C-fos Antisense Oligonucleotide Research Articles

Effects of Restraint Water-Immersion Stress-Induced Gastric Mucosal Damage on Astrocytes and Neurons in the Nucleus Raphe Magnus of Rats via the ERK1/2 Signaling Pathway

Restraint water-immersion stress (RWIS) consists of psychological and physical stimulation, and it has been utilized in the research of gastric mucosal damage. It has been shown by previous studies that the nucleus raphe magnus (NRM) is closely involved in the gastrointestinal function, but its functions on the stress-induced gastric mucosal injury (SGMI) have not been thoroughly elucidated to date. Consequently, in this research, we aim to measure the expression of astrocytic glial fibrillary acidic protein (GFAP), neuronal c-Fos, and phosphorylation extracellular signal regulated kinase 1/2 (p-ERK1/2) in the process of RWIS with immunohistochemistry and western blot methods. What is more, we detect the relation between astrocytes and neurons throughout the stress procedure and explore the regulation of the ERK1/2 signaling pathway on the activity of astrocytes and neurons after RWIS. The results indicated that all three proteins expression multiplied following peaked 3h substantially. The SMGI, astrocyte and neuron activity were affected after the astrocytotoxin L-A-aminohexanedioic acid (L-AA) and c-fos antisense oligonucleotide (ASO) injections. After the injection of PD98059, the gastric mucosal injury, astrocyte and neuron activity significantly fell off. These results suggested that RWIS-induced activity of astrocytes and neurons in the NRM may play a significant part in gastric mucosa damage via the ERK1/2 signaling pathway.

Requirement of an Early Activation of BDNF/c-Fos Cascade in the Retrosplenial Cortex for the Persistence of a Long-Lasting Aversive Memory.

During the past few years, there has been growing interest in the role of the retrosplenial cortex (RSC) in memory processing. However, little is known about the molecular changes that take place in this brain region during memory formation. In the present work, we studied the early post-training participation of RSC in the formation of a long-lasting memory in rats. We found an increase in c-Fos levels in the anterior part of the RSC (aRSC) after inhibitory avoidance (IA) training. Interestingly, this increase was associated with memory durability, since blocking c-Fos expression using specific antisense oligonucleotides (ASO) impaired long-lasting retention 7 days after training without affecting memory expression 2 days after training. In addition, we showed that BDNF is one of the upstream signals for c-Fos expression required for memory persistence, since blocking BDNF synthesis prevents IA training-induced increase in c-Fos levels in aRSC and affects memory persistence. In addition, we found that injection of BDNF into aRSC around training was sufficient to establish a persistent memory and that this effect was prevented by c-fos ASO infusion into the same structure. These findings reveal an early post-training involvement of aRSC in the processing of a long-lasting aversive memory.

Learning-facilitated long-term depression requires activation of the immediate early gene, c-fos, and is transcription dependent

De novo gene transcription is a prerequisite for long-term information storage in the brain. Learning-facilitated synaptic plasticity describes the ability of hippocampal synapses to respond with long-lasting synaptic plasticity to the coupling of afferent stimulation with a spatial learning experience. Strikingly, long-term depression (LTD) is facilitated by context-dependent spatial learning experiences suggesting it may play a role in information storage to enable spatial memory. Here, we investigated if learning-facilitated LTD requires the transcription factor, c-Fos and is transcription-dependent. Novel spatial learning about object-place configurations coupled with weak low frequency afferent stimulation induced robust LTD in control animals that persisted for >24h and was associated with elevations in hippocampal expression of c-Fos. Intracerebral application of a c-fos antisense oligonucleotide prevented the facilitation of LTD by novel spatial learning, inhibited elevations of c-Fos triggered by LTD and impaired spatial learning. The expression of the transcription factor zif268 was unaffected by the c-fos antisense oligonucleotide. Learning-facilitated LTD was prevented by a transcription inhibitor. These data support that learning-facilitated LTD requires elevations in c-Fos and is transcription dependent. The observation that LTD shares key regulatory mechanisms with learning and memory processes argues strongly for a role for this form of synaptic plasticity in long-term information storage in the hippocampus.

