anterior pituitary were reduced in a dose-dependent manner by dex administered either 5 d sc or 3 wk orally. In the brain, the highest dose dex suppressed CRH mRNA and CRH heteronuclear RNA in the paraventricular nucleus (PVN). However, in the peripherally active low-dose range of dex CRH mRNA and heteronuclear RNA showed resistance to suppression, and CRH mRNA expression in the PVN was in fact enhanced under the long-term treatment condition. In the PVN, c-fos mRNA was suppressed by the highest dose of dex, but this effect showed a degree of resistance after long-term oral treatment. c-fos mRNA responses in the anterior pituitary followed those in PVN and reflect central drive of the HPA axis even if corticosterone responses are strongly reduced. The results support the concept that low doses of dex can create a hypocorticoid state in the brain.

Mapping the areas sensitive to long‐term endotoxin tolerance in the rat brain: a c‐fos mRNA study

We have recently found that a single endotoxin administration to rats reduced the hypothalamic-pituitary-adrenal response to another endotoxin administration 4 weeks later, which may be an example of the well-known phenomenon of endotoxin tolerance. However, the time elapsed between the two doses of endotoxin was long enough to consider the above results as an example of late tolerance, whose mechanisms are poorly characterized. To know if the brain plays a role in this phenomenon and to characterize the putative areas involved, we compared the c-fos mRNA response after a final dose of endotoxin in animals given vehicle or endotoxin 4 weeks before. Endotoxin caused a widespread induction of c-fos mRNA in the brain, similar to that previously reported by other laboratories. Whereas most of the brain areas were not sensitive to the previous experience with endotoxin, a few showed a reduced response in endotoxin-pretreated rats: the parvocellular and magnocellular regions of the paraventricular hypothalamic nucleus, the central amygdala, the lateral division of the bed nucleus and the locus coeruleus. We hypothesize that late tolerance to endotoxin may involve plastic changes in the brain, likely to be located in the central amygdala. The reduced activation of the central amygdala in rats previously treated with endotoxin may, in turn, reduce the activation of other brain areas, including the hypothalamic paraventicular nucleus.

Divergent regulatory mechanisms governing BDNF mRNA expression in cerebral cortex and substantia nigra in response to striatal target ablation

We have studied the regulation of BDNF mRNA expression in the corticostriatal and nigrostriatal systems following neurotoxin ablation of striatal targets induced by quinolinic acid (QA) or 2S:2′R:3′R:-2-(2′3′-dicarboxycyclopropyl)glycine (DCG-IV) injections. Striatal lesions were verified by quantifying the loss of glutamic acid decarboxylase (GAD) mRNA expression. Levels of BDNF mRNA were analyzed at 6, 12, and 24 h postlesion. Both lesions paradigms highly induced BDNF mRNA in the ipsilateral cerebral cortex at 6 and 12 h postlesion to drop to control levels at 24 h postlesion. These inductions were mostly restricted to cortical layers II/III and V and ipsilateral insular and piriform cortices, which provide the main cortical inputs to the striatum. Analysis of neuronal activation on these areas demonstrated high levels of cFos mRNA in response to the excitotoxic striatal lesions. Dual labeling of cFos and BDNF mRNAs demonstrated a positive correlation between cortical neuronal activation and expression of BDNF mRNA. Consequently, expression of BDNF in cortical areas projecting to striatum is dependent on both target integrity and neuronal activity. Regulation of BNDF mRNA in substantia nigra, the second major source of BDNF to striatal cells, highly differed from that seen in cerebral cortex. Analysis of cellular expression alone or in combination of BDNF, cFos, tyrosine hydroxylase and GAD mRNAs demonstrated that expression of BDNF in substantia nigra is dependent on target integrity and independent of neuronal activity. In addition, we have studied regulatory mechanisms of BDNF mRNA in the subthalamic nucleus.

