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.     

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     

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.     

Expression of c-fos and jun protooncogenes in ovine trophoblasts in relation to interferon-tau expression and early implantation process

Expression of c-fos and jun protooncogenes was analyzed in the ovine extraembryonic trophoblast from days 14–18 of gestation, using Northern and Western blotting and immunohistochemistry. This study was carried out in relation to the early implantation process and the expression of interferon-tau, which is secreted in large amounts for a few days before implantation. Our results demonstrated that c-fos, c-jun, and junB were differently expressed in the ovine trophoblast around the time of implantation. The c-fos mRNA and protein were detected at high levels prior to attachment and decreased thereafter, following the pattern of expression of interferon-tau, whereas c-jun expression was maintained at relatively high levels during the implantation process. By contrast, the levels of junB mRNA and protein decreased prior to attachment. Immunohistochemical studies indicated that JunB, like C-Fos and interferon tau, was no longer expressed in the trophoblastic cells which had established cellular contacts with the uterine epithelium. A striking finding in this study is the temporal correlation between the accumulation of c-Fos and c-Jun proteins and the expression of the interferon-tau (days 14 and 15 of gestation). We also showed by gel-retardation assays that an AP-1-like site present in the promoter of one interferon-tau gene was functional in vitro, as judged by its ability to bind day-15 trophoblast nuclear protein extracts. Nuclear proteins binding to this site had the characteristics of AP-1, as judged by the ability to be competed efficiently by a consensus TRE (12.0-tetradecanoyl phorbol 13-acetate-responsive element)-site oligonucleotide and by antibodies to c-Fos and Jun proteins. These results suggest that Fos and Jun could form regulatory complexes of interferon-tau expression and/or are regulated by common mechanisms which are still unknown. Mol Reprod Dev 46:127–137, 1997. © 1997 Wiley-Liss, Inc.     

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 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.     

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.     

c-fos antisense RNA blocks expression of c-fos gene in F9 embryonal carcinoma cells

To study the function of proto-oncogene c-fos, we prepared an antisense plasmid that expresses in mammalian cells c-fos antisense RNA which is complementary to the endogenous c-fos mRNA. Upon transfection into undifferentiated F9 EC cells, the antisense plasmid directed constitutive expression of a large amount of c-fos antisense RNA. These cells were very low in the basal level of c-fos message and were unable to induce c-fos message when stimulated with interferon or phorbol ester. The failure to induce c-fos message led to the blockade of c-fos protein expression in these cells. Thus, these cells represented a c-fos defective phenotype. The blockade of c-fos gene expression seen in antisense-cells could be caused by rapid degradation of the c-fos message, since c-fos mRNA expression was rescued in these cells when treated with protein synthesis inhibitor, cycloheximide. We found that expression of c-myc gene was down-regulated in c-fos antisense-cells: Although control undifferentiated F9 cells constitutively expressed a high level of c-myc message, the antisense cells had a much lower amount of c-myc mRNA. Since p53 and heat shock gene 70 were expressed at comparable levels in control and antisense cells, c-myc gene expression appears to be regulated by c-fos gene in F9 EC cells. Lastly, these antisense cells grew as rapidly as control F9 cells and underwent differentiation after retinoic acid treatment, indicating that c-fos expression is not a prerequisite for differentiation of F9 cells.     

Bombesin stimulation of c-fos expression and mitogenesis in Swiss 3T3 cells: The role of prostaglandin E2-mediated cyclic AMP accumulation

Bombesin is a potent mitogen for Swiss 3T3 cells and can stimulate DNA synthesis in the absence of any other growth factor. This effect is mediated by multiple synergistic signaling pathways, including an accumulation of intracellular cyclic AMP (cAMP) and an increase in c-fos mRNA expression. The cyclooxygenase inhibitor indomethacin abolished prostaglandin E₂ release and substantially depressed cAMP levels induced by bombesin (EC₅₀ - 10 nM). In contrast, indomethacin at 1 μM did not affect 80K phosphorylation or Ca²⁺ mobilization by bombesin, indicating that cAMP synthesis can occur through a phospholipase C-independent pathway. Indomethacin caused a 30 to 35% decrease in c-fos induction and DNA synthesis in cells treated with bombesin (EC₅₀ - 40 nM). Significantly, the inhibitory effect of indomethacin was reversed in the presence of forskolin, a direct activator of adenylate cyclase. We conclude that cAMP plays a regulatory role in c-fos induction and mitogenesis in Swiss 3T3 cells treated with bombesin.