l-Dopa stimulates c-fos expression in dopamine denervated striatum by combined activation of D-1 and D-2 receptors

Administration of l-dopa to unilaterally 6-hydroxydopamine-lesioned rats, activates the early gene c-fos in the lesioned caudate-putamen. D-1 receptor blockade by SCH 23390, preventedl-dopa-induced Fos-like immunoreactivity in the whole caudate-putamen, while D-2 receptor blockade by raclopride reduced Fos-like immunoreactivity only in the dorso-lateral portion. The results suggest thatl-dopa induces c-fos primarily through an activation of D-1 receptors, while D-2 receptor stimulation plays a facilitatory influence on D-1 mediated c-fos expression.     

Developmental and stimulus-specific expression of the immediate-early genezif268 in rat spinal cord

Expression of the cellular immediate-early gene,zif268, was investigated using immunocytochemical methods in cervical spinal cord of neonatal and adult rats. The postnatal expression ofzif268 follows a specific temporal and spatial sequence in the spinal dorsal horn. Neurons immunoreactive for Zif268 protein were not present in cervical spinal cord before postnatal day (P) 6. At P6 they were occasionally observed in Rexed's lamina I. By P11, a few additional, faintly labeled, Zif268-positive neurons appeared in lamina III. Around P16, however, many immunoreactive neurons were found in laminae I–III and a few in laminae IV–VIl. The number of Zif268-immunoreactive neurons decreased markedly by P21 and was further reduced by P26 to become virtually absent in adult rats. In adults, surgical exposure of peripheral nerves alone enhanced Zif268 expression, but this effect largely disappeared in less than 6 h. Electrical stimulation of the nerves with high-frequency long trains, typical of those known to elicit long-term neural plasticity, induced a marked increase in Zif268 expression in the dorsal horn. Stimulation with single pulses had a much weaker effect. Zif268 may thus play a role in stimulus-induced, long-term neural plasticity in the spinal cord.     

Differential expression of Fos and Zif268 in the nigrostriatal system after methamphetamine administration in a rat model of Parkinson's disease

The goal of this study was to examine the topological specificity of methamphetamine-induced activation of the immediate-early gene proteins, Fos and Zif268, in the nigrostriatal system in a unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease with or without intrastriatal grafts of fetal ventral mesencephalon. Methamphetamine (3 mg/kg, i.p.) induced Fos-like immunoreactivity (FLI) dominantly in the striatum and the globus pallidus (GP) on the intact side as well as in the substantia nigra pars reticulata (SNr) on the lesioned side in the 6-OHDA rats. Lower levels of methamphetamine-induced FLI in the striatum and GP on the lesioned side were restored by intrastriatal grafts which could completely suppress the methamphetamine-induced rotation. In the striatum, a similar tendency could be observed between Fos and Zif268 immunoreactivity following methamphetamine. However, sparse immunoreactivity of Zif268 could be detected in the GP and SNr on both sides in the 6-OHDA rats. Intrastriatal grafts had little influence on Zif268 expression in these two regions. The differential expression of Fos and Zif268 was observed among the three regions of the nigrostriatal system following methamphetamine in the 6-OHDA rats. This may suggest that Fos and Zif268 therefore possess gene-specific and region-specific functions in the basal ganglia nuclei.     

Involvement of NMDA receptors in Zif/268 expression in the trigeminal nucleus caudalis following formalin injection into the rat whisker pad

We investigated the involvement of N-methyl-D-aspartate (NMDA) glutamate receptor in the expression of the proteins Zif/268 and c-Fos elicited by painful stimuli. To this purpose, the effect of the administration of MK-801, an NMDA receptor antagonist, on Zif/268 and c-Fos expression following a noxious stimulus, represented by formalin injection into the whisker pad of rats, was examined in neurons of the trigeminal nucleus caudalis. Furthermore, the co-localization of formalin injection-evoked Zif/268 and c-Fos expression and subunit 1 of the NMDA receptor (NR1) was studied in this nucleus. Zif/268 or c-Fos immunoreactivity elicited by formalin injection was significantly reduced by pretreatment with MK-801 in the superficial layer of the trigeminal nucleus caudalis; more than 40% of the neurons expressing Zif/268 and c-Fos in this layer were also immunolabeled by NR1. On the other hand, there was little effect of MK-801 administration on Zif/268 and c-Fos immunoreactivity in the nucleus proprius and deep lamina V of the trigeminal nucleus caudalis, while most neurons expressing Zif/268 or c-Fos in these two regions were labeled by NR1. These results point out differences between the superficial and deeper layers of the trigeminal nucleus caudalis in the involvement of NMDA receptor in the mechanisms underlying the expression of protein products of immediate early genes induced by painful stimuli.     

