Pattern Of C-fos mRNA Expression Research Articles

Reduced stress responsiveness in pregnancy: Relationship with pattern of forebrain c-fos mRNA expression

Stress-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis is known to be attenuated during late pregnancy and throughout lactation. To investigate the neural basis of this stress hyporesponsiveness we examined the changes in the restraint-induced HPA response and accompanying forebrain expression of c-fos mRNA that occur in rats between days 16 (D16) and 19 (D19) of gestation, times associated with declining levels of progesterone, a potential mediating factor. Compared to D16, the D19 group showed a significantly attenuated release of ACTH following 30min restraint. This reduced HPA response was accompanied by significantly lower levels of restraint-induced c-fos mRNA expression in the hypothalamic paraventricular nucleus. Other areas of the forebrain, including medial amygdala, piriform cortex, and ventrolateral septum, showed low c-fos mRNA expression in non-stressed (control) animals and a large increase following restraint, the magnitude of which was similar between D16 and D19 animals indicating no involvement in the differential HPA response to stress. However, a markedly different pattern of c-fos mRNA expression was observed in other brain areas, including barrel cortex and CA1 ventral and CA3 regions of the ventral hippocampus: D19 animals had low control expression which was increased by restraint, but D16 control animals had raised c-fos mRNA expression which was not further elevated by stress. These data demonstrate that region-specific changes in basal and stress-induced cellular activity occur during a period of late gestation coincident with attenuated HPA responsiveness. These changes in neuronal activity may contribute to the adaptive processes that prepare the mother for parturition and lactation.

Differential patterns of c-fos mRNA expression in the amygdaloid nuclei induced by chemical somatic and visceral noxious stimuli in rats

Pain includes a negative affective component, although the neural system is unclear. The amygdala including the lateral (La), basolateral (BL) and central (Ce) nuclei is thought to play a key role in emotional responses. In this study, we analyzed the c-fos mRNA expression, as a marker of neuronal activity, induced by two types of pain, chemical somatic and visceral noxious stimuli, in each amygdaloid nucleus in unanesthetized rats. We found that intraplantar injection of formalin as a chemical somatic noxious stimulus increased c-fos mRNA expression in the La and BL, but not Ce. On the other hand, intraperitoneal injection of acetic acid as a chemical visceral noxious stimulus induced it highly in the Ce, moderately in La and hardly in BL. These results suggest that distinct amygdaloid nuclei are activated by chemical somatic and visceral noxious stimuli, which might differentially contribute to emotional responses by them.

Distinct pattern of c-fos mRNA expression after systemic and intra-accumbens amphetamine and MK-801

Pharmacological manipulation of both dopamine and glutamate systems affects motor responses in laboratory animals. The two systems, however, seem to act in opposite ways, since direct or indirect activation of dopamine receptors induces similar stimulatory effects to those seen following blockade of N-methyl- D-aspartate receptors. In the present study we compared the pattern of c-fos activation induced by systemic and intra-accumbens administration of the non-competitive N-methyl- D-aspartate antagonist MK-801 and the indirect dopamine agonist amphetamine. Systemic MK-801 induced c-fos mRNA expression in the motor cortex and preferentially in the motor thalamus, i.e. ventrolateral nucleus. Systemic amphetamine, on the other hand, enhanced c-fos mRNA expression in the shell of the accumbens and in limbic thalamic nuclei such as the anteroventral and anterodorsal nuclei. The main effect observed after intra-accumbens administrations of either drug was enhanced c-fos expression in the thalamus, somewhat similar to what seen following systemic administration. In fact also in this case there was a preferential activation of the limbic thalamus by amphetamine and the motor thalamus by MK-801. The present results confirm that different neural substrates underlie behavioral effects induced by systemic administrations of N-methyl- D-aspartate receptor antagonists and dopamine agonists. Further they suggest that intra-accumbens manipulation of the two neural systems could affect different efferent pathways from this structure activating different thalamic targets.

