Differential forebrain c-fos mRNA induction by ether inhalation and novelty: evidence for distinctive stress pathways.

Previous studies indicate the existence of subtypes of stressors invoking distinct patterns of neuronal integration. Pathways activated by stress appear to depend on whether the perceived threat is processive/neurogenic or systemic in nature. To test this hypothesis, the present study compares magnitude and extent of c-fos mRNA induction in response to novelty (open field (OF), representing a processive stressor) or ether exposure (representing a systemic stressor). Exposure to the OF or ether fumes both produced increases in plasma corticosterone (CORT) levels; notably, peak levels of secretion were elevated in the ether group, suggestive of augmented HPA secretory activity to this stressor. In situ hybridization analysis of c-fos mRNA induction reveals common and divergent activational properties in the two stress groups. The extent of c-fos mRNA expression was similar in the paraventricular nucleus (PVN), despite stress-related differences in CORT secretion. Analysis of the dorsomedial hypothalamic nucleus, suprachiasmatic nucleus (SCN) and limbic sites, specifically, the lateral septum and medial amygdaloid nucleus, indicate greater c-fos mRNA induction in animals exposed to OF stress. The frontoparietal cortex was only forebrain region showing differential activation by ether. Differential c-fos induction was not observed in the medial preoptic area (ventrolateral quadrant), paraventricular thalamus, dorsolateral striatum or hippocampus. The results indicate that processive and systemic stressors differ not only in the patterning of neuronal activation in the CNS, but also in the extent to which selected stress-sensitive regions are induced.     

Stress-induced activation of CRF and c-fos mRNAs in the paraventricular nucleus are not affected by serotonin depletion

The role of serotonin in regulating the stress response is controversial. We have investigated the effects of serotonin depletion byp-chlorophenylalanine (PCPA) on corticotrophin-releasing factor (CRF) mRNA and c-fos mRNA responses in the paraventricular nucleus (PVN) together with circulating levels of ACTH and corticosterone to both physical and psychological stressors in the rat. PCPA pretreatment, which resulted in a 95% depletion in hypothalamic serotonin, had no effect on basal levels of ACTH or the increase in response to the physical stress of hypertonic saline. Plasma ACTH concentrations were also not affected by serotonin depletion in response to the predominantly psychological stress of restraint. Both basal and restraint stress-induced circulating corticosterone levels were however further stimulated in the PCPA-pretreated rats suggesting a possible inhibitory serotoninergic tone at the adrenal level. C-fos mRNA was undetectable in control animals. Activation of c-fos mRNA in response to stress was unaffected by serotonin depletion and the activation of magnocellular PVN and supraoptic nucleus cells was demonstrated to be stressor dependent. Basal and stress-induced levels of CRF mRNA were unaffected by PCPA pretreatment. It appears therefore that under these experimental conditions there is little if any involvement of serotonin in either basal levels or the stress-induced activation of the hypothalamo-pituitary-adrenal axis in vivo.     

The effect of adrenalectomy on stress-induced c-fos mRNA expression in the rat brain

Previously, we determined the pattern of stress-induced c-fos mRNA expression throughout the brain in order to gain further insight into the identification of the neural circuits mediating stress-induced regulation of the hypothalamic-pituitary-adrenal axis. In the present study, we determined if rapid effects of increased glucocorticoid levels after stress contribute to changes in c-fos mRNA expression. To this end, stress-induced c-fos expression was characterized in adrenalectomized (ADX) or adrenalectomized and corticosterone replaced (ADX/B) male rats. Animals were sacrificed 30 min post-onset of a 10 min swim stress, and in situ hybridization histochemistry was used to detect c-fos mRNA throughout the brain. The pattern of c-fos induction in the ADX and ADX/B animals was similar to that observed in the sham operated animals. Additionally, densitometric measurements were made to quantify the c-fos response in the paraventricular nucleus of the hypothalamus and the CA1/2 region of the hippocampus. We found that ADX did not alter the magnitude of the c-fos response to stress in these areas, but there was a slight dampening of the response in ADX/B animals. In sum, these results suggest that the pattern of c-fos expression observed 30 min post-stress is independent of stress-induced increases in circulating glucocorticoid concentrations.     