Fluctuations of interictal brain imaging in repeated 123I-IMP SPECT scans in an epileptic patient

Summary Single photon emission computed tomography (SPECT) brain scans with N-isopropyl-(iodine 123) p-iodoamphetamine (¹²³I-IMP) were performed three times in interictal periods in a 35-year-old man with intractable frontal lobe epilepsy and normal X-ray CT findings. The first scan showed decreased ¹²³I-IMP uptake in the right frontal lobe. This abnormal image was regarded as the primary focus of his epilepsy on the basis of its regional agreement with focal epileptic discharges on EEGs. In the second scan, he showed normal imaging, while the third scan showed the same abnormal image as before, in the right frontal lobe. The frequency of his clinical seizures was almost unchanged during the intervals between scans and further EEGs recorded soon after each scan showed almost no changes in the basic activities and frequency of the epileptic discharges. Such fluctuations in SPECT brain imaging suggest that the severity of functional inactivation underlying the focal hypoperfusion image as an epileptic focus may fluctuate considerably in the interictal state with no relation to the clinical features of epilepsy.     

Ethosuximide converts ictogenic neurons initiating absence seizures into normal neurons in a genetic model

Absence epilepsy is a form of generalized epilepsy commonly seen in children. The neuronal process by which ethosuximide (ETX), a first choice anti‐absence drug, prevents absence seizures is still unresolved. Recent clinical findings have indicated that focal cortical regions are involved during absence seizures. Consistently, it has been shown in genetic models of absence epilepsy that epileptic discharges arise from a delimited region of the cerebral cortex. Here, we made simultaneous in vivo electrocorticographic and intracellular recordings from the cortical focus of the genetic absence epilepsy rat from Strasbourg and examined the effects of systemic injection of ETX at a therapeutic concentration. We show that the interruption of seizures by ETX is correlated with a recovery, in the hyperactive focus neurons, of physiologic values of membrane potential, firing rate, and pattern, as measured in analogous neurons from nonepileptic rats. These data suggest that the anti‐absence action of ETX results from the conversion of ictogenic cortical neurons into normal cortical neurons.     

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.     

Modulatory role of catecholamines in the transsynaptic expression of c-fos in the rat medial prefrontal cortex induced by disinhibition of the mediodorsal thalamus: a study employing microdialysis and immunohistochemistry

We studied the interaction of catecholaminergic and thalamic afferents of the medial prefrontal cortex (PFC) by analyzing the effects of catecholamine depletion on thalamus-induced c-fos expression in the PFC of freely moving rats. Thalamic projections to the PFC were pharmacologically activated by perfusing the GABA-A receptor antagonist bicuculline (0.03 mM or 0.1 mM) through a dialysis probe implanted into the mediodorsal thalamic nucleus. Bicuculline perfusion induced Fos-like immunoreactivity in the thalamic projection areas, including the PFC, and in the thalamic nuclei surrounding the dialysis probe. 6-Hydroxydopamine lesions of the ventral tegmental area causing a 70–80% depletion of catecholamines in the PFC did not influence the increase in the number of Fos-like immunoreactive nuclei in the prefrontal cortex in response to thalamic stimulation. However, densitometric image analysis revealed that the intensity of Fos-like immunoreactivity in the PFC of lesioned rats perfused with 0.1 mM bicuculline was higher than in correspondingly treated controls. The behavioral activity to bicuculline perfusion, an increase of non-ambulatory activity (0.03 mM) followed by locomotion and rearing (0.1 mM), was not changed in 6-hydroxydopamine-lesioned rats. It is suggested that the thalamically induced c-fos response is directly mediated by excitatory, presumably glutamatergic, transmission and not indirectly by an activation of catecholaminergic afferents of the PFC. The increase in the intensity of Fos-like immunostaining in strongly stimulated, catecholamine-depleted rats suggests that catecholamines modulate the degree to which thalamic activity can activate the PFC of awake animals.     

