Induction Of c-Fos Immunoreactivity Research Articles

Activation of CRF1 receptors expressed in brainstem autonomic nuclei stimulates colonic enteric neurons and secreto-motor function in male rats.

Hypothalamic corticotropin-releasing factor (CRF) receptor 1 (CRF1 ) plays a role in acute stress-related stimulation of colonic motor function. Less is known on CRF1 signaling in the brainstem. We investigate CRF1 expression in the brainstem and the colonic response to 4th ventricle (4V) injection of CRF and urocortin (Ucn) 2 (3 µg/rat) in chronically cannulated male rats. Transcripts of CRF1 wild-type 1a and splice variants 1c, 1e, 1f, 1o along with three novel variants 1a-2 (desK-110 in exon 5), 1p (-exon 7), and 1q (exon 5 extension) were identified in the pons and medulla. The area postrema, nucleus tractus solitarius, dorsal motor nucleus of the vagus, locus coeruleus, and Barrington's nucleus isolated by laser capture microdissection expressed 1a, 1a-2, and 1p but not 1q. Compared to 4V vehicle, 4V CRF induced fecal pellet output (FPO) and diarrhea that were blocked by the CRF antagonist, astressin-B. CRF2 agonist, Ucn2 had no effect on basal or CRF-induced FPO. CRF actions were correlated with the induction of c-Fos immunoreactivity in myenteric neurons of the proximal and distal colon (pC, dC) and submucosal neurons of dC. c-Fos immunoreactivity occurred in 39% and 37% of myenteric cholinergic and 7% and 58% of nitrergic neurons in the pC and dC, respectively. CRF1a and its splice variants are expressed in brainstem nuclei, and activation of CRF1 signaling at the level of the brainstem stimulates colonic secretory-motor function through activation of colonic enteric neurons.

Induction of c-Fos immunoreactivity in the amygdala of mice expressing anxiety-like behavior after local perfusion of veratrine in the prelimbic medial prefrontal cortex.

Local perfusion of the sodium channel activator veratrine in mouse prelimbic medial prefrontal cortex (PL) induced c-Fos immunoreactivity in the sub-regions of amygdala. Co-perfusion of the NMDA receptor antagonist MK-801 diminished the c-Fos expression. Significant correlations were observed between c-Fos immunoreactivity and behavioral measures in the open-field test. The PL stimulation activates a neural network projecting to the amygdala via NMDA receptor-mediated glutamatergic neurotransmission. Anxiety-like behavior induced after the PL stimulation may be partly mediated through the activation of amygdala.

Effect of MTII on food intake and brain c-Fos in melanocortin-3, melanocortin-4, and double MC3 and MC4 receptor knockout mice

Mice with genomic knockout of either melanocortin type 3 receptors (MC3R−/−), type 4 receptors (MC4R−/−) or knockout of both (double knockout, DKO) were tested for their anorectic response to the mixed MC3/4R agonist, MTII, injected into the anterior cerebral ventricle. Wild type (WT) mice showed a strong anorexia and, as expected, DKO were completely unresponsive to MTII. In contrast, both MC3R−/− and MC4R−/− showed a partial anorectic response. Induction of c-Fos immunoreactivity by MTII was examined in brain regions including paraventricular hypothalamus (PVN) and area postrema (AP). Compared with WT, MC4R−/− showed no activation in AP but showed normal activation in PVN, whereas MC3R−/− showed reduced activation in PVN but not in AP. RT-PCR analysis showed that hypothalamic mRNA for MC3R in MC4R−/− and for MC4R in MC3R−/− was unaltered from WT levels. These data suggest that both receptor subtypes are involved in the behavioral action of MTII, and that the critical receptors are in different brain regions.

251 Potent and efficient testosterone suppression by chronic administration of novel metastin analogues, TAK-448 and TAK-683, in male rats

Metastin is a novel peptide that has been isolated from the human placenta as the cognate ligand of the G-protein-coupled receptor OT7T175 (or GPR54). However, its physiological functions have not yet been fully investigated. In the present study, we show that subcutaneous administration of metastin increased the plasma levels of gonadotropins (follicle-stimulating hormone and luteinizing hormone) and induced ovulation in prepubertal female rats that had been pretreated with pregnant mare serum gonadotropin to induce follicle maturation. Furthermore, metastin administration drastically increased the plasma levels of gonadotropins in male rats. This action was abolished by pretreatment with a GnRH antagonist, and was accompanied by induction of c-Fos immunoreactivity in GnRH neurons. These results suggest that s.c. administered metastin induces the release of gonadotropin via activation of the hypothalamic GnRH neurons.