Stress-Induced Sensitization of the Hypothalamic-Pituitary Adrenal Axis Is Associated with Alterations of Hypothalamic and Pituitary Gene Expression

We have previously reported that inescapable tail shock (IS) produces persistent changes in hypothalamic-pituitary-adrenal (HPA) axis function. These changes are manifest as an elevation in basal corticosterone (CORT) levels, a sensitization of adrenocorticotropin hormone (ACTH) and CORT responses to subsequent challenge, and a failure of dexamethasone to suppress both the ACTH and CORT responses to a subsequent challenge. The experiments presented here examine IS-induced alterations in the responsiveness of the HPA axis, particularly at the level of the anterior pituitary. The data presented show that adrenalectomy does not abolish the IS-induced sensitization of the HPA axis, suggesting that the sensitization is not solely caused by a defect in glucocorticoid negative feedback. Analysis of gene expression in the anterior pituitary revealed that IS exposure persistently elevated basal levels of proopiomelanocortin (POMC; the precursor to ACTH) mRNA and sensitized the POMC hnRNA and c-fos mRNA response to a subsequent challenge. Analysis of gene expression in the parvocellular division of the paraventricular nucleus of the hypothalamus (pPVN) after IS exposure revealed that basal levels of corticotropin-releasing hormone (CRH) mature mRNA are elevated and the c-fos mRNA response to a subsequent challenge is enhanced. Finally, a blunted in vitro ACTH response to CRH challenge is observed after IS exposure. These data suggest that the ultimate source of the IS-induced sensitization is not the anterior pituitary and implicate an increased drive on the anterior pituitary from the pPVN.

Long-term effects of a single exposure to immobilization on the hypothalamic-pituitary-adrenal axis: neurobiologic mechanisms.

In apparent contrast to previous results from other labs, we have found that a single exposure to a severe stressor such as immobilization (IMO) caused a long-term desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the homotypic stressor. Because such HPA desensitization was not found in response to heterotypic stressors, it seemed at first that we were describing a habituation process already observed after a single experience with the stressor. However, a more detailed analysis revealed two main properties incompatible with the interpretation of the results in terms of habituation: (1) The intensity of desensitization increases over the course of days to weeks with no additional exposures to the stressor, and (2) the degree of desensitization was greater with more severe stressors. The long-term effects were also observed after a single exposure to a high dose of a systemic stressor such as endotoxin but not after insulin-induced hypoglycemia, suggesting that not all severe systemic stressors can induce such long-term desensitization. Because systemic stressors are known to be processed in specific brain areas and because we have found changes in c-fos mRNA response to the homotypic stressor in some brain areas as a consequence of previous experience with IMO, we hypothesize that some severe stressors do not induce long-term desensitization because they are not processed in brain areas sensitive to previous experience with the stressor. The neurochemical mechanisms involved in the induction of long-term effects on the HPA axis are in process, but our results suggest only a partial role of glucocorticoids and NMDA receptors.

Cocaine pre-exposure produces a sensitized and context-specific c-fos mRNA response to footshock stress in the central nucleus of the AMYGDALA

In recent years, there has been growing interest in the putative relationship between stress and vulnerability to relapse in former drug addicts. In animal studies aimed at exploring this relationship, it has been shown that brief exposure to intermittent footshock stress produces reliable reinstatement of drug seeking after prolonged drug-free periods. Whereas footshock reinstates drug seeking, it does not reinstate behaviors maintained by non-drug reinforcers, suggesting that prior drug experience may produce a form of sensitization within neuronal systems that mediate stress-induced reinstatement. The primary objective of the present experiments was to determine whether pre-exposure to cocaine produces a long-lasting, sensitized neuronal response to footshock stress within two brain regions known to mediate footshock-induced reinstatement; the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST). In experiment 1, animals were injected for 7 days with cocaine (days 1 and 7 in test chambers; days 2–6 in homecages) or saline. After 21 drug-free days, they were exposed to footshock or no footshock. In experiment 2, rats were injected daily for 7 days with cocaine in one of two contexts and saline in the alternate context. After 21 drug-free days, they were given footshock either in the same context that they were given cocaine in or the alternate context. In CeA, footshock produced enhanced expression of c-fos mRNA in cocaine, but not saline, pre-exposed animals. Furthermore, this effect was gated by the environmental context in which cocaine was given; footshock only enhanced c-fos mRNA expression when it was given in a context that had previously been paired with cocaine. Although footshock induced c-fos mRNA expression in the BNST, its effects in this region were not dependent on drug history. The major findings are that a history of cocaine exposure produces sensitization to an acute stressor within CeA, and this effect is gated by environmental context.

