The effect of stimulus duration on noxious-stimulus induced c-fos expression in the rodent spinal cord

C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product,fos, has been proposed as an anatomical marker for neuronal activity following noxious peripheral stimulation. However, the literature on noxious-stimulus induced fos expression contains several puzzling observations on the time course and laminar distribution of neuronal labeling within the spinal cord. This study has analyzed the effect of stimulus duration on the expression of fos-like immunoreactivity (FLI) within the spinal cord of anesthetized rats. In order to examine the time course of fos expression following brief periods of stimulation, we required a type of stimulus that was intense enough to activate nociceptors but that did not produce tissue damage. We have therefore employed pulsed, high intensity electrical stimulation, with stimulus durations ranging from 3 s to 24 h. The results indicate that stimulus duration has a profound effect upon the number of labeled cells, the intensity of neuronal labeling, the laminar pattern of FLI, and the time course of fos expression. Brief stimulation periods induce relatively few and relatively lightly labeled neurons, located predominantly within the most superficial laminae of the dorsal horn. Maximal immunoreactivity appears approximately 2 h after stimulation has ceased, and disappears within hours. Continuous stimulation produces many more labeled cells, darker labeling, and FLI within both dorsal and ventral laminar regions. Maximal FLI is seen after approximately 4.5 h of continuous stimulation, with reduction in the number of labeled cells thereafter. These data indicate that the results of any study employing c-fos as a marker for neuronal activity may be affected by the duration of the exciting stimulus.     

Suppression of post-ischemic-induced Fos protein expression by an antisense oligonucleotide to c-fos mRNA leads to increased tissue damage

Activation of c-fos, an immediate early gene, and the subsequent upregulation of Fos protein expression occur following neural injury, including focal cerebral ischemia (fci). Fos and Jun form a heterodimer known as activator protein 1, which regulates the expression of many late effector genes. To study the downstream effects of c-fos expression following ischemia, we suppressed the translation of c-fos by administering an antisense oligonucleotide (AO) to c-fos mRNA. Eighteen hours prior to fci, male, Long Evans (LE) rats received intraventricular injections of AO, mismatched AO (MS) or artificial cerebrospinal fluid (aCSF). Fci was induced by permanent right middle cerebral artery occlusion. At 24-h post-occlusion, neurological function was assessed, and the animals were sacrificed. The brains were removed and stained with triphenyltetrazolium chloride for infarct volume determination. Fos immunohistochemistry was performed in separate animals to determine the effects of treatment on Fos expression number of Fos positive cells. AO administration reduced the number of cells with fci-induced Fos expression by 75%. No differences in neurological scores existed between any of the groups. AO-treated LE developed larger infarcts (40.1±1.0%, mean±S.D., p<0.001) than MS- or aCSF-treated controls (34.3±1.0%, 34.6±1.0%, respectively). These results suggest that c-fos activation and subsequent Fos protein expression exerts a neuroprotective effect, which is likely via upregulation of neurotrophins, following focal cerebral ischemia. This response, among others, may contribute to brain adaptation to injury that underlies functional recovery after stroke.     

Induction of c-fos gene expression by spinal cord transection in the rat

Sympathetic nerve activity is maintained after high spinal injury through circuits that remain in question. We evaluated patterns of c-fos gene induction as a monitor of spinal neurons responding to high spinal cord transection in the rat. Rats were anesthetized with isofluorane. Lower cervical or upper thoracic spinal segments were exposed, immersed in warm mineral oil and transected. Spinal cords were exposed but not transected in anesthetized controls. After 2.5 h, spinalized and control rats were perfused for immunocytochemistry. Cervical and thoracolumbar spinal segments and dorsal root ganglia were sectioned coronally. Tissues were incubated in primary, polyclonal antisera raised in rabbit or sheep against a peptide sequence unique to the N-terminal domain of Fos, and processed immunocytochemically. Neurons were induced to express Fos-like immunoreactivity (FLI), bilaterally, in the spinal gray, but not in primary sensory ganglia. Spinal cord transection induced neurons to express FLI in thoracic laminae I, IIo (outer substantia gelatinosa). Vre (lateral reticulated division), VII (lamina intermedia) and X, and the intermediolateral cell column. Lamina VIII was also labeled in spinal-injured but not in control animals. Immunolabeled nuclei were prominent in lumbar segments and were concentrated in the medial third of laminae I and IIo, and in laminae VII and X. Few cells were labeled in upper cervical or sacral segments. FLI was sparse in the spinal gray of controls and expressed mainly within the dorsal root entry zone of upper thoracic segments. Patterns of c-fos gene expression were site-specific and correlated with laminae that respond predominantly to noxious stimulation and that contain sympathetic interneurons. Laminae that are responsive to non-noxious stimuli and activated by walking, IIi, nucleus proprius, medial V and layer VI were not induced to express FLI. We conclude that neurons in specific spinal laminae that process high threshold afferents and that harbor neurons with sympathetic nerve-related activity are activated selectively by spinal cord transections. We hypothesize that peripheral afferents processed by spinal-sympathetic circuit neurons may regulate sympathetic discharge in the absence of supraspinal drive.     