Upregulation of AT1 receptor gene on activation of protein kinase Cβ/nicotinamide adenine dinucleotide diphosphate oxidase/ERK1/2/c-fos signaling cascade mediates long-term pressor effect of angiotensin II in rostral ventrolateral medulla

Angiotensin II induces the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2 via the activation of nicotinamide adenine dinucleotide diphosphate (NADPH) oxidase on stimulation of the angiotensin subtype 1 receptor (AT1R) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone and blood pressure are located. Angiotensin II-activated p38 MAPK in RVLM promotes a short-term pressor effect via augmented glutamatergic neurotransmission. We tested the hypothesis that the NADPH oxidase-dependent phosphorylation of ERK1/2 after the activation of conventional protein kinase C (PKC) mediates the AT1R-dependent long-term pressor effects of angiotensin II via transcriptional induction of the proto-oncogene c-fos gene in RVLM. In Sprague-Dawley rats, a microinjection of angiotensin II bilaterally into the RVLM induced membrane-bound translocation of the conventional PKCalpha, PKCbeta or PKCgamma isoform, phosphorylation of the p47 subunit of NADPH oxidase and ERK1/2, followed by phosphorylation of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB), and c-fos induction. The PKC inhibitor antagonized angiotensin II-induced p47 phosphorylation, and an antisense oligonucleotide (ASON) complementary to PKCbeta messenger RNA suppressed angiotensin II-induced ERK1/2 activation, phosphorylation or DNA binding activity of CREB, and upregulation of c-fos mRNA expression in the ventrolateral medulla. Furthermore, a microinjection of ERK1/2, CREB or c-fos ASON into the RVLM significantly reduced the long-term pressor effect and augmented AT1R expression in the ventrolateral medulla induced by intracerebroventricular infusion of angiotensin II. We concluded that the PKCbeta/NADPH oxidase/ERK1/2/CREB/c-fos cascade represents a novel signaling cascade that mediates the long-term pressor effect induced by angiotensin II in the RVLM.

Antisense oligonucleotide targeting c-fos mRNA limits retinal pigment epithelial cell proliferation; a key step in the progression of proliferative vitreoretinopathy

The purpose of this work was to investigate the effect of c-fos antisense oligonucleotide (c-fos-AS-ON) on proliferative vitreoretinopathy (PVR). Cultures of human retinal pigment epithelial (hRPE) cells were established from adult human corneal donors. These cells were positively stained for cytokeratins. C-fos-AS-ON effect on serum-stimulated cell proliferation was estimated by evaluating the incorporation of 5-bromo-2′-deoxy-uridine (BrdU) into cellular DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were respectively performed to quantify the serum-stimulated c-fos gene mRNA and protein expression in hRPE cells. Eight rabbits (16 eyes) were divided into c-fos-AS-ON treatment group and control group. 2.5 × 10 5 cultured hRPE cells were injected into the vitreous cavity of eyes to establish a PVR model. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection and by pathological study on days 28 post-injection. The results showed that blocking the expression of c-fos by the addition of c-fos-AS-ON to the culture medium significantly inhibited the hRPE cells proliferation. This effect of c-fos-AS-ON was found to be sequence specific (the use of a sense or a mismatch sense oligonucleotide had no such an effect) and dose-dependent (0.375 μM was the lowest effective dose tested). Growth inhibition by c-fos-AS-ON remained for at least 72 h. By using RT-PCR and Western blotting, we found that the c-fos-AS-ON could specifically inhibit c-fos mRNA and protein synthesis in cultured hRPE cells. Though the eyes injected with c-fos-AS-ON also developed features of PVR, the severities of days 14, 21 and 28 post-injection were significantly lower than those in the control eyes ( P < 0.05). We conclude that c-fos-AS-ON can inhibit cultured hRPE cell proliferation, which mechanism may relate to blocking the expression of c-fos and can reduce the prevalence of experimental PVR. These findings establish a rationale for investigating the potential use of a c-fos-AS-ON as a novel therapeutical tool in the treatment of PVR.