Age-related increase inc-fos expression in the sympathetic neurons of the rat superior cervical ganglion

C-fos expression was studied immunocytochemically in sympathetic neurons of the rat superior cervical ganglion (SCG). Fos-like immunoreactivity was confined to the principal neurons of the ganglion and was exclusively localized within their nuclei. In 2-month-old rats, the immunoreactivity was detected in 1.2% of the principal neurons with a density of 4.95 Fos-positive cells/mm² of ganglion area. This proportion increased with age and reached a value which was 6.5-fold higher in the 26-month-old rats than that in the young adult. A density of 24.5 Fos-positive cells/mm² of ganglion area was seen in the 26-month-old animals. The age-enhancedc-fos expression suggests that Fos may be involved in regulation of the genetic events associated with the adaptive changes in neuronal activity of the sympathetic ganglion during aging.     

Fos induction in the medullary dorsal horn and C1 segment of the spinal cord by acute inflammation in aged rats

In order to elucidate the effect of aging on nociceptive neurons in the central nervous system, c-fos was used as a marker of excitability of neurons in the medullary dorsal horn (MDH) and the first spinal segment (C1) following noxious stimulation of the lateral face of young and aged rats. The distribution of c-fos-positive cells was dense in the superficial laminae and sparse in the deep laminae of the MDH and C1 in both young and aged animals following subcutaneous injection of formalin into the lateral face, whereas few c-fos-positive cells were labeled after saline injection. The distribution of c-fos-positive cells in the superficial laminae of the aged rats was found to be denser and more rostro-caudally expanded compared to that in the young rats. C-fos-positive cells were distributed more rostro-caudally in aged than in young rats. There was no difference between young and aged rats in the distribution of c-fos-positive cells in the deep laminae. Substance P (SP), 5-HT and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) fibers and varicosities showed similar distribution density in the MDH and C1. Furthermore, many 5-HT-LI aberrant fibers and varicosities were observed in the MDH and C1 of the aged rats. The SP-LI and CGRP-LI cells in the trigeminal ganglion of aged rats were larger than those of young rats. These findings suggest that a deficit of the descending 5-HT inhibitory system produces the increment of c-fos-positive cells in the MDH and C1 of aged rats, resulting in the recruitment of a larger number of neurons in the superficial laminae of the MDH and C1 for conveying nociceptive sensory information to the central nervous system.     

PSA‐NCAM‐dependent GDNF signaling limits neurodegeneration and epileptogenesis in temporal lobe epilepsy

Polysialylated neuronal cell adhesion molecule (PSA‐NCAM), a polysialylated protein constitutively expressed in the hippocampus, is involved in neuronal growth, synaptic plasticity and neurotrophin signaling. In particular, PSA‐NCAM mediates Ret‐independent glial‐derived neurotrophic factor (GDNF) signaling, leading to downstream FAK activation. GDNF has potent seizure‐suppressant action, whereas PSA‐NCAM is upregulated by seizure activity. However, the involvement of Ret‐independent GDNF signaling in temporal lobe epilepsy (TLE) is not established. We tested the effects of PSA‐NCAM inactivation on neurodegeneration and epileptogenesis in a mouse model of TLE. In this model, unilateral intrahippocampal kainic acid (KA) injection induced degeneration of CA1, CA3c and hilar neurons, followed by spontaneous recurrent focal seizures. In the contralateral, morphologically preserved hippocampus, a long‐lasting increase of PSA‐NCAM immunoreactivity was observed. Inactivation of PSA‐NCAM by endoneuraminidase (EndoN) administration into the contralateral ventricle of KA‐treated mice caused severe degeneration of CA3a,b neurons and dentate gyrus granule cells in the epileptic focus, and led to early onset of focal seizures. This striking trans‐hemispheric alteration suggested that PSA‐NCAM mediates GDNF signaling, leading to transport of neuroprotective signals into the lesioned hippocampus. This hypothesis was confirmed by injecting GDNF antibodies into the contralateral hippocampus of KA‐treated mice, thereby reproducing the enhanced neurodegeneration seen after PSA‐NCAM inactivation. Furthermore, contralateral EndoN and anti‐GDNF treatment decreased GDNF family receptor α1 immunoreactivity and FAK phosphorylation in the epileptic focus. Thus, Ret‐independent GDNF signaling across the commissural projection might protect CA3a,b neurons and delay seizure onset. These findings implicate GDNF in the control of epileptogenesis and offer a possible mechanism explaining lesion asymmetry in mesial TLE.     