Expression of c-fos, fos B, and egr-1 in the medial preoptic area and bed nucleus of the stria terminalis during maternal behavior in rats

The spatial and temporal pattern of expression of the protein products of immediate early genes (IEGs) c-fos, fos B, and egr-1 were mapped in medial preoptic area (MPOA) and ventral bed nucleus of stria terminalis (VBST) during maternal behavior in rats. Immunocytochemical analysis indicated significant increases in the number of cells expressing c-Fos after 2 h of pup exposure, while Fos B levels showed a delayed response, reaching maximal levels after 6 h.     

Light-induced zif268 expression is dependent on noradrenergic input in rat visual cortex

In the present paper we investigated the role of the noradrenergic projection from the locus coeruleus on the expression of the immediate early gene zif268 in the visual cortex of rats exposed to ambient light stimulation. Local administrations of 6-hydroxydopamine (6-OHDA), a specific toxin directed against the catecholaminergic system, were performed in the locus coeruleus prior to visual stimulation. Animals were stimulated for 2 h by ambient light, after a 2-week dark adaptation period. Sham-operated controls displayed a massive increase in the number of zif268 positive cells after light stimulation. To the contrary, lesioned animals demonstrated a dramatic reduction in the number of zif268 positive nuclei across all cortical layers. A few scattered immunopositive nuclei were identified in cortical layer IV, however, this region also underwent a significant reduction in the number of zif268 immunopositive nuclei. Our results indicate that the noradrenergic system plays an important role in the expression of zif268 in the visual cortex of rats exposed to ambient light after dark isolation.     

Increased renal interstitial hydrostatic pressure causes c-fos expression in the rat''s spinal cord dorsal horn

To describe a sympathetic afferent circuit, interstitial hydrostatic pressure in the left kidney was increased in anesthetized rats for 1.5 h to activate renal mechanoreceptor afferents. Following renal afferent stimulation, the number of immunocytochemically stained cells for the immediate early gene c-fos was increased within the dorsal horn of the spinal cord. Relative to the surgical control procedure, increasing renal interstitial hydrostatic pressure produced more immunocytochemically stained cells per tissue section in laminae I and II of the dorsal horn both ipsilateral and contralateral to the stimulated kidney in the three most caudal thoracic spinal segments. Further, the number of c-fos immunocytochemically stained cells per section in the dorsal horn ipsilateral to the stimulated kidney was 28% greater than the number of stained cells contralateral to it. The staining patterns in the dorsal horns of stimulated and control animals were similar with most labeled cells in laminae I and II. These results indicate that (1) c-fos immunocytochemical staining may be useful for tracing specific sympathetic afferent pathways, (2) sensory pathways affected by increased renal interstitial hydrostatic pressure include spinal neurons located at lower thoracic levels, and (3) some of this sympathetic afferent pathway is located contralateral to the stimulated kidney. Neurons in the contralateral dorsal horn activated by renal stimulation may mediate renorenal reflexes.     

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.     