Y4 receptors and pancreatic polypeptide regulate food intake via hypothalamic orexin and brain-derived neurotropic factor dependent pathways

Gut-derived peptides are known to regulate food intake by activating specific receptors in the brain, but the target nuclei and neurons influenced are largely unknown. Here we show that peripherally administered pancreatic polypeptide (PP) stimulates neurons in key nuclei of the hypothalamus critical for appetite and satiety regulation. In the lateral hypothalamic area (LHA), also known as the feeding center, neurons expressing the orexigenic neuropeptide orexin co-localize with the early neuronal activation marker c-Fos upon i.p. injection of PP into mice. In the ventromedial hypothalamus (VMH), also known as the satiety center, neurons activated by PP, as indicated by induction of c-Fos immunoreactivity, express the anorexigenic brain-derived neurotrophic factor (BDNF). Activation of neurons in the LHA and VMH in response to PP occurs via a Y4 receptor-dependent process as it is not seen in Y4 receptor knockout mice. We further demonstrate that in response to i.p. PP, orexin mRNA expression in the LHA is down-regulated, with Y4 receptors being critical for this effect as it is not seen in Y4 receptor knockout mice, whereas BDNF mRNA expression is up-regulated in the VMH in response to i.p. PP in the fasted, but not in the non-fasted state. Taken together these data suggest that PP can regulate food intake by suppressing orexigenic pathways by down-regulation of orexin and simultaneously increasing anorexigenic pathways by up-regulating BDNF.

Leptin Directly Activates SF1 Neurons in the VMH, and This Action by Leptin Is Required for Normal Body-Weight Homeostasis

Leptin, an adipocyte-derived hormone, acts directly on the brain to control food intake and energy expenditure. An important question is the identity of first-order neurons initiating leptin's anti-obesity effects. A widely held view is that most, if not all, of leptin's effects are mediated by neurons located in the arcuate nucleus of the hypothalamus. However, leptin receptors (LEPRs) are expressed in other sites as well, including the ventromedial hypothalamus (VMH). The possible role of leptin acting in "nonarcuate" sites has largely been ignored. In the present study, we show that leptin depolarizes and increases the firing rate of steroidogenic factor-1 (SF1)-positive neurons in the VMH. We also show, by generating mice that lack LEPRs on SF1-positive neurons, that leptin action at this site plays an important role in reducing body weight and, of note, in resisting diet-induced obesity. These results reveal a critical role for leptin action on VMH neurons.

Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat

Metastin is a novel peptide that has been isolated from the human placenta as the cognate ligand of the G-protein-coupled receptor OT7T175 (or GPR54). However, its physiological functions have not yet been fully investigated. In the present study, we show that subcutaneous administration of metastin increased the plasma levels of gonadotropins (follicle-stimulating hormone and luteinizing hormone) and induced ovulation in prepubertal female rats that had been pretreated with pregnant mare serum gonadotropin to induce follicle maturation. Furthermore, metastin administration drastically increased the plasma levels of gonadotropins in male rats. This action was abolished by pretreatment with a GnRH antagonist, and was accompanied by induction of c-Fos immunoreactivity in GnRH neurons. These results suggest that s.c. administered metastin induces the release of gonadotropin via activation of the hypothalamic GnRH neurons.

