Massive activation of c-fos in forebrain after mechanical stimulation of the locus coeruleus

Brief implantation of a 33-ga cannula in the locus coeruleus (LC) of the rat caused widespread and intense ipsilateral activation of c-fos throughout the forebrain. Areas showing heavy staining included the cingulate, piriform, parietal, frontal cortex, and the olfactory tubercle. Prior lesion of the LC with 6-hydroxydopamine (6-OHDA) abolished the response. It is concluded that the mechanical stimulation and/or trauma involved in the implantation of a cannula in the LC is sufficient to cause widespread activation of noradrenergic neurotransmission throughout the forebrain. The use of this procedure for drug delivery should therefore be reevaluated.     

Acoustically activated c-fos expression in auditory nuclei of the anaesthetised guinea pig

The spatial expression of the immediate-early gene c-fos in central auditory nuclei of the anaesthetised guinea pig was investigated following exposure of the animal to acoustic stimulation. Accurate control of both the spectra and the level of the stimulus was designed so that the presumed excitation of central auditory nuclei was similar across animals. For unstimulated anaesthetised control animals, levels of labelling were significantly higher when compared with unanaesthetised controls. This appeared to be a result of the combination of the experimental manipulations and also the use of the anaesthetic. A surprising finding was that unstimulated control animals placed in an anechoic chamber demonstrated the highest levels of fos-like immunoreactivity (Fos-LIR). When anaesthetised animals were exposed to acoustic stimuli the total number of cells showing Fos-LIR was elevated when compared to anaesthetised, but unstimulated animals. There was no evidence at any level of the auditory pathway that these animals demonstrated spatially restricted Fos-LIR which may have suggested place-frequency mapping. In contrast, spatially restricted labelling was found in awake animals exposed to an identical stimulus.     

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.     

Different populations of cells in the suprachiasmatic nuclei express c-fos in association with light-induced phase delays and advances of the free-running activity rhythm in hamsters

Circadian rhythmicity is controlled by a light-entrainable pacemaker located in the suprachiasmatic nuclei (SCN) of the mammalian hypothalamus. Brief light exposure during the subjective night causes phase shifts of the free-running activity rhythm and expression of c-fos-related proteins (Fos) among a population of cells in the hamster SCN. Light exposure (30 lux for 15 min) during the early subjective night (CT13) causes phase delays (-60 +/- 12 min), while exposure at mid-subjective night (CT18) causes phase advances (114 +/- 48 min) of the free-running activity rhythm. Light exposure at mid-subjective day (CT6) does not cause phase alterations of the rhythm. Similarly, only light exposure at CT13 or CT18 induces Fos expression in the SCN. The distribution of Fos-immunoreactive cells in the SCN is more widespread in animals stimulated with light at CT18. In addition, a group of cells located dorsal and anterior to the SCN express Fos only after stimulation at CT18. The data are consistent with the hypothesis that Fos expression represents an event in the signal transduction pathway leading to light-induced alterations in circadian pacemaker function. Furthermore, the data raise the possibility that different populations of cells in the suprachiasmatic hypothalamus may participate in light-induced phase advances and delays of the circadian pacemaker.     

Stimulant-mediated c-fos induction in striatum as a function of age, sex, and prenatal cocaine exposure

Induction of the immediate-early gene c-fos by the stimulants cocaine and amphetamine (AMPH) was analyzed by Fos immunocytochemistry at different ages in the brains of prenatally cocaine-treated and control rats. Cocaine and AMPH induced c-fos in patches of striatal neurons during the first postnatal week, and thereafter produced a progressively more homogeneous pattern that was more dense medially. Quantification of Fos-immunoreactive cells in older rats revealed differences related to sex and prenatal cocaine treatment. Both cocaine and AMPH produced dose-dependent increases in the number of Fos-immunoreactive cells in striatum. Prenatal cocaine exposure resulted in increased Fos in males in response to AMPH (2 mg/kg) at P18 and cocaine (10 mg/kg) at 1–2 months. In females, prenatal cocaine treatment resulted in a reduced response to cocaine at 1–2 months. Increased c-fos induction was observed in control females compared to control males in response to low doses of stimulants; no such sex difference was observed in prenatally cocaine-treated rats. The dopamine D1 antagonist SCH23390 blocked cocaine-mediated c-fos induction in all groups. The NMDA antagonist MK-801 blocked cocaine-mediated c-fos induction in the medial striatum. In females only, MK-801 pretreatment resulted in a dramatic increase in the number of Fos-immunoreactive cells in lateral striatum. These findings indicate differences in the neural basis of c-fos induction in males and females, and changes in stimulant-mediated c-fos induction resulting from prenatal cocaine exposure.     