C-fos mRNA expression associated with PGF 2α-induced nest-building behaviour in female pigs

Domestic pigs, Sus scrofa, build a maternal nest on the day before parturition. A model for nest building has been established in pigs, in which exogenously administered prostaglandin (PG) F 2α may be used to elicit nesting behaviour in cyclic, pseudopregnant and pregnant pigs. The central mechanisms mediating this response are unknown. The present study determined regional brain activity using semi-quantitative analysis of c-fos mRNA, after induction of nest-building behaviour by PGF 2α in Large White pseudopregnant pigs. Oestradiol valerate injections (5 mg/day) were given on days 11–15 of the oestrous cycle to induce pseudopregnancy. The pigs were housed individually in pens (2.8×1.7 m) containing straw. On the test day (day 46 or 47 of pseudopregnancy) animals were injected with 3 ml saline ( n=5) or 15 mg of PGF 2α (Lutalyse, Upjohn; n=6) intramuscularly. Pigs treated with PGF 2α, but not saline, displayed bouts of rooting, pawing and gathering straw, which we interpret as nest building behaviour. The pigs were killed 65 min after treatment, which was 30 min after peak nest building activity, and the brain, uterus and ovaries removed for processing using in situ hybridisation. Saline-treated pigs had elevated levels of c-fos mRNA, compared to background, in the pituitary, corpus luteum and uterus, and a lower, but elevated, level of expression in cerebellum, cortex, hippocampus and olfactory bulb. PGF 2α-treated pigs had significantly higher levels of c-fos mRNA expression than saline-treated pigs in the parvocellular and magnocellular regions of the hypothalamic paraventricular nucleus, the supraoptic nucleus (including the pars dorso-medialis), the neural lobe of the pituitary gland and the cerebellum. PGF 2α-treated pigs also had significantly higher c-fos induction in corpus luteum. These data show that the pattern of c-fos mRNA expression in specific brain areas is different between pigs that show PGF 2α-induced nest building and saline-injected controls.

Expression of fos and jun Proto-oncogenes in Benign versus Malignant Human Uterine Tissue

Objective. The objective of this study was to evaluate expression of fos and jun proto-oncogenes in benign human uterine tissue compared with malignant uterine tissue.Methods. Forty-two endometrial tissue specimens were obtained at the time of hysterectomy. Tissue samples from different phases of the menstrual cycle and from postmenopausal patients were stained using immunohistochemical methods to detect Fos and Jun proteins, estrogen and progesterone receptor status, and Ki67 (detects a nuclear antigen associated with proliferating cells). Tissue was examined microscopically for nuclear staining in endometrial epithelium and stroma. The endometrium was based on the patient's last menstrual period, pathologic dating, and proliferative versus nonproliferative status as determined by Ki67. Benign and malignant specimens were subjected to Northern blot analysis to evaluate levels of expression of c-fos, c-jun, and jun-B mRNA. The pattern of c-fos mRNA expression in malignant samples was further evaluated using in situ hybridization.Results. In proliferative, secretory, postmenopausal, and progesterone-influenced, uterine specimens immunohistochemically stained and examined, the endometrial and stromal nuclei stained for both Fos and Jun in varying intensities. However, no pattern was found in the variation of intensity according to the phase of the endometrium. Similarly, in malignant and benign endometrial tissue examined by Northern blot and in situ hybridization analyses, expression of proto-oncogene mRNAs was readily detectable, but no statistical correlation between type of tissue examined, grade of adenocarcinoma, and stage of endometrial cancer was found in this study.Conclusions. In rodent models, control of uterine cell proliferation is related to change in expression of fos and jun proto-oncogenes. Our results indicate that hormonal control is likely to be different in human endometrium and probably involves genes other than the proto-oncogenes under study. Expression of Fos and Jun do not correlate with endometrial cancer stage and grade.

Differential expression of c-fos mRNA in the rat neocortex by in situ hybridization

The c-fos mRNA expression pattern in rat neocortex, was determined in the rat kept in a 12:12 light/dark cycle, in constant dark, or in constant light by in situ hybridization. At the beginning of the light period, c-fos mRNA was induced both in the neocortex and suprachiasmatic nucleus (SCN). Transiently increased c-fos mRNA expression was detected from 0830 to 0900 and soon declined to basal levels. Immediately prior to the beginning of the dark period, c-fos mRNA expression also increased and remained elevated in the neocortex following the dark period. In the constant dark group, c-fos mRNA expression showed no transient elevation at the beginning of the light period. On the other hand, c-fos mRNA expression in the constant light group increased during their subjective dark period as well as normal light/dark cycle. These results demonstrate a circadian pattern of c-fos mRNA expression in the neocortex which is similar to that observed previously in the inner and outer nuclear layers of the retina.