Sexual stimulation activates c-fos within estrogen-concentrating regions of the female rat forebrain

Regions of the brain that concentrate estrogen and progesterone are thought to regulate female sexual behavior by altering gene expression and neural sensitivity to afferent stimulation. We used immunocytochemistry and in situ hybridization to examine c-fos gene expression within estrogen-concentrating regions of the forebrain following various types of sexual stimulation with or without hormone treatment. Ovariectomized rats received injections of estradiol benzoate 48 h and progesterone 4 h before testing. Control rats that had been ovariectomized at least 5 months before testing did not receive hormone treatment. Rats were then either placed into bilevel testing chambers with sexually vigorous males, received manual stimulation of the flanks, received vaginocervical stimulation with a glass rod, or were left in their home cages. Copulation with intromission and ejaculation in hormone-treated rats, or stimulation of the vaginal cervix in both hormone-treated and control rats, produced a dramatic induction of c-fos mRNA and Fos-like immunoreactivity in estrogen-concentrating regions, such as the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus, ventromedial hypothalamus, lateral habenula, and medial amygdala, in addition to regions that do not readily concentrate estrogen, such as the neocortex, thalamus, and striatum. Mechanical stimulation of the flanks produced a smaller induction of Fos in these rats, whereas hormone treatment alone had no effect. These data demonstrate that afferent sensory stimulation, but not estrogen or progesterone, regulates c-fos gene expression within different estrogen-concentrating and non-concentrating regions of the female rat forebrain.     

Repeated administration of methamphetamine blocked cholecystokinin-octapeptide injection-induced c-fos mRNA expression without change in capsaicin-induced junD mRNA expression in rat cerebellum

In the cerebellum, there are numerous cholecystokinin (CCK-8)-containing fibers. Since systemic CCK-8 injection-induced anxiety (psychological stress) activates the locus coeruleus cells that send mossy fiber inputs to the cerebellum, we examined whether systemic CCK-8 injections activate the rat and mouse cerebellum. First, injections of CCK-8 were found to induce c-fos mRNA expression in a vague patchy pattern that is different from single methamphetamine-induced Zebrin band-like c-fos mRNA expression, suggesting that the CCK-8 activating mossy fibers induce gene expression differently from the dopamine-containing mossy fibers in the ventral tegmental area. Second, since CCK-8 facilitates neural activity of dopamine in the midbrain, we examined whether repeated methamphetamine administration that induced behavioral sensitization had similar effects on the cerebellar CCK system. Repeated administration of methamphetamine suppressed the CCK-8-induced c-fos mRNA expression in the rat cerebellum. Third, capsaicin injections (physical stress) into a hind limb of the rat increased junD mRNA expression with no effect on c-fos mRNA expression, and repeated methamphetamine injections had no effect on the capsaicin-induced expression of junD mRNA. Fourth, either single injection of methamphetamine or CCK-8 to mice increased c-fos mRNA expression in the locus coeruleus, and so noradrenalin, but not dopamine, might interact with CCK-8-activating system. However, we considered the possibility unlikely. Thus, we conclude that repeated methamphetamine administration though dopamine selectively inhibits the c-fos mRNA expression after CCK-8 injection in the cerebellum.     