Increased c-fos expression in spinal neurons induced by electrical stimulation of the ureter in the rat

The spinal processing of afferent input from the ureter was examined using an immunocytochemical technique to detect the expression of c-fos, an immediate early gene. Proximal and distal sites in one ureter were electrically stimulated separately or together at intensities that elicited a pseudo-affective response (an increase in arterial pressure). Very few Fos + cells (range: 0.6–6.6 cells/half section were present in the L₁-L₂, L₅-S₂ spinal segments in sham operated control animals; however, following stimulation of the ureter, a significant increase in the numbers of Fos + cells was detected at spinal levels L₁-L₂ (mean 24.5–33.1 cells/half section) and L₆-S₁ (mean 17.4–33.0 cells/half section). In L₆-S₁,the numbers of Fos + cells were significantly greater ipsilateral (mean 25.2 cells/half section) vs. contralateral (12.3 cells/half section) to stimulation; whereas in L₁-L₂, the numbers were similar on both sides of the spinal cord. In L₁-L₂, a greater percentage of Fos + cells was present in superficial medial (MDH, 49.7%) and lateral dorsal hom (LDH, 40.8%); whereas in L₆-S₁, the cells were more numerous in sacral parasympathetic nucleus (SPN, 38.7%) and LDH (25.6%) regions. This distribution of Fos + cells varies in a number of respects from that noted in previous experiments after chemical irritation of the urinary bladder and urethra which activated neurons only in L₆-S₁ and primarily in the DCM and MDH. The results indicate that nociceptive afferent inputs from different areas of the urinary tract are processed in different regions of the spinal cord.     

Neurons in the superficial dorsal horn of the rat spinal cord projecting to the medullary ventrolateral reticular formation express c-fos after noxious stimulation of the skin

The nociceptive nature of the neurons of the superficial dorsal horn (laminae I–III) which project to the medullary ventrolateral reticular formation is studied in the rat. Medullary injections of Fluoro-Gold showed exclusive retrograde labeling of laminae I–III cells when the tracer filled a zone intermediate between the lateral tip of the lateral reticular nucleus and the spinal trigeminal nucleus, pars caudalis. This zone is here called VLMlat. Following noxious mechanical or thermal stimulation of the skin, double-labeled neurons, which stained retrogradely and were Fos-immunoreactive, prevailed in laminae I and IIo. Double-labeled neurons were few in lamina IIi after thermal stimulation and entirely lacking in lamina III after the two kinds of stimulation. Findings in lamina I confirm previous electrophysiological data (see Menétrey et al.,J. Neurophysiol., 52 (1984) 595–611) showing that lamina I cells projecting to the ventrolateral reticular medulla convey noxious messages. The occurrence of numerous double-labeled cells in lamina IIo suggests that this lamina is also involved in nociceptive transmission to the VLMlat.     

Effects of hypoxia-ischemia and seizures on neuronal and glial-like c-fos protein levels in the infant rat

Unilateral carotid ligation in immature rats, followed by 2 h of hypoxia led to ischemic cell change from 2 h after the insult, on the ligated side of the brain. There was a time-dependent induction of immunoreactive c-fos protein in neurones but not glia or ependyma on the non-ligated side of the brain. Induction only occurred in rats that had seizures post hypoxia-ischemia. In the ligated hemisphere c-fos protein was induced in glial-like cells in the corpus callosum, fornix/fimbria and internal capsule and in ependymal cells lining the lateral ventricle starting from 2 h after hypoxia but subsiding by 3 days. No neuronal c-fos induction was seen in areas showing neuronal damage. MK-801 or carbamazepine, which prevented hypoxia-ischemia-induced seizures, also prevented c-fos induction in the non-ligated hemisphere while MK-801 was associated with increased c-fos induction in hippocampal neurones from the ligated side, as well as in glial-like and ependymal cells.These results suggest several processes are involved following the hypoxic-ischemic insult. Firstly, severe hypoxia-ischemia is associated with a reduction in neuronal c-fos protein levels, probably as a result of neuronal failure and death. Secondly, post hypoxic seizures cause c-fos induction in surviving neurones. Thirdly, glial-like from regions in which there is neural loss also exhibit induction of c-fos, which may be important for their subsequent proliferation or for the production of growth factors.     

