At Least Three Neurotransmitter Systems Mediate a Stress-Induced Increase in c-fos mRNA in Different Rat Brain Areas

1. Protooncogene c-fos mRNA levels were determined in the rat cerebral cortex, hippocampus, and cerebellum after exposure to a combined forced swimming and confinement stress. The stress resulted in an increase in c-fos mRNA levels in all three brain areas.2. In an effort to elucidate the neurotransmitter systems involved in this stress-induced increase, animals were injected, prior to exposure to the stress, with either diazepam, MK-801, or propranolol.3. In both the cerebral cortex and the hippocampus the stress-induced increase in c-fos mRNA was inhibited by MK-801, suggesting that it is mediated via NMDA receptors. In the hippocampus, propranolol had a similar effect, indicating that -adrenergic receptors are also involved in the stress-induced increase in c-fos mRNA.4. On the other hand, the increase in c-fos mRNA produced by the stress of the injection was inhibited in the cerebral cortex by diazepam or propranolol and in the hippocampus only by diazepam. Furthermore, administration of MK-801 resulted in an increase in c-fos mRNA in the hippocampus of the nonstressed animals. In the cerebellum no one of the three drugs employed affected c-fos mRNA levels in either stressed or nonstressed animals.5. Our results thus show that various forms of stress activate, in different brain areas, neurons with either NMDA, -adrenergic, and/or GABA-A receptors.     

Midazolam and theN-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid (AP-7) attenuate stress-induced expression of c-fos mRNA in the dentate gyrus

Summary 1. The effects of restraint stress on c-fos mRNA expression in the dentate gyrus were investigated byin situ hybridization.2. Confirming previous findings, c-fos mRNA expression increased after 30 min of forced restraint.3. This effect was attenuated by a previous i.c.v. injection of the anxiolytic benzodiazepine midazolam (20 nmol/2 µl) or theN-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid (AP-7; 5 nmol/2 µl).4. These results suggest that the dentate gyrus is activated during restraint stress and that this activation may be modulated by benzodiazepine -aminobutyric acid_A (GABA_A) or NMDA receptors.     

c-Fos Expression by Dopaminergic Receptor Activation in Rat Hippocampal Neurons

While dopamine is likely to modulate hippocampal synaptic plasticity, there has been little information about how dopamine affects synaptic transmission in the hippocampus. The expression of IEGs including c-fos has been associated with late phase LTP in the CA1 region of the hippocampus. The induction of c-fos by dopaminergic receptor activation in the rat hippocampus was investigated by using semiquantitative RT-PCR and immuno-cytochemistry. The hippocampal slices which were not treated with dopamine showed little expression of c-fos mRNA. However, the induction of c-fos mRNA was detected as early as 5 min after dopamine treatment, peaked at 60 min, and remained elevated 5 h after treatment. Temporal profiles of increases in c-fos mRNA by R(+)-SKF-38393 (50 M) and forskolin (50 M) were similar to that of dopamine. An increase in [cAMP] was observed in dopamine-, SKF-, or forskolin-treated hippocampal slices. By immunocytochemical studies, control hippocampal cells showed little expression of c-Fos immunoreactivity. However, when cells were treated with dopamine, an increase in the expression of c-Fos immunoreactivity was observed after treatment for 2 h. The treatment of hippocampal neurons with R(+)-SKF38393 (50 M) or forskolin (50 M) also induced a significant increase in c-Fos expression. These results indicate that the dopamine D1 receptor-mediated cAMP dependant pathway is associated with the expression of c-Fos in the hippocampal neurons. These data are consistent with the possible role of endogenous dopamine on synaptic plasticity via the regulation of gene expression. Furthermore, these results imply that dopamine might control the process of memory storage in the hippocampus through gene expression.     