NMDA Receptor Overstimulation Triggers a Prolonged Wave of Immediate Early Gene Expression: Relationship to Excitotoxicity

Exposure of the rodent striatum to quinolinic acid (QA,N-methyl-d-aspartate receptor agonist) induces immediate early gene (IEG; c-fos, c-jun, jun-B, zif/268) expression that may extend 12–24 h after injection. In order to determine the specificity of the prolonged IEG response to the QA injection, the temporal pattern of c-fos mRNA expression was examined during the first 4 h after administration of saline or QA (40 μg). As early as 30 min after intrastriatal injection, both saline and QA increased c-fos mRNA levels. In the saline group, this increase in IEG expression was only transient and returned to baseline by 1 h. In contrast, c-fos mRNA levels within QA-injected animals continued to rise significantly at 1 and 4 h. In a second experiment, rats received 4 ng- to 40-μg injections of QA followed by sacrifice at 6 h to determine if increasing QA doses caused the appearance of the prolonged IEG response phase. The prolonged IEG response was evident at 6 h only in animal groups that received higher dose ranges (4–40 μg) of QA. A final experiment was undertaken to determine if blockage of NMDA receptor stimulation would also inhibit the prolonged IEG response at 6 h in relationship to neuronal sparing evidenced at 24 h post-QA injection. The NMDA receptor antagonist, MK-801, blocked the prolonged IEG response at 6 h following QA (40 μg) injection while also preventing striatal neuropeptide mRNA decline by 24 h. Delaying the MK-801 administration for 1–2 h post-QA injection revealed that the intensity of the prolonged IEG mRNA response may be predictive of neuronal demise within the QA lesion site. These results suggest that prolonged IEG expression is associated with QA excitotoxicity of the rodent striatum and subsequent neuronal degeneration.

Differential patterns of c-fos mRNA expression in amygdala during successive stages of odor discrimination learning.

Expression of the activity-dependent gene c-fos was used to assess relative levels of neuronal activation in the amygdala and related structures of rats at different stages of odor discrimination learning. In situ hybridization was used to evaluate c-fos mRNA content within the amygdalar subdivisions, the bed nucleus of the stria terminalis, and the hippocampus. After initial exploration of the test apparatus, c-fos mRNA levels were increased in the medial and, to lesser extent, basolateral subdivisions and remained low in the central division. The balance of amygdala to hippocampal labeling favored hippocampus. Rats engaged in familiar nose-poke responses had comparably elevated labeling in the medial and basolateral divisions and low labeling densities in the central division. The ratio of hippocampal to amygdala labeling was at control levels. Rats required to switch from ad libitum responding to cued responding to odors had high basolateral to medial labeling ratios. This was in marked contrast to the medial dominance found in control and exploration rats. Hybridization was substantially more dense in basolateral amygdala than in hippocampal CA1; this imbalance was unique to the group required to form first associations between odors and rewards. Rats performing an overtrained odor discrimination had the least differentiation between amygdalar subdivisions of any behavioral group. The hippocampus-to-amygdala labeling ratio favored hippocampus and was nearly identical to the ratio in exploration rats. These results demonstrate that the balance of activity within the between limbic structures shifts according to behavioral demands. It is suggested that the balances reflect the availability of pertinent afferent cues, interactions between hippocampus and the extended amygdala, and relative levels of activity in the diffuse projections to the limbic system.

Region-specific reduction in stress-induced c-fos mRNA expression during pregnancy and lactation

Hypothalamo-pituitary-adrenal (HPA) responses to stress are dramatically attenuated during lactation. To examine whether this is due to diminished stress-induced activation of specific areas of the brain involved in HPA responses, c-fos mRNA expression was employed as a marker of stress-induced neuronal activation. Regional levels of expression were quantified in female rats exposed to 30 min immobilisation stress during late pregnancy (days 19–21), early lactation (days 3–4) and mid-lactation (days 10–14), and compared with the levels in virgin females. Stress-induced levels of corticosterone were significantly lower in late pregnant and early lactating rats compared with the levels in virgin females, and this correlated with a marked attenuation of stress-induced c-fos mRNA expression in the parvocellular division of the PVN. This reduced activation suggests that neuroendocrine hyporesponsiveness during lactation may arise from an effect on afferent pathways to the PVN. Extrahypothalamic areas known to be important for HPA activation displayed three patterns of c-fos mRNA expression: (i) in the ventral tegmental area, dorsal vagal complex, pyriform cortex and all areas of the hippocampus (CA1, CA2, CA3, dentate gyrus), expression levels did not vary significantly with reproductive status; (ii) in the locus coeruleus (A6 catecholaminergic group), a peak of expression was detected in late pregnant animals; and (iii) in the medial amygdala, ventral part of the lateral septum and cingulate cortex expression was significantly reduced in pregnant and lactating animals, with a nadir in early lactation. The decreased expression of c-fos mRNA in these latter areas correlated with that in the parvocellular PVN, and suggests that their interaction may contribute to the reduced neuroendocrine responses of lactating rats.