Expression of c-fos and hsp70 mRNA in neonatal rat cerebrocortical slices during NMDA-induced necrosis and apoptosis

Respiring neonatal rat cerebrocortical slices were exposed for 30 min to toxic concentrations of N-methyl-d-aspartate (NMDA; 100 μM, 500 μM and 1000 μM). In situ hybridization was used to study c-fos and hsp70 mRNA before, during, and for 8 h after NMDA exposure. Cell swelling and nuclear morphology were assessed using Cresyl violet (Nissl) staining. Possible evidence for apoptosis was examined using in situ terminal transferase d-UTP nick-end labeling (TUNEL) staining and agarose–gel electrophoresis of extracted slice DNA. After NMDA administration c-fos and hsp70 mRNA expression increased, with maxima occurring, respectively, at 1 h and 4 h after NMDA exposure. When treatment with dizocilpine (MK-801; 10 μM), a non-competitive NMDA antagonist, was started before NMDA exposures, expression of both c-fos and hsp70 mRNA was decreased to values near control, indicating that activation of NMDA receptors induces both genes. Only a minority of induced cells expressed FOS protein and no HSP70 protein expression was seen. These apparent failures of translation might be related to the stress response. Histologically, 1000 μM NMDA produced substantial necrosis, with no evidence of apoptosis. Evidence for apoptosis was found at the two lower NMDA concentrations, which produced TUNEL-positive fragmented nuclei and faint ladder patterns in DNA electrophoresis. Dizocilpine pre-treatment blocked NMDA-induced necrosis and attenuated TUNEL-positive staining in slice parenchyma. TUNEL-positive staining with a different morphology was found in the injury layer, a region 50-μm thick where mechanical trauma was inflicted when slices were cut from brain. When slices received dizocilpine immediately after decapitation, TUNEL-positive staining no longer occurred in the injury layer, in agreement with previous cell culture studies that implicated NMDA receptor activation after mechanical trauma to neurons. We conclude that at the toxic doses studied, NMDA receptor activation results primarily in necrosis. However, data at low NMDA concentrations are consistent with a small amount of apoptosis.     

A comparison of Δ-THC and anandamide induced c-fos expression in the rat forebrain

Rats were injected with the cannabinoid receptor agonists Δ⁹-THC (5 mg/kg) or anandamide (20 mg/kg) and assessed for changes in body temperature and locomotor activity. Their brains were then examined for the expression of the immediate early gene c-fos. Similar reductions in body temperature and locomotor activity were seen with Δ⁹-THC and anandamide although there was evidence, in line with previous reports, to suggest a shorter duration of action of anandamide. Δ⁹-THC and anandamide caused equally high levels of c-fos expression in the paraventricular nucleus of the hypothalamus and the lateral septum. Both drugs also increased c-fos expression in the central nucleus of the amygdala although the effect was greater with Δ⁹-THC. Only Δ⁹-THC caused significant increases in c-fos expression in the nucleus accumbens and caudate-putamen. These differences may be linked to differential activation of cannabinoid receptor subtypes or to differences in efficacy in activating second messenger systems linked to cannabinoid receptors. These findings complement evidence of qualitative differences in the actions of anandamide and Δ⁹-THC emerging from tests of drug discrimination, cross-tolerance, conditioned place preference and anxiety.     

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.     

Combined c-fos and C-2-deoxyglucose method to differentiate site-specific excitation from disinhibition: analysis of maternal behavior in the rat

On the basis of evidence that ¹⁴C-2-deoxyglucose (2-DG) autoradiography indicates activity at axonal terminals, whereas c-fos immunocytochemistry indicates activity of neuronal cell bodies, we combined these techniques in adjacent histological brain sections to assess excitatory and disinhibitory synaptic relations in selected sites in female rats in which maternal behavior was elicited by natural parturition, sensitization (7- to 10-day cohabitation with foster pups), or hysterectomy. All individuals in these three groups expressed maternal behavior immediately before 2-DG injection. Controls were non-maternal virgins. Parturient and Hysterectomized groups: elevation (compared with controls) in both 2-DG and c-fos activity in medial preoptic area (MPOA) indicated an increase in its input and output activity, i.e., an excitatory interaction; the MPOA was previously shown to be critical for maternal behavior. Sensitized group: a decrease in 2-DG activity of vomeronasal nuclei (bed nucleus of the accessory olfactory tract, BAOT, and medial amygdala, ME, replicating our previous study) and an elevation in c-fos activity, jointly indicate disinhibition of these nuclei, that were previously shown to modulate pup-chemostimulation-induced sensitization. All other sites showed evidence of excitatory input–output relationships (i.e., joint increase in both 2-DG and c-fos activity), e.g., bed nucleus of the stria terminalis (BNST), lateral habenula (LHAB), central gray (CG), thalamus (THAL), septum (SEPT), and ventral tegmental area (VTA). The present study demonstrates the feasibility of measuring 2-DG and c-fos activity jointly in adjacent sections of the same brain, thereby providing evidence to distinguish between localized excitation and disinhibition.     