Nitric oxide induces apoptosis via AP-1-driven upregulation of COX-2 in rat pheochromocytoma cells

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, is induced in many cells by numerous inflammatory mediators, including nitric oxide (NO). Upregulation of COX-2 expression has been implicated in the pathophysiology of neuronal cell death. In the present study, we have found that the NO-induced upregulation of COX-2 via activation of activator protein-1 (AP-1) signaling leads to apoptotic cell death. Cultured rat pheochromocytoma (PC12) cells treated with sodium nitroprusside (SNP), a NO-releasing compound, exhibited marked induction of COX-2 expression, which was associated with apoptotic cell death as evidenced by internucleosomal DNA fragmentation, cleavage of poly(ADP-ribose) polymerase, activation of caspase-3, accumulation of p53, increased Bax/Bcl-X L ratio, and dissipation of mitochondrial membrane potential. In addition to the upregulation of COX-2 expression, SNP treatment led to activation of AP-1. Pretreatment of PC12 cells with c-fos antisense oligonucleotide abolished the NO-induced increase in DNA binding of AP-1 and upregulation of COX-2 expression. Furthermore, pretreatment with a selective COX-2 inhibitor (SC58635) rescued the PC12 cells from the apoptotic cell death induced by NO. Similar results were obtained when the NO-induced upregulation of COX-2 expression was blocked by the siRNA interference. These results suggest that excessive NO production during inflammation induces apoptosis in PC12 cells through AP-1-mediated upregulation of COX-2 expression.

Role of c-Fos protein on glutamate toxicity in primary neural hippocampal cells

The hippocampus is extremely sensitive to microenvironmental signals and toxic events, including massive glutamate release. Despite the extensive literature related to the cascade of molecular events triggered in postsynaptic neurons, the distinction between proapoptotic and survival pathways is still being discussed. In this study, we have investigated the role of c-Fos in glutamate-induced toxicity in primary cultures of hippocampal neurons by using antisense oligonucleotide (ASO) technology. Exposure of cells (5 days in vitro; DIV) to glutamate 0.5 mM for 24 hr caused massive nuclear alteration. An increase in the number of caspase-3-positive cells was also observed 24 hr after glutamate treatment. The expression of c-fos and c-jun immediate-early genes was increased 30 min after glutamate exposure. The study of c-Fos and c-Jun protein expression revealed an increase in the number of cells positive for both antibodies. To investigate whether the expression of c-Fos protein after glutamate treatment was related to cell death activation or cell survival pathways, cells were exposed to 5 microM of c-fos ASO at 4 DIV, 24 hr before glutamate treatment. The presence of the ASO in the medium significantly decreased the number of altered nuclei, and this was associated with a significant reduction in the number of c-Fos-positive cells after glutamate treatment. Exposure of cells to the c-fos ASO under the conditions described above decreased caspase-3 immunostaining induced by glutamate. These results suggest that the synthesis of c-Fos protein after glutamate exposure favors cell death pathway activation in which caspase-3 is also involved.

C-fos antisense oligonucleotides increase firing rate of striatal neurons in the anaesthetized rat

Extracellular electrophysiological recordings were used to study the firing rate of striatal neurons before and up to 4 h after intrastriatal c-fos antisense oligonucleotide injections in urethane-anesthetized rats. A four-fold increase from baseline neuronal activity was observed between 1 and 3 h upon antisense treatment, but not after control oligonucleotide injections. We conclude that, under urethane anesthesia, which here does not affect c-fos expression in the striatum by itself, neuronal activity appears to be tonically suppressed by basal striatal c-fos expression.

Protein Kinase C ϵ Dependence of the Recovery from Down-regulation of S1P1 G Protein-coupled Receptors of T Lymphocytes

Sphingosine 1-phosphate (S1P) from mononuclear phagocytes and platelets signals T cells predominantly through S1P1 G protein-coupled receptors (Rs) to enhance survival, stimulate and suppress migration, and inhibit other immunologically relevant responses. Cellular S1P1 Rs and their signaling functions are rapidly down-regulated by S1P, through a protein kinase C (PKC)-independent mechanism, but characteristics of cell-surface re-expression of down-regulated S1P1 Rs have not been elucidated. T cell chemotactic responses (CT) to 10 and 100 nm S1P and inhibition of T cell chemotaxis to chemokines (CI) by 1 and 3 microm S1P were suppressed after 1 h of preincubation with 100 nm S1P, but recovered fully after 12-24 h of exposure to S1P. Late recovery of down-regulated CT and CI, but not early down-regulation, was suppressed by PKC and PKCepsilon-selective inhibitors and was absent in T cells from PKCepsilon-null mice. The same PKCepsilon inhibitors blocked S1P-evoked increases in T cell nuclear levels of c-Fos and phosphorylated c-Jun and JunD after 24 h, but not 1 h. A mixture of c-Fos plus c-Jun antisense oligonucleotides prevented late recovery of down-regulated CT and CI, without affecting S1P induction of down-regulation. Similarly, S1P-elicited threonine phosphorylation of S1P1 Rs was suppressed by a selective inhibitor of PKCepsilon after 24 h, but not 1 h. Biochemical requisites for recovery of down-regulated S1P1 Rs thus differ from those for S1P induction of down-regulation.