Area postrema removal abolishes stimulatory effects of intravenous interleukin-1β on hypothalamic-pituitary-adrenal axis activity and c-fos mRNA in the hypothalamic paraventricular nucleus

This study examined the role of the area postrema (AP) in transducing peripheral immune signals, represented by intravenous (i.v.) interleukin-1β (IL-1), into neuroendocrine responses. The AP, a circumventricular organ with a leaky blood–brain barrier, lies adjacent to the nucleus of the solitary tract (NTS) in the medulla. The AP was removed by aspiration, and 2 weeks later, AP-lesioned or sham-lesioned rats were injected i.v. with 0.5 μg/kg IL-1 or sterile saline. After 30 min, brains were removed and analyzed for c-fos mRNA levels in various structures implicated in the hypothalamic-pituitary-adrenal axis response to peripheral cytokine challenge. The sham-lesioned animals responded to IL-1 with large elevations in adrenocorticotropic hormone (ACTH) and corticosterone levels in the plasma and c-fos mRNA levels in cells of the AP, NTS, central nucleus of the amygdala, bed nucleus of the stria terminalis, hypothalamic paraventricular nucleus (PVN), and meninges. Prior AP removal abolished the IL-1-induced increases in ACTH and corticosterone in the plasma and c-fos mRNA levels in the NTS and PVN. However, AP removal had no effect on IL-1-induced increases in c-fos mRNA levels in the other areas examined. The selective AP lesion effects suggest that the AP and adjacent NTS play a pivotal role in transducing a circulating IL-1 signal into hypothalamic-pituitary-adrenal axis activation by a pathway that may be comprised of known anatomical links between the AP, NTS, and corticotropin-releasing hormone neurons of the PVN.     

Cycloheximide prevents kainate-induced neuronal death and c-fos expression in adult rat brain

The present study was directed at evaluating the possible involvement of protein synthesis in excitotoxin-induced neuronal damage and prolonged expression of the proto-oncogene, c-fos. Kainic acid-induced seizure activity elicited varying degrees of neuronal damage and cell loss in selectively vulnerable regions of the adult rat limbic system. Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter behavioral seizure characteristics, but markedly attenuated damage to susceptible neuronal populations. A prolonged increase in c-fos mRNA was observed byin situ hybridization up to 16 h after the onset of seizures in regions exhibiting neuronal death. Pretreatment with cycloheximide did not affect the transient induction of c-fos observed in numerous structures, but significantly reduced the prolonged expression of c-fos mRNA in kainatevulnerable regions. Despite producing massive seizure activity, systemic kainic acid administration during the early postnatal period did not induce any neuronal death, and did not result in prolonged c-fos expression in any brain structures. The developmental onset of selective neuronal vulnerability coincided with that of prolonged c-fos expression in susceptible neuronal populations. In adult rats, seizure activity induced by pentylenetetrazole did not produce neuronal damage nor did it produce prolonged c-fos expression. These results not only demonstrate that kainate-induced neurotoxicity and the prolonged expression of c-fos are both prevented by cycloheximide, but also strengthen the idea that prolonged c-fos expression is a marker of neuronal death.     

The pattern of brain c-fos mRNA induced by a component of fox odor, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), in rats, suggests both systemic and processive stress characteristics

Predators to rodents and their associated odors are increasingly chosen to study the neural mechanisms of stress and anxiety. Specifically, predatory odors are believed to elicit responses based on the perceived threat (psychological or processive), rather than to any direct systemic effects (pain, blood loss, infection, etc.) of the stimulus, which are mediated by distinct neural pathways. The hypothesis that a chemical component from fox feces, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), elicits stress responses by specific activation of processive neural pathways was tested. Different amounts of TMT (range: 0-600 micromol) or the control odor butyric acid (0-1200 micromol) were presented to male Sprague-Dawley rats for 30 min. Immediately after odor presentation, rats were sacrificed, blood levels of adrenocorticotropic hormone (ACTH) and corticosterone were measured, and brains were rapidly harvested to measure regional brain c-fos mRNA induction by in situ hybridization. Presentation of TMT (> or =75 micromol), but not butyric acid (up to 1200 micromol), significantly increased ACTH and corticosterone release. TMT presentation, especially with amounts (> or =75 micromol) producing endocrine activation, induced c-fos mRNA in several brain areas, including the olfactory bulb, lateral septal nucleus, septohypothalamic nucleus, anteromedial and oval nuclei of the bed nucleus of the stria terminalis, the central nucleus of the amygdala, the anteroventral, anterodorsal, and medial preoptic nuclei, the anterior, dorsomedial, lateral, supramammillary, dorsal premammillary and paraventricular hypothalamic nuclei, the external lateral parabrachial nucleus, the locus coeruleus, and the nucleus of the solitary tract. Interestingly, these brain regions represent a mix of regional c-fos mRNA induction pattern not reported previously with any other single stressor. These results suggest that TMT elicits stress responses through a relatively unique and complex mix of brain regions associated with both processive and systemic neural pathways, unlike those seen in response to cat odors.     