Dopamine-dependent increases in phosphorylation of cAMP response element binding protein (CREB) during precipitated morphine withdrawal in primary cultures of rat striatum.

Chronic morphine leads to compensatory up-regulation of cAMP signaling pathways in numerous brain regions. One potential consequence of up-regulated cAMP signaling is increased phosphorylation of cAMP response element binding protein (CREB), a transcription factor that may regulate neuroadaptations related to morphine dependence. Altered gene expression within the nucleus accumbens (NAc), a ventral component of the striatum that receives substantial dopaminergic input, may play a role in some of the motivational aspects of opiate withdrawal. To determine if morphine withdrawal leads to increased CREB phosphorylation in striatal tissues, we examined the effects of naloxone-precipitated morphine withdrawal on CREB phosphorylation in primary cultures of rat striatal neurons. Precipitated morphine withdrawal was associated with enhanced dopamine-, SKF 82958 (D1 receptor agonist)-, and forskolin-induced CREB phosphorylation. During precipitated withdrawal, D1 receptor-mediated CREB phosphorylation was dependent on cAMP-dependent protein kinase (PKA). Precipitated withdrawal also led to up-regulation of c-fos mRNA in response to SKF 82958. CREB protein levels were not altered by acute or chronic morphine. These results suggest that D1 receptor-mediated signal transduction is enhanced during morphine withdrawal. Furthermore, they are consistent with in vivo evidence suggesting that increased CREB activation in portions of the striatum (e.g. the NAc) is related to dysphoric states associated with drug withdrawal.

Group II metabotropic glutamate receptor modulation of DOI-induced c-fos mRNA and excitatory responses in the cerebral cortex.

Recent studies have demonstrated that the hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) enhances glutamatergic transmission in the prefrontal cortex. This increase can be suppressed by metabotropic glutamate2/3 (mGlu2/3) receptor activation. In addition to enhancing glutamatergic transmission, DOI increases cortical c-fos expression. We tested if a reduction in glutamate release produced by mGlu2/3 receptor activation attenuates DOI-induced c-fos expression in the cortex. Similar to previous studies, DOI produced a robust increase in c-fos mRNA throughout the cortex, including the prefrontal, frontoparietal, and somatosensory regions. Pretreatment with the mGlu2/3 agonist LY379268 attenuated the DOI-induced increase in the prefrontal cortex. This suppression was blocked by the mGlu2/3 antagonist LY341495. In contrast, the DOI-induced increase in c-fos mRNA in the frontoparietal and somatosensory cortex was unaffected by the mGlu2/3 agents. These findings suggest that Group II metabotropic glutamate receptor agonists are capable of modulating postsynaptic function preferentially in the limbic cortex under conditions of enhanced glutamate release.

Interactions between heterotypic stressors and corticosterone reveal integrative mechanisms for controlling corticotropin-releasing hormone gene expression in the rat paraventricular nucleus.

Although the convergence of neural and humoral afferent information onto paraventricular neuroendocrine corticotropin-releasing hormone (CRH) neurons is a major determinant for adaptive stress responses, the underlying integrative mechanisms are poorly understood. To dissect the relative contributions made by neural afferents and corticosterone to these processes, we determined how the concurrent application of two heterotypic physiological stressors, chronic dehydration (produced by drinking hypertonic saline) and sustained hypovolemia (produced by subcutaneous injections of polyethylene glycol), is interpreted by the synthetic and secretory activity of CRH neurons using in situ hybridization and plasma ACTH measurements. These two stressors are encoded by relatively simple, distinct, and well defined sets of neural afferents to CRH neurons. Both increase plasma corticosterone, but they have opposing actions on CRH gene expression when applied separately. In the first experiment, we showed that chronic dehydration suppresses CRH gene transcription after hypovolemia, but not the preproenkephalin and c-fos mRNA responses or ACTH secretion. In the second, we showed that negative feedback actions of corticosterone do not suppress CRH gene activation after hypovolemia, but instead determine the prestress lower limit of a range within which the CRH gene then responds. Collectively, these data show that at least two processes are integrated to control how the CRH gene responds to multiple stimuli. First, the presence of corticosterone, which although permissive for appropriately activating the CRH gene during hypovolemia, does not mediate the suppressed gene response. Second, neural afferent-driven processes that encode dehydration play a central role in suppressing CRH activation.