Amyloid-β Induces Smac Release via AP-1/Bim Activation in Cerebral Endothelial Cells

Insoluble fibrils of amyloid-beta peptide (Abeta) are the major component of senile and vascular plaques found in the brains of Alzheimer's disease (AD) patients. Abeta has been implicated in neuronal and vascular degeneration because of its toxicity to neurons and endothelial cells in vitro; some of these cells die with characteristic features of apoptosis. We used primary cultures of murine cerebral endothelial cells (CECs) to explore the mechanisms involved in Abeta-induced cell death. We report here that Abeta(25-35), a cytotoxic fragment of Abeta, induced translocation of the apoptosis regulator termed second-mitochondria-derived activator of caspase (Smac) from the intramembranous compartment of the mitochondria to the cytosol 24 hr after exposure. In addition, we demonstrated that X chromosome-linked inhibitor-of-apoptosis protein (XIAP) coimmunoprecipitated with Smac, suggesting that the two proteins bound to one another subsequent to the release of Smac from the mitochondria. Abeta(25-35) treatment also led to rapid AP-1 activation and subsequent expression of Bim, a member of the BH3-only family of proapoptotic proteins. Bim knockdown using an antisense oligonucleotide strategy suppressed Abeta(25-35)-induced Smac release and resulted in attenuation of CEC death. Furthermore, AP-1 inhibition, with curcumin or c-fos antisense oligonucleotide, reduced bim expression. These results suggest that Abeta activates an apoptotic cascade involving AP-1 DNA binding, subsequent bim induction, followed by Smac release and binding to XIAP, resulting in CEC death.

Hyperdynamic circulation in portal-hypertensive rats is dependent on central c-fos gene expression

Portal hypertension is associated with hyperdynamic circulation, but the pathogenesis remains unclear. To clarify the role of central cardiovascular regulatory mechanisms, several protocols were conducted in rats with portal hypertension due to portal vein stenosis (PVS). Neuronal activation was quantified by immunohistochemical staining for Fos, the protein product of the c-fos gene. Fos expression in several brain nuclei with cardiovascular-regulatory roles was examined at 1, 3, 5, and 10 days following PVS surgery. This was correlated with development of cardiovascular changes measured at the same time points. Finally, Fos expression in the nucleus tractus solitarius (NTS) was blocked by local microinjection of c-fos antisense oligonucleotides twice daily for 5 days following PVS. The results showed that Fos-positive neurons were significantly increased in the paraventricular nucleus of hypothalamus, supraoptic nucleus, ventrolateral medulla, and NTS, detectable at day 1 and persistently increased at every day examined in the PVS rats. However, the hyperdynamic circulation developed between days 3 to 5. Administration of c-fos antisense oligonucleotides eliminated the hyperdynamic circulation in PVS rats, but had no effect on sham-operated controls. We conclude that the activation of central cardiovascular-regulatory nuclei, through a c-fos–dependent pathway, is necessary for development of hyperdynamic circulation in portal-hypertensive rats. (H EPATOLOGY 2002;35:159-166.)

Effects of c-Fos antisense and mis-sense oligonucleotides on Fos expression in the vestibular nucleus and vestibular compensation following unilateral labyrinthectomy

The aim of the present study was to investigate the effects of an antisense ologodeoxynucleotide (AS-ODN), complementary to the initiation codon of rat c-fos mRNA, on Fos protein expression in the medial vestibular nuclei (MVN) and behavioural recovery ('vestibular compensation') following unilateral labyrinthectomy (UL) in guinea pig. The AS-ODN (1 mM), which was infused into the IVth ventricle close the the MVN beginning at 1 h pre-UL and continuing to 5 h post-UL, inhibited expression of the Fos protein in the MVN; however, a mis-sense ODN (MS-ODN) had a similar effect, suggesting a non-sequence-specific mechanism of action. Neither the AS-ODN nor the MS-ODN had any significant effect on vestibular compensation relative to vehicle controls.