Oxytocin neurons in the rat hypothalamus exhibit c-fos immunoreactivity upon osmotic stress

In order to evaluate the responses to osmotic stress of oxytocinergic neurons in vivo, we have studied oxytocin (OXY) and c-fos protein expression in the brain by means of double-immunostaining. C-fos immunoreactivity was detected in a subset of OXY neurons, as well as in other neurons non-immunoreactive for OXY, as early as 90 min after intraperitoneal injection of a hypertonic saline solution. C-fos expression was found in approx. 70% of OXY-immunoreactive neurons in the supraoptic (SON), lateral subcommisural (LSN) and paraventricular (PVN) nuclei, and not in OXY neurons in other hypothalamic areas. The expression of c-fos may be used as a means to map the circuitry by which osmotic stimulation activates OXY-containing neurons, and thus provide further insights into the functions with which OXY may be associated.     

Focal cortical dysplasia: pathophysiological approach

Overview Clinical and experimental studies on focal cortical dysplasia (FCD) were carried out.Materials and methods For the experimental study, an experimental FCD model of rats was developed. Twenty Wistar rats at 0–2 days after birth were used for the study. Kainic acid (KA) solution was injected stereotaxically into medial and lateral sites of the sensori-motor cortex. Bipolar electrodes were inserted in five rats. Their behavior and electroencephalogram (EEG) were recorded using a digital-video-EEG monitoring system. After observation periods of 1, 2, and 6 months, rats were perfused for pathological study. FCD was observed adjacent to the site of KA injection in all rats more than 1 month after the injection.Results and discussions EEG recording demonstrated focal spike discharges in and around the site of injection. However, clinical seizure was not observed. Pathological studies showed decrease in GABA-A receptors and increase in GABA-B receptors not only in the lesion but also in perilesional areas. Fifteen surgical cases of FCD with intractable epilepsy were subjected to the clinical study. Neuro-imaging studies including high-resolution magnetic resonance imaging and single-photon emission computed tomography were performed. Conventional EEG studies demonstrated focal EEG abnormalities with epileptic phenomena. At surgery, intraoperative electrocorticography (ECoG) was performed to localize epileptic foci under neuroleptoanalgesia. Thirteen patients showed epileptiform discharges on preresection ECoG. All foci in non-eloquent areas were resected. Pathological studies including immunohistochemical staining were performed, and the characteristics of the FCD in relation to EEG findings were analyzed. Patients in whom total lesionectomy with complete focus resection was performed had favorable postoperative courses. Nine patients (64.3%) have been seizure-free with reduced medication, and significant improvement was achieved in two patients (14.3%). Electrophysiological examination revealed epileptogenecity not only in the lesions but also in perilesional areas. The immunohistochemical studies showed a decrease in GABA-A receptors and an increase in GABA-B receptors in both the lesions and perilesional areas, but N-methyl-d-aspartate receptors were almost negative in both areas. Glutamate R1 was decreased in both areas, but glutamate R2 was increased in both areas. These findings support the results of a electrophysiological study.Conclusions In conclusion, not only the epileptic property of experimental focal cortical dysplasia but also perilesional epileptogenesis was demonstrated. These findings supported the results of surgery for patients with focal cortical dysplasia. In cases of FCD, total removal of the lesion and resection of the perilesional epileptic focus are needed for a good outcome.     

Vomeronasal organ-mediated induction of fos in the central accessory olfactory pathways in repetitively mated female rats