Intracerebroventricular administration of β-endorphin increases the expression of c-fos and of corticotropin-releasing factor messenger ribonucleic acid in the paraventricular nucleus of the rat

We evaluated the effects of intracerebroventricular (i.c.v.) administration of β-endorphin and naloxone, an opioid antagonist, on the induction of c-fos and corticotropin-releasing factor (CRF) mRNA to clarify the effects of β-endorphin on cellular activity and CRF gene expression in the paraventricular nucleus (PVN) of the rat using in situ hybridization. A significant induction of c-fos mRNA was noted in the PVN after i.c.v. injection of β-endorphin, compared to control. This induction was inhibited by the administration of naloxone. A significant increase in CRF mRNA levels in the PVN was observed 120 min after the i.c.v. injection of β-endorphin. This increase was partially, but significantly, inhibited by naloxone administration. In addition, i.c.v. administration of β-endorphin increased plasma ACTH concentration in freely moving rats, which was inhibited by intravenous injection of CRF antiserum. These results suggest that the i.c.v. injection of β-endorphin increases the neuronal activity and the biosynthesis of CRF in the PVN, and stimulates the secretion of ACTH by increasing CRF secretion. This effect on the PVN was mediated, at least in part, via the opioid receptor.     

Opposite effects of rough and gentle handling with repeated saline administration on c-fos mRNA expression in the rat brain

Summary The rough handling with repeated saline administration (1.2 ml/kg s.c. for 7 days) enhanced cortical c-fos mRNA expression in the rat brain after a single saline stimulation (1.2 ml/kg s.c.) due to increasing baseline c-fos mRNA levels, whereas the gentle handling with repeated saline administration declined c-fos mRNA expression after a single injection due to decreasing the baseline of c-fos mRNA levels. These two types of handling with the repeated injection led to diametrically opposite results on c-fos mRNA expression after a single stimulation. Neither two types of handling with repeated saline injections affected the net increment of c-fos mRNA induction after a single stimulation, therefore, the effects of handling with repeated treatment on c-fos mRNA expression might be independent of the effects of a single saline stimulation. The present study suggests that c-fos mRNA induction after a single stimulation might be affected by the types or intensities of handling and that care must be taken to estimate c-fos mRNA induction.     

Glutamate Induces c-fos Proto-oncogene Expression and Inhibits Proliferation in Oligodendrocyte Progenitors: Receptor Characterization