Distribution of c-fos expressing dorsal horn neurons after electrical stimulation of low threshold sensory fibers in the chronically injured sciatic nerve

The distribution of proto-oncogene c-Fos protein-immunoreactive cells in the spinal cord dorsal horn was studied after electrical stimulation at Aα/Aß-fiber intensity of normal and previously injured sciatic nerves in urethane anesthetized rats. No or only occasional Fos protein-like immunoreactive cells were seen after stimulation of the normal uninjured nerve or after nerve transection without stimulation. Electrical nerve stimulation at 3, 12, and 21 days after sciatic nerve transection resulted in substantial increases in the numbers of Fos protein-like immunoreactive cell nuclei in each of Rexed's laminae I–V. Combined demonstration of Fos protein-like immunoreactivity and of glial fibrillary acidic protein-like immunoreactivity (astroglia) or OX-42 immunoreactivity (microglia), indicated that the observed Fos protein-like response was confined to neurons and not to astroglia or microglia. Combined demonstration in the spinal cord of Fos protein-like immunoreactive neurons and neurons labeled retrogradely with Fluoro-Gold from the gracile nucleus showed that some of the Fos protein-like immunoreactive neurons in Rexed's laminae III and IV contributed to the postsynaptic dorsal column pathway. The results indicate that stimulation at Aα/Aß-fiber intensity of a previously injured nerve gives rise to an abnormally increased activation pattern of postsynaptic neurons in the dorsal horn, some of which contribute to the postsynaptic dorsal column pathway.     

Expression of c-fos and inducible hsp-70 mRNA following a transient episode of focal ischemia that had non-lethal effects on the rat brain

Expression of c-fos and inducible hsp-70 mRNA was studied with in situ hybridization techniques at different times following an episode of middle cerebral artery (MCA) occlusion not resulting in any apparent lethal effect on the rat brain. hsp-70 and c-fos mRNA were found in the ipsilateral striatum and adjacent cortex. In the striatum, levels of hsp-70 mRNA increased from 1 to 2 and 4 h of reperfusion, whereas levels of c-fos mRNA decreased from 1 to 4 h of reperfusion. These results demonstrate that following non-lethal focal ischemia the brain areas within the MCA territory show high c-fos and hsp-70 mRNA expression response, illustrating the concomitant induction of these mRNAs in cells that survive the ischemic insult.     

Induction of c-fos gene expression by spinal cord transection in Sus scrofa

Functional responses of primary sensory afferents and spinal cord were monitored in swine subjected to a high cervical (C1) spinal transection. Two and a half hours after transection, dorsal root ganglia and cervical and thoracolumbar spinal segments were processed immunocytochemically for the c-fos gene product, Fos and related antigens. In spinal-transected animals, Fos-like immunoreactivity (FLI) was induced in spinal laminae I, V, VII and X and the intermediolateral cell column but not in sensory ganglia as compared to controls: spinal-intact age-matched littermates. Spinal laminae expressing FLI harbor sympathetic and somatic interneurons and may aid in maintaining sympathetic outflow.     

Clonidine diminishes c-jun gene expression in the cardiovascular sensitive areas of the rat brainstem

The present study investigated the effect of clonidine on the basal and inducible c-jun and c-fos mRNA expression in the nucleus tractus solitarius (middle, mNTS, and rostral, rNTS) and the rostral ventrolateral medulla (caudal, cRVLM, and rostral, rRVLM). Conscious rats received saline, clonidine (30 μg/kg, i.v.), saline plus sodium nitroprusside (NP), or clonidine plus NP. Under basal conditions (saline-infused rats), c-jun mRNA was expressed in the mNTS and rRVLM but not in the rNTS or cRVLM whereas c-fos mRNA was not detectable. Clonidine attenuated the increases in c-fos in the mNTS and cRVLM and c-jun gene expression in the mNTS and rRVLM caused by NP-evoked hypotension and also reduced the basal expression of c-jun mRNA in the mNTS and rRVLM. These findings establish a causal link between clonidine inhibition of c-fos expression in brainstem and its hypotensive action, and provide the first evidence that clonidine attenuates the expression of the closely linked c-jun gene in neurons implicated in centrally mediated hypotension.     