Effects of c-Fos antisense and mis-sense oligonucleotides on Fos expression in the vestibular nucleus and vestibular compensation following unilateral labyrinthectomy

Effects of c-Fos antisense and mis-sense oligonucleotides on Fos expression in the vestibular nucleus and vestibular compensation following unilateral labyrinthectomy

Light exposure activates retina ganglion cell lysophosphatidic acid acyl transferase and phosphatidic acid phosphatase by a c-fos-dependent mechanism.

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in ganglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in vitro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light; no increase was observed in preparations obtained from light-exposed animals treated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.

Antisense oligonucleotides to C-fos reduce postictal seizure susceptibility following fully kindled seizures in rats

In a previous study we demonstrated that increased FOS expression in the amygdala induced by partial kindling seizures could be attenuated by administering c-Fos specific antisense oligonucleotides. In addition, we found that the administration of c-Fos antisense oligonucleotides at the beginning of the amygdala kindling process facilitated the appearance of stage V kindled seizures. In the present study, we evaluated the effect of the suppression of FOS on the expression and severity of the generalized fully kindled seizures as well as on the susceptibility to additional ictal events during the postictal and interictal periods. We observed that the administration of c-Fos antisense oligonucleotides did not modify the behavioral and electrographic manifestations of generalized stage V kindled seizures. However, c-Fos antisense oligonucleotides significantly reduced the susceptibility to additional ictal events during the postictal refractory period. Hence, the increased FOS protein induced by generalized tonic clonic seizures may participate in postictal mechanisms.

Corticosteroid-resistant bronchial asthma is associated with increased c-fos expression in monocytes and T lymphocytes.

Unstimulated peripheral blood mononuclear cells (PBMCs) from corticosteroid-resistant (CR) but not corticosteroid-sensitive (CS) asthmatics demonstrate increased activating peptide-1 (AP-1)- and decreased glucocorticoid receptor (GR)-DNA binding. We test whether these abnormalities are associated with excessive generation of c-fos, the inducible component of AP-1. The c-fos transcription rate, mRNA and protein levels, and GR-DNA binding were quantitated in PBMCs, T cells, and monocytes from CS, CR, and nonasthmatic subjects. There was a 1.7-, 4.2-, and 2.3-fold greater increase in the baseline c-fos transcription rate, mRNA expression, and protein levels, respectively, in PBMCs derived from CR compared with CS patients. At optimal stimulation with PMA, there was a 5.7-, 3.4-, and 2-fold greater increase in the c-fos transcription rate, mRNA accumulation, and protein levels, respectively, in CR compared with CS PBMCs. These abnormalities were detected in both the T cell and monocyte subpopulations. PMA stimulation converted PBMCs from a CS to a CR phenotype and was associated with direct interaction between c-Fos and the GR. Pretreatment of PBMCs from CR patients with c-fos antisense oligonucleotides enhanced GR-DNA binding activity in CR PBMCs stimulated with dexamethasone. We suggest that increased c-fos synthesis provides a major mechanism for the increased AP-1- and decreased GR- DNA binding seen in CR asthma.

Functional neuroanatomy of the basal ganglia as studied by dual-probe microdialysis

Dual probe microdialysis was employed in intact rat brain to investigate the effect of intrastriatal perfusion with selective dopamine D 1 and D 2 receptor agonists and with c-fos antisense oligonucleotide on (a) local GABA release in the striatum; (b) the internal segment of the globus pallidus and the substantia nigra pars reticulata, which is the output site of the strionigral GABA pathway; and (c) the external segment of the globus pallidus, which is the output site of the striopallidal GABA pathway. The data provide functional in vivo evidence for a selective dopamine D 1 receptor-mediated activation of the direct strionigral GABA pathway and a selective dopamine D 2 receptor inhibition of the indirect striopallidal GABA pathway and provides a neuronal substrate for parallel processing in the basal ganglia regulation of motor function. Taken together, these findings offer new therapeutic strategies for the treatment of dopamine-linked disorders such as Parkinson’s disease, Huntington’s disease, and schizophrenia.