The application of antisense oligonucleotide technology to the brain: Some pitfalls

Summary 1. Amphetamine-induced c-fos andegr-1 expression in the striatum was used as a model in which to study the effects of antisense oligodeoxynucleotides (ODNs) directed at c-fos. Using direct infusions of ODNs into the striata of animals we have demonstrated that c-fos antisense ODNs retain most of their biological activity with 2- or 3-base substitutions. The c-fos antisense and mismatch ODNs attenuated Fos immunoreactivity but had little effect on Egr-1 immunoreactivity.2. In another group of studies examining the role of c-fos in amygdala kindling, we have demonstrated that ODNs cause neurotoxic damage following repeated daily infusions into the amygdala. The damage observed was greatly diminished when the time interval between infusions was extended.     

c-fos expression in the amygdala:In vivo antisense modulation and role in anxiety

Summary 1. The amygdaloid complex is a key structure in mechanisms of fear and anxiety. Expression of the immediate-early gene c-fos has been reported in the central nucleus of the amygdala following various stressors, but the functional role of this phenomenon has remained unknown.2. c-fos expression was observed in the central nucleus when rats were subjected to a pharmacologically validated animal model of anxiety, the Vogel conflict test, but not after mere exposure to the test apparatus. Bilateral amygdala injection of a 15-mer phosphorothioate c-fos antisense oligodeoxynucleotide prior to testing blocked conflict-induced c-fos expression and had behavioral effects similar to those of established antianxiety drugs.3. Separate experiments determined that antisense treatment did not affect conflict behavior by acting on shock thresholds or drinking motivation.4. These findings provide evidence that neuronal activation and c-fos induction in the amygdala may be of importance for mechanisms of fear and anxiety.     

Co-localization of ecdysteroid receptors and c-fos-like protein in the brain of Manduca sexta larvae

Summary The presence of c-fos, a marker for cell activation, was investigated in cerebral neurons actively expressing ecdysteroid receptors during larval-pupal development in the tobacco hornworm, Manduca sexta. Colocalization was accomplished by ecdysteroid autoradiography using the tritiated high affinity 20-hydroxyecdysone agonist ponasterone A and immunocytochemistry with an antibody to a peptide sequence which is highly conserved in both human and murine c-fos. Immunoreactivity to a c-fos-like protein(s) was present in nuclei of many neurons of all the developmental stages examined. However, with the exception of the optic lobe, cells expressing nuclear ecdysteroid receptors were more immunoreactive than non-ecdysteroid-binding neurons. These data suggest that ecdysteroid-induced gene activation and translation may involve c-fos expression. Offprint requests to: H.-J. Bidmon     

Secretin Activates Visceral Brain Regions in the Rat Including Areas Abnormal in Autism

1. The aim of this study was to determine whether central networks are involved in the presumptive behavioral and autonomic regulatory actions of secretin, a gut hormone that has been reported to have ameliorative effects in autistic children.2. Central neural responses monitored by regional c-fos gene expression were examined in response to intracerebroventricular secretin injection in awake, freely-moving Sprague-Dawley rats. Tissue sections were incubated in an antibody to the c-fosgene product, Fos, and processed immunohistochemically.3. Qualitative differences in Fos immunoreactivity in stress adaptation and visceral representation areas of the brain were observed between secretin- and vehicle-infused age-matched pairs (n = 4 pairs). Secretin-activated regions include the area postrema, dorsal motor nucleus, medial region of the nucleus of the solitary tract and its relay station in the lateral tegmentum, locus ceruleus, ventral periaqueductal gray, periventricular thalamic nucleus, paraventricular hypothalamus magnocellularis, medial and central amygdala, lateral septal complex as well as ependymal and subependymal nuclei lining the third ventricle. Specific areas of the cerebral cortex were heavily labeled in secretin-treated rats, as compared to controls: the medial bank of the anterior prefrontal cortex, orbitofrontal cortex, the piriform cortex, and the anterior olfactory nucleus. Secretin attenuated Fos immunoreactivity in the dorsal periaqueductal gray, intralaminar thalamus, medial parvicellular compartment of the hypothalamus, supraoptic nucleus of the hypothalamus, lateral amygdala, motor cortex, and the somatosensory and association areas of the parietal cortex.4. Secretin alters the activity of structures involved in behavioral conditioning of stress adaptation and visceral reflex reactions. This study predicts a possible cellular mechanism, activation of third ventricular ependymal and subependymal cells, as well as central regulatory actions of secretin. The physiological effects of secretin on behavioral, endocrine, autonomic and sensory neuronal activation patterns, together, contribute to central c-fos activation. Secretin alters the activity of structures involved in behavioral conditioning of stress adaptation and visceral reflex reactions. This study predicts a possible cellular mechanism, activation of third ventricular ependymal and subependymal cells, and central regulatory actions of secretin. The physiological effects of secretin on behavioral, endocrine, autonomic and sensory neuronal activation patterns, together, contribute to central c-fos activation. These findings mandate further investigation of secretin as a brain/gut stress regulatory hormone.     

