Differential expression of the c-fos protein and synaptophysin in zebrin II positive and zebrin II negative cerebellar cortical areas in 4-aminopyridine seizures.

The present study examined temporal activation patterns of rat cerebellar cortical neurons in 4-aminopyridine induced seizures, using c-fos protein as a marker of neuronal activity. C-fos-containing cells were counted in each cerebellar cortical layer, and cell count was compared between zebrin II positive and zebrin II negative bands of the lobules of the vermis and cerebellar hemispheres. We found significant activation of granule cells and interneurons of the molecular layer in zebrin II positive bands. The Purkinje cells, in contrast, exhibited non-significant, scattered c-fos immunoreactivity across all bands. Fluctuation of synaptophysin expression in the mossy fibre rosettes of the granular layer was determined via light microscopic immunohistochemistry. We detected a transient, significant decrease in synaptophysin staining density following 4-aminopyridine seizures, which may indicate short-term synaptic depression. We also identified different timing of increased c-fos expression in the neurons of the cerebellar cortex in different cortical zones. In particular, the activation pattern of the interneurons of the molecular layer reflected the climbing fibre distribution, reflecting the zonal olivo-cortico-nuclear organization. Seizure-induced activation of the granule cells corresponded with the zebrin II positive zones. This observation raises the possibility that zebrin II positive compartments may be more susceptible to cerebellar convulsions.

Non-competitive antagonists of NMDA and AMPA receptors decrease seizure-induced c-fos protein expression in the cerebellum and protect against seizure symptoms in adult rats.

The aim of the present study was to examine the role of ionotropic glutamate receptors in the cerebellum during generalized seizures. Epileptic neuronal activation was evaluated through the immunohistochemical detection of c-fos protein in the cerebellar cortex. Generalized seizures were precipitated by the intraperitoneal injection of 4-aminopyridine. The animals were pretreated with the NMDA receptor antagonists MK-801 (2 mg/kg), amantadine (50 mg/kg), and the AMPA receptor antagonist GYKI 52466 hydrochloride (50 mg/kg). Two hours after 4-aminopyridine injection, the number of c-fos immunostained cell nuclei was counted in serial immunohistochemical sections of the cerebellar vermis. The number of c-fos immunostained cell nuclei in the granular layer decreased significantly in animals pretreated with the glutamate receptor antagonists compared to the untreated animals having convulsion. We can conclude that mossy fiber stimulation exerts its seizure-generating action mainly through the ionotropic glutamate receptors of the mossy fiber synapses. Both NMDA and AMPA receptor antagonists are effective in reducing glutamate-mediated postsynaptic effects in the cerebellar cortex.

Interstrain differences of ionotropic glutamate receptor subunits in the hippocampus and induction of hippocampal sclerosis with pilocarpine in mice.

Rodent strains used in epilepsy research have various neurological characteristics. These differences were suggested to be attributed to the diverse densities of the ionotropic glutamate receptor (iGluR) subunits. However, previous studies failed to find interstrain differences in the hippocampal receptor levels. We supposed that a detailed layer-to-layer analysis of the iGluR subunits in the hippocampus might reveal strain-dependent differences in their base lines and reactions induced by pilocarpine (PILO) between two mouse strains without documented ancestors. Levels of iGluR subunits in Balb/c and NMRI mice were compared using semiquantitative immunohistochemistry. The alterations in the neuronal circuitry were validated by neuropeptide Y (NPY) and neuronal nuclear antigen (NeuN) immunostainings. Immunohistochemistry showed interstrain laminar differences in some subunits of both the control and PILO-treated animals. The seizure-induced irreversible neuronal changes were accompanied by reduced GluA1 and GluA2 levels. Their changes were inversely correlated in the individual NMRI mice by Pearson's method. Increase in NPY immunoreactivity showed positive correlation with GluA1, and negative correlation with GluA2. The NMRI strain was susceptible to PILO-induced hippocampal sclerosis, while the Balb/c animals showed resistance. Basal levels of iGluRs differ in mouse strains, which may account for the interstrain differences in their reactions to the convulsant.

Immunohistochemistry of cerebellar seizures: mossy fiber afferents play an important role in seizure spread and initiation in the rat.