Expression of c-fos protein in rat brain after electrical stimulation of the aortic depressor nerve

To reveal central nervous system (CNS) structures involved in the baroreceptor reflex we studied the distribution of Fos protein-like immunoreactivity in the rat brain after one hour of electrical stimulation of the aortic depressor nerve (ADN). In 13 male Wistar rats under urethane the ADN was cut on both sides and the central ends were placed on stimulating electrodes. Intermittent (11 s on, 6 s off) electrical stimulation at parameters set to elicit a drop in mean arterial pressure of 15-30 mmHg was applied to one, both or neither ADNs for 1 h. CNS sections were incubated for 48 h in anti-Fos antibody and prepared for visualization of the reaction product using the ABC immunoperoxidase technique. Label was found in several discrete brain nuclei primarily on the side ipsilateral to the side of stimulation. In the medulla labelled nuclei were found in the nucleus tractus solitarius, area postrema, rostral and caudal ventrolateral medulla, nucleus ambiguus and medullary reticular formation. In the pons labelled neurons were found in the lateral and ventrolateral parabrachial nucleus, locus coeruleus, pontine reticular field and A5 region. In the forebrain labelled nuclei were observed in the peri- and paraventricular hypothalamus, supraoptic nucleus, subfornical organ, preoptic area, central nucleus of the amygdala, median preoptic area, horizontal limb of the diagonal band, bed nucleus of the stria terminalis and islands of Calleja. In control animals moderate amounts of label were present in the supraoptic nucleus and periventricular hypothalamus bilaterally. These results define central pathways involved in mediating the baroreceptor reflex.     

Peripheral bombesin induces c-fos protein in the rat brain

Bombesin injected intraperitoneally induces c-fos protein-like immunoreactivity in the medial nucleus tractus solitarius and the parvocellular part of the paraventricular nucleus of the hypothalamus in the rat brain. C-fos expression induced by bombesin is less densely represented compared with CCK. Capsaicin pretreatment did not influence c-fos-immunoreactivity induced by bombesin and significantly reduced that induced by CCK.     

Induction of the immediate early genes egr-1 and c-fos by methamphetamine in mouse brain

Methamphetamine (METH) is one of the most commonly abused psychostimulant, and is known to induce dopaminergic neurotoxicity by generating oxidative stress and free radicals. In the present study we investigated the effects of METH on egr-1 and c-fos immediate early gene induction in different regions of mouse brain, at different doses and different time courses. We also measured the tissue levels of monoamines in order to correlate their changes with gene expression. A single injection of METH (40 mg/kg) significantly increased egr-1 and c-fos mRNA expression within 30 min in frontal cortex, nucleus accumbens, caudate putamen, septum and CA1 region of hippocampus. Time course studies showed that in most cases, both genes were expressed within 30 min and decreased after 60 min. METH produced a significant decrease in striatal dopamine level, reaching a very low level after 24 h. Striatal serotonin level significantly increased and returned to control levels after 2 h. These data show that METH induced egr-1 and c-fos mRNA expression in selective brain areas, which correlated with an alteration in monoamines.     

NGF treatment potentiates c-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid

The induction of the c-fos gene in the rat brain by NGF was studied in a model of acute cholinergic hypofunction, i.e., the lesion of the nucleus basalis magnocellularis (NBM) with quisqualic acid. Choline acetyltransferase and Fos immunoreactivity (IR) in the NBM were analyzed at different times after the excitotoxic lesion. NGF treatment induced a potentiation of Fos expression 4 and 24 h after lesion. The possibility is discussed that c-fos induction is one of the early mechanisms of the neuroprotective action of NGF.     

C-fos gene expression in rat brain around birth: effect of asphyxia and catecholamines

At birth, the mammalian nervous system must adapt rapidly to the new conditions it encounters in the extra-uterine environment. One aspect of this adaptation, known as arousal, is mediated by catecholamines, the levels of which in the brain increase rapidly after birth. The pattern of gene expression also changes. Shortly after birth, expression of the immediate early gene c-fos, known to reflect general neural activity, is up-regulated. Furthermore, asphyxia often occurs in connection with birth. In order to examine the effects of this phenomenon on the expression of c-fos, as well as on the rate of noradrenaline (NA) turnover, asphyxia was induced in rat pups delivered by caesarean section. Northern blot analysis and in situ hybridization revealed that the increase in expression of c-fos in certain areas of the brain was greatly enhanced by asphyxia of moderate duration; whereas more prolonged asphyxia lowered the level of c-fos mRNA. Asphyxia had a similar effect on the rate of NA turnover. Adrenergic receptor antagonists administered prior to birth attenuated the birth-related induction of c-fos mRNA. However, the potentiation of c-fos expression by asphyxia was not altered by these antagonists. Therefore, we propose that while catecholamines play an important role in the induction of c-fos in the brain at birth, the effects of asphyxia involve a different mechanism.