Psychomotor stimulant- and opiate-induced c-fos mRNA expression patterns in the rat forebrain: Comparisons between acute drug treatment and a drug challenge in sensitized animals

Amphetamine-, cocaine-, and morphine-induced c-fos expression patterns were examined following an injection protocol that has previously been shown to produce behavioral sensitization and enhanced dopamine release in the striatal complex. Drug-specific c-fos patterns were observed in both acute and sensitization injection paradigms. A sensitization pretreatment schedule did, however, alter the c-fos expression patterns induced by all the drugs in the caudate putamen, nucleus accumbens, and the cerebral cortex. In some striatal and cortical regions, there was an increase or recruitment of cells expressing c-fos whereas in others there was an apparent decrease or inhibition. The somatosensory cortex was one area where pretreatment with all three drugs increased c-fos expression. The results suggest that the neuronal networks that are modulated by systemic drug injections in the sensitized animal differ from those affected by the initial drug exposure; areas of overlap may indicate common ‘sensitization’ circuits. Special issue dedicated to Dr. Eric J. Simon.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

c-fos antisense reduces expression of Krox 24 in rat caudate and neocortex

Summary 1. The aim of this study was to investigate the neurochemical effects and measure the anatomical spread of infusion of c-fos antisense (AS) DNA into the striatum.2. Rats were anesthetized and infused in opposing striata with c-fos AS and c-fos sense (S) DNA. Ten hours later they were injected with apomorphine (2 mg/kg, i.p.) and 20 min later they were overdosed with sodium pentobarbital and their brains either perfused or frozen. Vibratome-cut sections were immunostained for the detection of c-fos, JunB, Krox 24, somatostatin, substance P, dynorphin, tyrosine hydroxylase, and enkephalin. Cryostat-cut sections from the caudate were immunostained for the detection of c-fos, JunB, and Krox 24, as well asin situ hybridization for proenkephalin mRNA. Sections from the globus pallidus were used for the autoradiographic localization of D2 dopamine and A2_a adenosine receptors. Sections from the substantia nigra were used for the autoradiographic localization of D1 dopamine and cannabinoid receptors. A second group of rats was injected in opposing striata with biotin-labeled c-fos AS DNA and c-fos S DNA. Ten hours later they were challenged with apomorphine (2 mg/kg, i.p.) and 20 min later brains were either perfused or frozen. Sections from these brains were cut throughout the rostral-caudal extent of the forebrain and the biotin labeled AS DNA was localized.3. Krox 24 was expressed at high levels on the sense side of the brain in the striatum and overlying neocortex. However, on the AS-injected side there was a reduction in Krox 24 expression in striatum and overlying cortex. The biotin-labeled AS studies confirmed that the striatal infusion spread throughout the dorsal striatum as well as the overlying neocortex. We did not detect any changes in neurotransmitter receptors, neuropeptides, or tyrosine hydroxylase in AS/S-injected rat brains.4. These results demonstrate that c-fos AS reduces Krox 24 expression in striatal and neocortical neurons but does not change the expression of a number of other proteins involved in basal ganglia function. Whether this effect is due to nonspecific actions of c-fos AS or to its effects on a component of the transduction pathway responsible for basal Krox 24 expression (NMDA receptors?) is unknown.     