Effects of intracerebroventricular ethanol on ingestive behavior and induction of c-Fos immunoreactivity in selected brain regions

The early changes in the central nervous system (CNS) following drinking of ethanol (ETOH) are poorly understood. It is known that chronic intracerebroventricular (ICV) administration of ethanol to rats induces preference for imbibed alcohol solutions. These results suggest that ICV ethanol could alter taste preference. In the present study, we tested whether ETOH{ICV} could induce a conditioned taste preference (CTP) or aversion (CTA) and alter c-Fos immunoreactivity (c-Fos-IR) in brain regions associated with feeding, aversion, and/or reward. Acute ETOH{ICV}, as tested in the ETOH-naı̈ve rat, did not induce CTA nor affect the amount of water imbibed by treated rats. The effects of ETOH{ICV} on intake and preference were determined using a novel palatable (i.e. sweet) noncaloric 0.1% saccharin solution. A single dose of ETOH{ICV} in the ETOH-naı̈ve animal induced a CTP for saccharin. ETOH{ICV} significantly increased c-Fos-IR in a number of brain sites associated with feeding and reward including the bed nucleus of the stria terminalis, lateral dorsal area (BSTLD); nucleus accumbens, shell area (AcbSh); hypothalamic paraventricular nucleus (PVN); and lateral septum, ventral area (LSV). Thus, ETOH induced a CTP, not CTA, via central mechanisms; it increased c-Fos-IR in specific sites associated with feeding and reward.

Application of Bicuculline to the Gerbil Somatosensory Cortex Induces Repetitive Ear Movements Accompanied by Induction of c-Fos Immunoreactivity in the Thalamus.

We previously established a seizure-sensitive strain of Mongolian gerbil, MGS/Idr, in which seizure-inducing stimuli often elicit repetitive backward jerks of the ears at the beginning of seizures, and found that unilateral application of (−)-bicuculline methiodide (BMI) to an area of the somatosensory cortex in this strain induced similar, but long-lasting, repetitive movements of the contralateral ear. In the present study, we have extended this observation by using immunohistochemistry to look for areas of the brain in which c-Fos protein, an immediate early gene product, appeared unilaterally following BMI application. At 4 hr after BMI application, the cerebral cortex under the application site showed intense c-Fos immunostaining. In addition, c-Fos immunoreactivity appeared in restricted areas of the ipsilateral thalamus, including the ventroposterior nucleus, posterior nucleus, and reticular nucleus. Control saline-treated gerbils did not show unilateral staining in the corresponding thalamic areas. These areas may form a network with the somatosensory cortex that is involved in the recurrent phenomena, as proposed for other areas of the cerebral cortex.

C-FOS expression in the rat brain in response to substance P and neurokinin B

Substance P, the principal neurokinin peptide in the mammalian brain and the natural ligand for the NK 1 tachykinin receptor, plays an integrative role in the regulation of cardiovascular, neuroendocrine and behavioural responses to stress. In rats, stimulation of periventricular NK 1 receptors in the forebrain induces a distinct pattern of cardiovascular responses which is accompanied by intense grooming behaviour. Ligands for NK 3 receptors induce a different pattern of cardiovascular and behavioural responses which comprises an increased release of vasopressin from the posterior pituitary and wet-dog shakes behaviour. To define the brain areas in the rat which respond to stimulation of forebrain NK 1 and NK 3 receptors and participate in the generation of these responses, the induction of c-Fos immunoreactivity was examined in brains following intracerebroventricular injections of substance P and neurokinin B in conscious rats. Stimulation of central NK 1 receptors by substance P (25, 100 and 500 pmol) injected into the lateral ventricle elicited grooming behaviour (face washing and hind limb grooming) and resulted in a marked c-Fos expression in the paraventricular, dorsomedial and parabrachial nuclei and in the medial thalamus. At 25 pmol, substance P did not significantly increase c-Fos expression, at 100 pmol, maximal c-Fos activation was induced in all four brain regions which responded to the peptide. Intracerebroventricular pretreatment of rats with the selective and high-affinity, non-peptide NK 1 receptor antagonist, RP 67580 (500 pmol), but not with its inactive enantiomer, RP 68651, completely abolished the behavioural response to substance P and reduced the substance P-induced c-Fos expression in all brain areas to nearly control levels. Intracerebroventricular injection of the natural ligand for NK 3 receptors, neurokinin B (500 pmol), elicited wet-dog shakes behaviour and activated c-Fos expression in localized regions of the forebrain including the organum vasculosum laminae terminalis, subfornical organ, median preoptic nucleus, paraventricular, supraoptic and anterior hypothalamic nuclei, medial thalamus and in the ventral tegmental area. These results demonstrate that the neurokinins, substance P and neurokinin B, induce specific and different patterns of c-Fos expression in distinct regions of the rat brain. Brain areas which selectively responded to substance P have been traditionally linked to the central regulation of cardiovascular and neuroendocrine reactions to stress or involved in the processing of nociceptive responses. On the other side, brain areas activated by neurokinin B are known to be involved in the central regulation of blood pressure, water and salt homeostasis or control of behaviour.