Regional patterns of c-fos mRNA expression in rat hippocampus following exploration of a novel environment versus performance of a well-learned discrimination

Previous studies using c-fos cRNA in situ hybridization demonstrated a differential involvement of hippocampal subfields CA1 and CA3 in the acquisition of an olfactory discrimination (Hess et al., 1995). The present experiments employed the same method to examine changes in neuronal activity associated with two related behaviors: (1) initial exploration of the training apparatus and (2) performance of a well-learned odor discrimination. Rats in the two groups had similar labeling patterns within hippocampus indicating increased expression in all three major subfields with the greatest effect being in CA1. This pattern of "CA1 dominance" was notably different from that produced during early stages of two-odor discrimination learning in prior experiments. Hippocampal labeling in exploration and performance rats differed in that (1) hybridization was greater in CA1, CA3, and dentate gyrus in the former group and (2) a tendency for labeled cells to occur in clusters was more evident in exploration animals. Levels of c-fos mRNA in olfactory and visual structures were not predictive of expression patterns within hippocampus although labeling in piriform cortex and dentate gyrus was correlated in rats performing a well-practiced discrimination. Moreover, the pattern of hybridization in olfactory bulb was found to be behaviorally dependent. These results, together with those from previous studies, indicate that hippocampus has multiple patterns of regional activation but that one of these is common to very different behavioral circumstances. It is hypothesized that this common pattern emerges whenever the animal responds to distant cues using species-specific or well-learned behaviors and involves coordinated temporal convergence of sensory and septal/brainstem inputs.

Temporal variation of c-Fos proto-oncogene expression during osteoblast differentiation and osteogenesis in developing rat bone.

To delineate the implication of c-fos protooncogenic in the osteogenie process, we have investigated the temporal pattern of c-fos mRNA expression in fetal and neonatal rat bone during intramembranous and endochondral bone formation. Northern blot analysis of mRNA extracted from calvaria and femur showed that expression of c-fos, Histone H4, and osteocalcin mRNAs followed a temporal sequence during bone development. The levels of histone H4 mRNA, a marker of cell proliferation, were high at early stages of fetal development of calvaria and femur, and decreased until birth. In both the postnatal calvaria and femur, c-fos mRNA levels increased transiently at birth and preceded a rise in osteocalcin transcripts, a marker of the mature osteoblast phenotype. The immunohistochemical analysis showed that c-Fos protein was expressed in osteoprogenitor cells in the perichondrium and periosteum, and not in mature osteoblasts which expressed markers of differentiated osteoblasts such as type-I collagen, bone sialoprotein, and osteocalcin. Thus, the transient c-fos proto-oncogene expression during the postnatal life that precedes the osteocalcin expression may be involved in the transition from the precursor state to mature osteoblasts. These results suggest that c-fos proto-oncogene may play an important role in osteogenesis during rat postnatal life.

Systemic interleukin-1 induces early and late patterns of c-fos mRNA expression in brain