Expression of c-fos and c-myc protooncogenes in an immortalized hepatocyte line harbouring SV40 T antigen and hGH as transgenes

A clonal hepatocyte line (FMH-202-2), derived from livers of fetal transgenic mice harbouring human growth hormone (hGH) and SV40 T antigen as transgenes, was used in the investigation of protooncogene expression involved in liver-specific growth control and/or in hepatocellular transformation. In this model system, representing an immortalized, yet untransformed phenotype, the transgenes hGH and SV40 T antigen were expressed constitutively. The c-fos protooncogene was induced by incubation with insulin, epidermal growth factor (EGF) and insulin-like growth factor (IGF-I) in a transient manner comparable to its expression in primary murine hepatocytes. Elucidation of second messenger mechanisms demonstrated that c-fos induction by hepatotrophic growth factors was not mediated by protein kinase C. In contrast to primary hepatocytes, the c-myc protooncogene exhibited a constitutive expression pattern which was independent of growth factor stimulation. These results indicate that apart from hGH and SV40 T antigen, c-myc may play a role in cellular immortalization, but that constitutive expression of these genes, even in combined coexpression, does not suffice to induce the transformed phenotype.     

Differential Mechanisms of Glutamate-Stimulated Perturbations in the Kinetics of c-fos mRNA Induction Are Associated with Maturation of Cerebellar Granule Cells in Primary Culture

In further exploring proposals for the measurement of early gene (c-fos mRNA) levels as a predictive index for in vitro excitotoxicity, this study, using immature (2 days in vitro) cultures of mouse cerebellar granule cells as an experimental model system, was undertaken to determine the effect of glutamate (Glu) i) in stimulating increases in intracellular free-calcium ([Ca²⁺]_i), ii) on cell viability and iii) on induction of steady-state c-fos mRNA levels. In parallel experiments the action of agents (viz. 55 mM KCl and the calcium ionophore, A23187) that mediate Ca²⁺ entry into cells via different routes was also evaluated. Glu was unable to induce excitotoxicity in granule cells at this stage of development in culture, but did stimulate a concentration-dependent and marked increase in [Ca²⁺]_i levels while also mediating a dramatic concentration-dependent perturbation in the kinetics of c-fos mRNA induction that appeared to arise solely from NMDA receptor-mediated Ca²⁺ influx. The results are presented in comparison to the actions of KCl and A23187 and considered in relation to earlier studies undertaken using mature (7 days in vitro) cultures of cerebellar granule cells.     

Decreases in local hormone biosynthesis and c-fos gene expression accompany differentiation of porcine preadipocytes

Summary To better understand possible autocrine or paracrine mechanisms involved in adipose tissue development, we have studied the biosynthesis of insulinlike growth factor I (IGF-I) and prostaglandin E₂ (PGE₂) by cultured porcine preadipocytes in response to factors known to modulate cell growth and differentiation. The expression of c-fos was also monitored because of the potential role of that proto-oncogene in coordination of growth and differentiation. Preadipocytes were grown to confluence and then maintained in one of three media treatments: a) standard medium supplemented with 10% fetal bovine serum (FBS), b) FBS supplemented with dexamethasone (Dex), c) FBS supplemented with dibutryladenosine 3′–5′-cyclic monophosphate. Indirect measurements of growth indicated that cell proliferation did not differ due to media type. Histochemical and enzymatic measurements of adipocyte development revealed that differentiation occurred only in those cultures exposed to Dex. The increase in adipocyte differentiation in response to Dex was associated with a decrease in c-fos and actin RNA expression whereas the decrease in c-fos RNA expression in response to Dex was small (approximately 40%); immunocytochemical analysis indicated that induction of Fos protein occurred only in undifferentiated cells. Thus, the cells responsible for the decrease in c-fos RNA expression are possibly those signaled to differentiate into adipocytes. Expression of IGF-I RNA and secretion of IGF-I and PGE₂ were also decreased in response to Dex treatment. These data provide the first demonstration that biosynthesis of IGF-I by preadipocytes can be modulated by a potent inducer of adipocyte differentiation. The combined results indicate that glucocorticoids may stimulate adipocyte differentiation by suppressing intracellular and putative intercellular mitogenic signals. This work was supported in part by grant HD 18447 from the National Institutes of Health, Bethesda, MD (G. J. H.). Mention of a trade mark, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture or University of Georgia and does not imply its approval to the exclusion of other products that may be suitable.