Abdominal vagi mediate c-Fos expression induced by X-ray irradiation in the nucleus tractus solitarii of the rat

The mechanism of induction of emesis by X-ray irradiation remains largely unknown. The purpose of the present research was to clarify the neuronal basis of the induction of nausea induced by X-ray irradiation analyzing c-Fos expression in the nucleus tractus solitarii (NTS) as a marker of cellular excitation. We confirmed that the dose of X-ray irradiation (4 Gy) used for the present research could actually induce nausea by preliminary measurement of kaolin intake. Induction of c-Fos immunoreactivity in the NTS was observed in the animals that received X-ray irradiation of the whole body. The mean number of c-Fos positive cells in the animals that received irradiation was significantly larger than that in the non-irradiated animals. Partial exposure of the abdomen to X-rays showed significantly greater c-Fos expression than that of the head. These results indicated the presence of a certain route for transmitting information from the periphery toward the central nervous system by X-ray irradiation. The number of c-Fos positive cells induced by X-ray irradiation in animals vagotomized at the subdiaphragmatic level was lower than that in sham-operated animals. Animals receiving a serotonin subtype three (5-HT 3, 5-hydroxytryptamine) receptor antagonist (tropisetron, ICS 205-930, 3-tropanyl-indole-3-carboxylate) showed a significant reduction in c-Fos protein expression compared to animals receiving a vehicle. These results strongly suggested that X-ray irradiation activates 5-HT 3 receptors on the terminals of the abdominal vagal nerves to excite the afferent pathway, thereby inducing emesis.

Estradiol induces a phasic Fos response in the hippocampal CA1 and CA3 regions of adult female rats.

Previous studies have shown that estradiol induces structural and functional changes in hippocampal CA1 pyramidal cells of the adult female rat. Estradiol increases the density of dendritic spines and axospinous synapses on CA1 pyramidal cells, and increases these cells' sensitivity to NMDA receptor-mediated synaptic input. Curiously, while estradiol effects are observed in CA1 pyramidal cells, the majority of the evidence indicates that these cells lack genomic estradiol receptors. In contrast, genomic estradiol receptors are expressed in at least some hippocampal interneurons in CA1. The goal of the present study was to determine which hippocampal neuronal populations are activated by estradiol, as determined by induction of c-Fos immunoreactivity, as well as the time-course of this activation. We quantified c-Fos expression in each of the major subdivisions of the hippocampus in adult female rats at various time points during the same estradiol treatment regimen known to regulate dendritic spines and synapses on CA1 pyramidal cells. Our results show a phasic estradiol-induced c-Fos response in the pyramidal cell layers of both CA1 and CA3. c-Fos was induced within 2 h of treatment, decreased at 6 and 12 h, and subsequently increased again at 24 h after treatment with estradiol. Double labeling for c-Fos and GAD 65 or GAD 67 suggests that c-Fos is induced primarily in principal cells, though a small proportion of GABAergic cells is also labeled. These estradiol-induced changes in c-Fos expression may reflect phasic neuronal activation and coupling to gene expression, which could be involved in estradiol's effects on excitatory synaptic connectivity in the hippocampus.

Differential effects of haloperidol and two anxiolytic drugs, buspirone and lesopitron, on c-Fos expression in the rat striatum and nucleus accumbens

We have studied the effects of the neuroleptic haloperidol and the non-benzodiazepine anxiolytics buspirone and lesopitron on the expression of c-Fos immunoreactivity in the rat forebrain. Haloperidol and buspirone administration resulted in a significant quantitative increase in the number of Fos-immunoreactive neurons in the lateral striatum and a presumable qualitative increase in the nucleus accumbens. In contrast, lesopitron did not lead to a significant increase in the c-Fos expression in the striatum. The induction of c-Fos immunoreactivity by buspirone is compatible with an interaction of this compound with D2 dopamine receptors, as documented for haloperidol. The lack of effects after lesopitron administration suggests that, in contrast with buspirone, this compound has no dopaminergic blocking activity.