The effect of glutamate on c-fos expression in oligodendrocyte progenitors was investigated by Northern blot analysis. Glutamate caused rapid and transient induction. Both 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), two competitive non-NMDA ionotropic receptor antagonists, reduced glutamate-induced c-fos expression, whereas the NMDA antagonist MK-801 was ineffective. In addition, the glutamate receptor agonists (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) and kainate strongly induced c-fos. However, the metabotropic receptor agonist trans-(±)-1-amino-(1S,3R)-cyclopentanedicarboxylic acid (trans-(±)-ACPD) did not increase c-fos mRNA level and the antagonist L-(+)-2-amino-3-phosphonopropionic acid did not block glutamate-induced c-fos mRNA. These findings indicate that c-fos induction in oligodendrocyte progenitors is mediated through the AMPA/kainate receptors, while NMDA and metabotropic receptor subtypes are not involved. Chelation of extracellular calcium by EDTA prevented glutamate-induced c-fos expression. Similarly, the protein kinase C inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine dihydrochloride (H7) and down-regulation of protein kinase C by prolonged exposure to phorbol-12-myristate 13-acetate blocked c-fos induction. These results suggest that induction of c-fos through AMPA/kainate receptors is dependent on extracellular calcium influx and involves downstream activation of phorbol ester-sensitive protein kinase C. The effect of glutamate on oligodendrocyte progenitor proliferation was assessed by [³H]thymidine incorporation. Glutamate and the agonists kainate and AMPA, but not trans-(±)-ACPD, caused a dose-dependent decrease in [³H]thymidine incorporation. All these pharmacological agents were not toxic to oligodendrocyte progenitors. CNQX reversed the inhibitory effects produced by glutamate and the various agonists. These results suggest that glutamate may modulate the growth and differentiation of oligodendrocytes in the central nervous system.     

Muscarinic cholinergic receptor-mediated modulation on striatal c-fos mRNA expression induced by levodopa in rat brain

Summary To clarify the interactions between dopamine receptors and muscarinic cholinergic receptors by which neurotransmitters may affect genetic responses, we studied the effects of the muscarinic cholinergic agonist, carbachol, and the muscarinic cholinergic antagonist, trihexyphenidyl, on levodopa-induced c-fos messenger RNA (mRNA) expression in rat striatum. Animals were administered levodopa (levodopa with one-tenth dosage of carbidopa), carbachol or thrihexyphenidyl alone or administered in combination as levodopa (100 mg/kg) + carbachol, or levodopa + trihexyphenidyl given as a single bolus. Levodopa given alone increase the expression of c-fos mRNA. Although carbachol or trihexyphenidyl alone was ineffective in inducing c-fos mRNA, the combination of levodopa and carbachol ( 0.1 mg/kg) significantly suppressed the induction of c-fos mRNA as compared with levodopa given alone. The combined administration of levodopa and trihexyphenidyl showed a trend toward an additive effect on the induction of c-fos mRNA vs levodopa alone. These findings suggest that the muscarinic cholinergic system may modulate the levodopa-induced c-fos mRNA expression which then regulates the expression of other mRNAs.     

Induction of the c-fos proto-oncogene during opiate withdrawal in the locus coeruleus and other regions of rat brain

Opiate regulation of the nuclear proto-oncogene c-fos was studied in the locus coeruleus (LC) and other regions of rat brain by immunoblotting, northern blotting, and in situ hybridization procedures. Precipitation of opiate withdrawal in rats, which is known to increase LC firing rates 4-fold, led to a two- to three-fold increase in levels of mRNA and protein for c-fos in the LC 1–2 h after initiation of withdrawal. In contrast, levels of c-fos expression were decreased in LC from rats treated acutely or chronically with morphine but not experiencing withdrawal, conditions under which LC firing rate are depressed. Similar regulation of c-fos expression during opiate withdrawal was found in the amygdala, ventral tegmentum, nucleus accumbens, neostriatum, and cerebral cortex, but not in a number of other brain regions studied, which included the hippocampus, dorsal raphe, periaqueductal gray, and paragigantocellularis. In the LC and some other brain regions, induction of c-fos during opiate withdrawal was associated with a parallel induction of c-jun, another nuclear proto-oncogene, which, like c-fos, is expressed rapidly in brain in response to certain extracellular stimuli. The results demonstrate a novel use of c-fos in neuropharmacology, namely to map neuronal pathways and neuronal cell types activated in response to acute and chronic opiate administration and during opiate withdrawal, as well as in response to other psychotropic drug treatments.