This study was designed to examine the mechanisms by which systemic interleukin-1 affects neuroendocrine systems in the brain. Intraperitoneal injections of interleukin-1 beta (1.25 micrograms/rat) were administered to rats. One or three hours after injection, the expression levels of the immediate-early gene c-fos and of genes for several neuropeptides, receptors, and enzymes were examined by in situ hybridization histochemistry. In the brainstem at 1 hr, c-fos mRNA was elevated in the area postrema and nucleus of the solitary tract, but not in the locus coeruleus. At 3 hr, the c-fos mRNA levels had increased further in the nucleus of the solitary tract. Rostrally, elevations in c-fos mRNA levels were found in the hypothalamic and thalamic paraventricular nuclei, central nucleus of amygdala, bed nucleus of the stria terminalis, and medial preoptic area, peaking at 1 hr and diminishing at 3 hr. In addition, at 3 hr a new pattern of c-fos activity emerged--the arcuate nucleus and cells at the external margins throughout the brain now expressed c-fos mRNA. Corticotropin-releasing hormone mRNA levels were doubled in the paraventricular nucleus at 1 and 3 hr, concomitant with elevations in plasma adrenocorticotrophic hormone (ACTH) and corticosterone. Tyrosine hydroxylase mRNA levels in the brainstem did not change. The c-fos mRNA induction patterns reveal a temporally dynamic response to interleukin-1 administration. We propose that the early set of structures responding to interleukin-1 initiates the neuroendocrine response to cytokines. Coactivation of the area postrema and nucleus of the solitary tract may reflect entry into the brain and neural transduction of the peripheral signal. The late set--including the nucleus of the solitary tract, arcuate nucleus, and the brain's edge--may reflect cellular activation along the diffusion routes traveled by interleukin-1 or a bioactive transduction product, because the pattern of edge labeling is similar to the autoradiographic pattern of flow lf radiolabeled tracer substances in the cerebrospinal fluid. The late c-fos mRNA response to interleukin-1, therefore, may represent a demonstration of information transfer in the parasynaptic mode, also known as volume transmission.

Intracerebroventricular administration of corticotropin-releasing factor inducesc-fos mRNA expression in brain regions related to stress responses: comparison with pattern ofc-fos mRNA induction after stress

Centrally administered corticotropin-releasing factor (CRF) produces a number of physiological and behavioral changes akin to those elicited by exposure to acute stress. However, the specific brain site of action responsible for the centrally activating property of CRF has not been precisely determined. In this study, we used in situ hybridization histochemistry forc-fos mRNA to map potential neuronal structures activated after intracerebroventricular (i.c.v.) injection of CRF and compared the distribution ofc-fos mRNA with that after stress. Wistar male rats were sacrificed 30, 60, 120 and 180 min after the i.c.v. injection of 1 μg ovine CRF or vehicle alone. Another group of rats was exposed to immobilization stress for 60 min or electrical foot-shock stress (1.5 mA, 1-s duration, 30×) for 15 min and sacrificed before and 30, 60 120 and 180 min after the beginning of stress. Centrally administered CRF rapidly (30–60 min) inducedc-fos mRNA expression in most of the areas that showed hybridization signals forc-fos after stress: the limbic structures, including the piriform cortex, cingulate cortex, the lateral septal nucleus, the hippocampus, the anterior corticomedial and the medial amygdaloid nuclei, the hypothalamic nuclei, such as the paraventricular nucleus, the supraoptic nucleus (SO) and the dorsomedial nucleus (DMD), and some brainstem nuclei like the pontine nucleus, the locus ceruleus (LC) and Barrington's nucleus. The granular layer of the cerebellum, some thalamic nuclei and the habenula also showed by hybridization signals after i.c.v. injection of CRF and stress. However, c-fos induction in the bed nucleus of the stria terminalis, the central nucleus of the amygdala (CeA) and the nucleus tractus solitarius (SOL) was seen only after i.c.v. administration of CRF; in the septohypothalamic nucleus and the superior olive, however, c-fos mRNA expression was observed only after stress. There were no differences in the pattern ofc-fos mRNA expression between the two stress paradigms. In contrast, i.c.v. injection of saline-induced expression ofc-fos mRNA in the piriform cortex, neocortex, cingulate cortex and the amygdala was much less than that seen after i.c.v.-administered CRF as evident in the intensity of the signals. These results suggest that CRF producesc-fos mRNA expression in the brain areas related to stress response, ant that CRF may induce behavioral and neuroendocrine responses through activating these brain structures, such as the limbic system and the hypothalamic nuclei. It is also suggested that the CeA, LC and SOL may be involved in the expression of cardiovascular and autonomic actions as well as an increase in arousal induced by CRF.

Mouse brain c-fos mRNA distribution following a single electroconvulsive shock.

The regional distribution of c-fos mRNA in the mouse brain has been investigated by in situ hybridization autoradiography after seizures induced by an acute electroconvulsive shock (ECS). ECS led to a widespread induction of the proto-oncogene c-fos in the brain, with highest concentrations in discrete areas within the limbic system and also in the hypothalamus and cerebellum. The mild stress of sham treatment in earclipped animals induced a weaker and qualitatively different pattern of c-fos mRNA expression involving the cortex, hippocampus, and cerebellum. These data suggest the usefulness of c-fos in situ hybridization as a marker of neuronal stimulation and in mapping a range of effects from a mild stress to the robust changes of an electroconvulsive seizure.