Effect of central and continuous intravenous injection of interleukin-1 beta on brain c-fos expression in the rat: involvement of prostaglandins.

Utilizing immunohistochemistry for the c-fos protein to detect neuronal activity, we examined the effects of continuous intravenous and intracerebroventricular infusion of interleukin (IL)-1 beta in the rat brain, and the involvement of prostaglandins (PGs) in IL-1 beta-induced c-fos expression. Continuous intravenous infusion of IL-1 beta (10 ng/min) markedly augmented c-fos expression in the paraventricular (PVN) and the supraoptic (SON) nuclei of the hypothalamus as well as in the central amygdaloid nucleus (CeA). The number of IL-1 beta-induced c-fos-positive cells in the PVN and SON was significantly lower in rats pretreated with indomethacin than in vehicle-treated rats. However, the number of IL-1 beta-induced c-fos-positive cells in the CeA remained unchanged. c-fos protein was induced after intracerebroventricular infusion of IL-1 beta (200 ng) in the PVN, SON, and arcuate nuclei of the hypothalamus, and in the CeA. The induction of c-fos immunoreactivity by central administration of IL-1 beta was blocked by indomethacin (500 micrograms/rat), except in the CeA. These findings suggest that PGs are involved in the complex transmission of signals from circulating or central IL-1 beta to hypothalamic neurons.

Seizure-induced c-fos expression in rat medulla oblongata is not dependent on associated elevation of blood pressure

This study was performed to determine whether c-fos immunoreactivity (IR) induced in medulla oblongata by pentylenetetrazole seizures is a consequence of seizure-associated blood pressure elevation and activation of baroreceptor afferent pathways, or occurs independently of hypertension. Immunohistochemical study of sections of medulla oblongata revealed that seizures are followed by induction of c-fos IR in nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMN 10), and ventrolateral medulla (VLM), while there is negligible c-fos IR after saline sham injections. Seizures were associated with blood pressure elevation peaking at 31 ± 17% (±SD) above baseline. Experimental hypertension at a similar level induced by i.p. phenylephrine also resulted in induction of c-fos IR in NTS. When seizures were preceded by antihypertensive treatment with the α-adrenergic antagonist, phentolamine, peak blood pressure tended to remain near the baseline level and lower than sham-injected controls. Normotensive seizures were associated with c-fos IR in NTS, DMN 10, and VLM similar to the pattern following hypertensive seizures. Seizure-induced activation of c-fos IR occurred despite normal blood pressure, and thus can be attributed to a direct effect of the seizure, and not to an indirect effect mediated by hypertension.

Activation of brainstem catecholaminergic neurons by conditioned and unconditioned aversive stimuli as revealed by c-Fos immunoreactivity

In an attempt to define areas of the brain that respond to stressors and influence immune function, we have previously identified stress-induced, c-Fos-immunoreactive areas of the diencephalon. We found that c-Fos was strongly expressed in cells of the paraventricular nuclei (some of which contain corticotropin-releasing hormone (CRH) and other hypothalamic areas directly associated with autonomic function. To further characterize the presumptive pathways mediating stress-induced immune alterations, including the assessment of brainstem catecholaminergic neuron involvement, the induction of c-Fos immunoreactivity was examined in the brainstems of rats exposed to conditioned and unconditioned, immunomodulating stimuli. In response to electric footshock (the unconditioned stimulus (US)), c-Fos immunoreactivity was strongly induced in the noradrenergic neurons of the locus ceruleus (A6), the nucleus of the solitary tract (A2/C2), the ventral lateral medulla (A1/C1), A5, and A7, as well as in unidentified neurons of the dorsal and ventral subdivisions of the periqueductal gray (PAG), and in the serotonergic neurons of the dorsal raphe nuclei. Conditioned animals re-exposed to the conditioned stimulus showed c-Fos induction in these same areas but to a lesser degree. Control animals exposed only to the conditioning stimulus (CS) (electronic tone) in the absence of the US, expressed very little, if any, c-Fos activity in the above loci except for a small degree of baseline expression in the PAG. These results further confirm the role of autonomic and endocrine pathways as mediators of the stress response and will help to more fully characterize the pathways of stress-induced immune alteration.