In vivo administration of c-Fos antisense oligonucleotides accelerates amygdala kindling

Repeated subconvulsive electrical stimulation of the amygdala leads to generalized seizures and provides an experimental model of epileptogenesis. Following electrical kindling stimulation the expression of c-Fos is rapidly induced. To evaluate the role of FOS protein in epileptogenesis, we used an antisense oligonucleotide strategy designed to inhibit its expression in the brain. Experimental and control oligonucleotides were delivered directly into the amygdala just prior to electrical stimulation. Immunocytochemical analysis showed that the administration of c-Fos antisense (but not sense) oligonucleotides inhibited expression of FOS in the amygdala following electrical stimulation. Behaviorally, treatment with c-Fos antisense oligonucleotides significantly accelerated the development of fully kindled (stage V) seizures. These data suggest that the increased FOS expression following electrical stimulation may be part of a protective mechanism which acts to inhibit epileptogenesis in the amygdala.

C-fos Antisense oligonucleotide microinjected into the medial preoptic area modulates body temperature during heat and cold exposure

The analgesic activity of acetyl-L-carnitine (ALCAR) in neuropathic pain is well established. By contrast, its potential efficacy in the relief of acute pain has not been reported. The antinociceptive effect of ALCAR was, therefore, examined in the mouse hot-plate and abdominal constriction tests, and in the rat paw-pressure test. ALCAR (100 mg kg−1 s.c. twice daily for seven days) produced an increase of the pain threshold in both mice and rats. ALCAR was also able to reverse hyperalgesia induced by kainic acid and NMDA administration in the mouse hot-plate test. The antinociception produced by ALCAR was prevented by the unselective muscarinic antagonist atropine, the M1 selective antagonists pirenzepine and S-(-)-ET126, and by the choline uptake inhibitor hemicholinium-3 (HC-3). By contrast the analgesic effect of ALCAR was not prevented by the opioid antagonist naloxone, the GABAB antagonist CGP 35348, the monoamine synthesis inhibitor (α)-methyl-p-tyrosine, and the Gi-protein inactivator pertussis toxin. Moreover, ALCAR antinociception was abolished by pretreament with an antisense oligonucleotide (aODN) against the M1 receptor subtype, administered at the dose of 2 nmol per single i.c.v injection. On the basis of the above data, it can be postulated that ALCAR exerted an antinociceptive effect mediated by a central indirect cholinergic mechanism. In the antinociceptive dose-range, ALCAR did not impair mouse performance evaluated by the rota-rod and hole-board tests.

Medial prefrontal cortical injections of c-fos antisense oligonucleotides transiently lower c-Fos protein and mimic amphetamine withdrawal behaviours

Prefrontal cerebral cortical areas display decreased expression of several transcription factor/immediate-early genes, including c-fos, during amphetamine withdrawal.[52]Antisense strategies can help to test possible roles for this prefrontal c-fos down-regulation in the behavioural correlates of amphetamine withdrawal. Medial prefrontal cortical injections delivering 1.7nmoles of anti c-fos oligonucleotides revealed an approximately 3h half-life for phosphothioate and a 15min half-life for phosphodiester oligonucleotides. Antisense phosphothioates complementary to the c-fos translational start site reduced levels of c-Fos protein, while exerting modest and variable effects on c-fos messenger RNA levels. Neither missense phosphorothioate nor antisense phosphodiester oligonucleotides significantly reduced levels of either c-fos messenger RNA or protein. Animals injected with anti c-fos phosphothioate oligonucleotides into the medial prefrontal cortex displayed marked reductions in linear locomotor activity and repetitive movements measured in a novel environment, effects not seen when missense oligonucleotides were used or when animals were accustomed to the activity monitor prior to antisense oligonucleotide injection.Behavioural changes produced by prefrontal cortical injections of c-fos antisense oligonucleotides closely mimic alterations recorded during amphetamine withdrawal. Prefrontal c-fos could thus conceivably play roles in the neurobiological underpinnings of psychostimulant withdrawal and of responses to stressors such as exposure to novel environments.