Removal of the VNO significantly reduced the enhancement of lordosis and the induction of fos immunoreactivity in luteinizing hormone-releasing hormone (LHRH) neurons in ovariectomized estrogen-primed rats. There was a significant positive correlation between the two variables. In the accessory olfactory bulb (AOB) of the repetitively mated rats, the number of fos-positive cells in the granule (G) cell layer was significantly lower in the VNO-removed rats, whereas that in the mitral (M) cell layer was not significantly different between VNO-removed and VNO-sham females. The G/M ratio (calculated by dividing the mean number of fos-positive cells in the G cell layer by that in the M cell layer), taken as an estimate of the output of the AOB, was relatively larger in the VN-sham as compared with the VNO-removed rats. There were significant positive correlations between G/M ratio and the increase in LQ and between the G/M ratio and the percentage of fos-positive LHRH cells. The positive correlation between the number of fos-positive cells in the posterodorsal medial amygdala (PDMA) and the increase in LQ and that between the number of fos-positive cells in the PDMA and the percentage of fos-positive LHRH cells were significant, supporting the role of the medial nucleus of amygdala in lordosis. However, the correlation between G/M ratio and the number of fos-positive cells in the PDMA was not significant, indicating that fos immunoreactivity in the PDMA is not directly related to that in the AOB. In conclusion, the results support the involvement of the accessory olfactory system in mediating the facilitatory effects of repeated mating on lordosis in female rats and suggest that the influence of the accessory olfactory system is mediated likely through the LHRH neuronal system. Integration and filtering of sensory information may take place at various levels of the brain, such as the AOB and the medial amygdala, before being transmitted to higher brain centers controlling lordosis behavior in female rats.     

Differences in c-fos immunoreactivity due to age and mode of seizure induction

The aim of this study was to determine whether the regional distribition and time course of immunoreactivity to the c-fos protein varies with maturation and method of seizure induction. The effect of the two chemical convulsants, pentylenetetrazol (PTZ) and flurothyl, on the spatial and temporal pattern of c-fos-like immunoreactivity in immature (postnatal day (P) 10) was compared to that in adult rats. Patterns of c-fos-like immunoreactivity following O₂ deprivation were also evaluated at the 2 ages because hypoxia is acutely epileptogenic in immature animals but not adults. C-fos-like immunoreactivity was examined at 2, 4, and 6 h after onset of chemically induced seizures or O₂ deprivation at both ages. After PTZ or flurothyl seizures, both ages exhibited similar patterns of IR in amygdala, pyriform cortex, and hypothalamus. Age-dependent regional differences were most prominent in cortex: superficial layers of retrosplenial, cingulate, and neocortex stained in adults; staining was confined to deep layers of neocortex in P10 rats. Intense staining of dentate gyrus and hippocampus occurred with more prolonged seizures, but not brief seizures. PTZ administration resulted in staining at 2 h after seizure onset and was reduced by 4 h in adults, but immunoreactivity was not seen until 4 and 6 h after seizure onset in immature rats, indicating an age effect on the time course of IR. In immature rats, immunoreactivity patterns after hypoxia were markedly different from PTZ or flurothyl; staining was confined to layer VI of neocortex in these animals, and rarely involved limbic structures. These differences in the pattern of c-fos immunoreactivity suggest that the neuronal populations involved in epileptogenesis are influenced by age as well as seizure phenotype and intensity.     

Control of scar formation in experimentally induced epilepsy

Penfield proposed that the meningocerebral scar that forms following trauma to the brain plays an important role in the development of posttraumatic epilepsy. Although the epileptogenic scar has come to be widely accepted as a cause of epilepsy, there is no direct evidence that scar formation contributes to epileptogenesis. This current study showed that procedures that control the development of collagen in a fibroblastic scar may modify the development of epilepsy. Epilepsy induced in the guinea pig by injection of metallic aluminum powder into the cerebral cortex was used as a model of posttraumatic epilepsy. Following application of aluminum and implantation of epidural electrodes, animals received either daily injections of prednisolone or an ascorbic acid-deficient diet to block scar formation. Control animals also had an injection of aluminum, but afterward received saline injections or a normal diet. Control animals developed epileptic spikes and often exhibited focal seizures. All manifestations of epileptogenesis were markedly reduced in animals treated with prednisolone or the ascorbic acid-deficient diet. The reduction in epileptiform activity corresponded to reduced collagenous scar formation in the treated animals. Although effective when given prophylactically, prednisolone did not inhibit the activity of an already established epileptic focus whether induced by aluminum or by amygdala kindling, nor did it block pentylenetetrazol-induced seizures. The finding that epileptogenesis is blocked by two procedures that inhibit scar formation but show no evidence of a direct anticonvulsant effect, suggests that scar formation is a significant factor in epileptogenesis induced by metallic aluminum. The collagenous component appears to be more significant than the glial component of the scar.