Induction of c-FOS immunoreactivity in the hippocampus following potassium stimulation

In microdialysis procedures high potassium ion concentrations are generally used to induce neurotransmitter release. However, the widespread effects, if any, of such a treatment have not been described. In order to establish a possible link between c-mfos expression and stimulating conditions for neurotransmitter release in microdialysis procedures we administered KCl (100mM) into the hippocampus. Proto-oncogene c-FOS-like immunoreactivity is upregulated in granule cells of the dentate gyrus, pyramidal cells of the hippocampus, cingulate, piriform and frontoparietal cortices at 2 h, but not 24 h after K + administration. Neither implantation of the probes nor perfusion with artificial cerebrospinal fluid resulted in similar patterns of c-FOS immunoreactivity. In addition, we investigated whether the impairment of the cholinergic septohippocampal pathway would modify the K +-induced expression of the immediate early gene c-fos in the hippocampus. The expression of c-fos induced by KCl was not altered in the animals with fimbria-fornix lesion despite the marked decrease in acetylcholine release in the hippocampus. Glutamate concentrations measured in the same superfusates showed that a significantly greater glutamate release occurs in denervated hippocampi. Furthermore, abolishment of seizure-like activity (induced by KCl) in anaesthetized animals did not alter expression of c-FOS immunoreactivity in the K +-stimulated hippocampi. The results from these studies confirm that most of the releasable acetylcholine of the hippocampus is linked to the fimbrial input and may suggest that c-FOS upregulation in this model does not respond to any cholinergic input from the medial septum via the fimbria-fornix.

Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli

The protein product of the c- fos proto-oncogene was immunocytochemically localized in forebrain regions of adult male Lewis rats subjected to a physically aversive footshock stimulus or a Pavlovian-conditioned, non-aversive, auditory stimulus. Animals receiving the conditioned stimulus were first conditioned by repeatedly pairing electric footshock, the unconditioned stimulus (US), with an auditory cue, the conditioned stimulus (CS). These animals were later tested with the CS in the absence of the US, a procedure which, like footshock itself, suppresses immune function. In animals exposed to the conditioned or unconditioned stressor, c-Fos was strongly expressed in cells of the paraventricular nuclei (PVN) of the hypothalamus, some of which contain corticotropin-releasing hormone (CRH), and other forebrain areas directly associated with autonomic function, the ventral lateal septal nuclei (LSV), the medial amygdaloid nuclei (AME), the sensorimotor cortex, the basal ganglia and thalamic nuclei. Control animals exhibited very little or no c-Fos in the above areas. The identified forebrain nuclei can now be targeted for further study aimed at elucidating their role in stress-induced immune alteration.

Neuroprotective effects of L-kynurenine on hypoxia-ischemia and NMDA lesions in neonatal rats.

Kynurenic acid is the only known endogenous excitatory amino acid receptor antagonist in the central nervous system. In the present study, we examined whether increasing brain concentrations of kynurenic acid by loading with its precursor L-kynurenine, or blocking its excretion with probenecid, could exert neuroprotective effects. Neuroprotective effects were examined in a neonatal model of hypoxia-ischemia, and following intrastriatal injection of N-methyl-D-aspartate (NMDA). Seven-day-old rats underwent unilateral ligation of the carotid artery, followed by exposure to 8% oxygen for 1.5 h. L-kynurenine administered 1 h before the hypoxia-ischemia showed a dose-dependent significant neuroprotective effect, with complete protection at a dose of 300 mg kg-1. The induction of c-fos immunoreactivity in cerebral cortex was also blocked by this dose of L-kynurenine. Probenecid alone had moderate neuroprotective effects, while a combination of a low dose of probenecid with doses of 50-200 mg kg-1 of L-kynurenine showed significant dose-dependent neuroprotection. Kynurenine dose-dependently protected against NMDA neurotoxicity in 7-day-old rats. Neurochemical analysis confirmed that L-kynurenine with or without probenecid markedly increased concentrations of kynurenic acid in cerebral cortex of 7-day-old rats. These results show for the first time that pharmacologic manipulation of endogenous concentrations of kynurenic acid can exert neuroprotective effects.