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.     

Effects of intracerebroventricular dizocilpine (MK801) on dehydration-induced dipsogenic responses, plasma vasopressin and c-fos expression in the rat forebrain

This study determined the interaction between glutamate receptors and dehydration-induced drinking, vasopressin (AVP) release, plasma osmolality and c-fos expression in the brain of conscious rats. The NMDA receptor antagonist dizocilpine (100 nmol infused into the cerebral ventricles) suppressed drinking following either 22 h water deprivation or intragastric injection of hypertonic saline (1.5 M), attenuated the increased plasma vasopressin induced by dehydration, but had no effects on peripheral hyperosmolality caused by either water deprivation or injections of hypertonic saline. Dizocilpine had no inhibitory effects on feeding after 24 h food deprivation. Dizocilpine also suppressed c-fos expression induced by dehydration in the median preoptic nucleus (MPN), the supraoptic and paraventricular nuclei (SON and PVN), but did not influence c-fos expression in the subfornical organ (SFO). The non-NMDA receptor antagonists CNQX (400 nmol) or DNQX (60 nmol) affected neither the animals' drinking nor c-fos expression induced by dehydration. Double staining showed that suppression of c-fos expression following dizocilpine occurred in the NMDA R1 receptor containing neurons in the hypothalamus. These results suggest that the NMDA-type glutamate receptors may be involved in dehydration induced dipsogenic and neuroendocrinological responses. They complement our earlier findings that dizocilpine also attenuated drinking and c-fos expression following intraventricular infusions of angiotensin II.     

Environmental context modulates the ability of cocaine and amphetamine to induce c-fos mRNA expression in the neocortex, caudate nucleus, and nucleus accumbens

We reported previously that environmental novelty enhances the acute psychomotor activating effects of amphetamine, its ability to induce behavioral sensitization, and its ability to induce c-fos mRNA in the striatum and other structures, relative to when amphetamine is given in the home cage. The purpose of the present experiment was 2-fold: to determine (1) whether environmental novelty has a similar effect on the ability of cocaine to induce c-fos mRNA, and (2) whether this effect is seen in neurologically-intact rats (in previous experiments we studied the intact hemisphere of rats with a unilateral 6-OHDA lesion). In the dorsal portion of the caudate putamen, core and shell of the nucleus accumbens, and in several cortical regions, both amphetamine (1.5 mg/kg) and cocaine (15 mg/kg) induced higher levels of c-fos mRNA expression when administered in a novel environment, relative to when they were administered in the home cage. The ability of environmental context to modulate psychostimulant drug-induced immediate early gene expression may be related to its ability to modulate forms of drug experience-dependent plasticity, such as behavioral sensitization.     

c-fos Protooncogene expression in rat hippocampus and entorhinal cortex following tetanic stimulation of the perforant path

The elevated expression of the c-fos protooncogene has been proposed to be a marker of cell activation leading to a long term cellular response. In this communication we compared the c-fos mRNA accumulation in the hippocampus (i.e. postsynaptic cells) and entorhinal cortex (i.e. presynaptic cells) following high (tetanic) and low frequency electrical stimulation of the perforant path. Using Northern blot analysis we have found that high frequency stimulation elevates c-fos expression in both hippocampus and entorhinal cortex, and the increase of c-fos mRNA levels in the entorhinal cortex is less pronounced, but longer lasting, than in the hippocampus. Slight increase of c-fos mRNA levels has been also observed in low frequency treated animals in the entorhinal cortex, but not in the hippocampus. These findings raise the question about differences in mechanisms involved in c-fos activation in both parts of the brain after stimulation which evokes long term potentiation (LTP) of synaptic efficacy.     

Dopamine agonists and stress produce different patterns of Fos-like immunoreactivity in the lateral habenula

In rats treated systemically with either amphetamine, amfonelic acid or apomorphine, large numbers of cells displaying Fos-like immunoreactivity (FLI) could be seen in the lateral zone of the lateral habenula. The induction of FLI by amphetamine could be blocked either by pretreatment with haloperidol or by 6-hydroxydopamine lesions of ascending dopamine fibers at the level of the lateral hypothalamus. In contrast, a variety of stressors selectively induced FLI in the most medial portion of the lateral habenula. These findings support the concept of a functional differentiation of the medial and lateral regions of the lateral habenula and provide further evidence for involvement of the habenula in the circuitry of the basal ganglia.