Clinical reports suggest the participation of the cerebellum in epilepsy. Mossy fibers are the main excitatory afferents of the cerebellar cortex; most of them use glutamate and strongly excite granule cells through NMDA and AMPA receptors. The role of the ponto-cerebellar mossy fibers in cerebellar neuronal hyperactivity was investigated in the present study in experimental adult Wistar rats. We detected neuronal hyperactivity through the expression of the glutamate-induced c-fos protein, by means of immunohistochemistry and immunoblotting in the vermis and in the hemispheres. Generalized seizures were induced by means of intraperitoneal 4-aminopyridine injections. Following the 4-aminopyridine seizures, the c-fos expression of cerebellar granule cells was significantly elevated at 1.5h in every lobule. Maximum c-fos expression was seen at 3h. The role of the ponto-cerebellar mossy fiber afferents in the induction of c-fos expression was examined after the transection of the middle cerebellar peduncle on the left side. Immunohistochemical analysis 14 days after the surgery revealed that the synapsin I immunoreactivity was significantly reduced in the cerebellar cortex on the operated side, compared to the sham-operated controls and to the non-operated cerebellar hemisphere of the operated animals, indicating the degeneration of mossy fiber terminals. Transection of the middle cerebellar peduncle suppressed cerebellar c-fos expression in the vermis and in the hemispheres significantly. These findings suggest the strong involvement of the middle cerebellar peduncle and the ponto-cerebellar mossy fibers in the pathophysiology of cerebellar epilepsy.

Single-dose and chronic corticosterone treatment alters c-Fos or FosB immunoreactivity in the rat cerebral cortex.

The aim of this study was to examine the effects of single-dose and chronic corticosterone treatment on the inducible transcription factor c-Fos and FosB, and thereby to estimate the effects of high-doses of corticosterone on calcium-dependent neuronal responses in the rat cerebral cortex. At the same time we investigated the distribution of interneurons containing calretinin (CR), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) in chronically treated animals in order to collect data on the involvement of inhibitory neurons in this process. Adult male rats were injected subcutaneously with 10mg corticosterone, whereas controls received the vehicle (sesame oil). The animals were fixed by transcardial perfusion 12 and 24h following single corticosterone injection, and the brains were processed for c-Fos and FosB immunohistochemistry. To investigate the effects of repeated corticosterone administration, rats were daily treated with the same amount of corticosterone (10mg/animal, subcutaneously) for 21 days. Controls were injected with vehicle. At the end of the experiment, the rats were perfused and immunohistochemistry was used to detect the presence of the FosB protein, CR, VIP and NPY. Quantitative evaluation of immunolabelled cells was performed in the neocortex and the hippocampus. The number of immunoreactive nuclei per unit area was used as a quantitative measure of the effects of corticosterone. It was found that a single-dose administration of corticosterone resulted in a significant, time-dependent increase of c-Fos protein immunoreactivity in the granule cell layer of the dentate gyrus, as well as in regions CA1 and CA3 of the hippocampus 12 and 24h post-injection with respect to control animals. Significant enhancement of c-Fos immunoreactivity was also observed in the neocortex at 12 and 24h post-injection. Single-dose treatment did not significantly alter FosB immunolabelling. Repeated administration of corticosterone produced a complex pattern of changes in FosB immunolabelling: significant increase in FosB immunoreactivity was detected in the granule cell layer of the dentate gyrus, with no significant changes in the CA1 and CA3 layers of the hippocampus and in the neocortex. However, a significant decrease of FosB induction in the neocortex was observed in chronically treated rats in comparison to single-dose injected animals (12h before immunohistochemistry). Analysis of immunohistochemical detection of interneuronal markers revealed a significant reduction of the CR immunolabelling in the CA3 area of the hippocampus. No changes in VIP or NPY immunoreactivity were found in the Ammon's horn 3 weeks following daily corticosterone treatment. NPY immunoreactivity was significantly attenuated in the neocortex. The present data suggest that single-dose corticosterone treatment increases immunoreactivity of c-Fos protein in a time-dependent manner, 12 and 24h post-injection in the rat hippocampus and the neocortex, whereas chronic corticosterone treatment influences FosB immunoreactivity, primarily in the dentate gyrus. Chronic corticosterone administration seems to affect CR levels in the CA3 area of the hippocampus.

Late expression of FosB transcription factor in 4-aminopyridine-induced seizures in the rat cerebral cortex.

In this study, the immunolocalization of FosB transcription factor was investigated in acute and chronic experimental models of seizures induced by 4-aminopyridine. Wistar rats were injected intraperitoneally daily with 5mg/kg 4-aminopyridine for 1, 4, 8 and 12 days and sacrificed 24h after the last injection. Corresponding control groups received the solvent of 4-aminopyridine. Immunohistochemistry revealed an increase in FosB immunolabelling in the frontal cortex in 4-aminopyridine-treated animals compared to controls, both in acute and chronic time course groups. The dentate gyrus displayed elevated FosB immunopositivity only after repeatedly applied convulsant (4-aminopyridine), i.e. following 4, 8 and 12 days of treatment, but no significant immunolocalization was observed in the hippocampus proper. The neuronal localization of FosB after 12 days of 4-aminopyridine-induced convulsions was analysed by means of FosB-parvalbumin double immunolabelling. The increased number of double-labelled cells was significant in the frontal cortex, hilum of the dentate fascia and region CA1 of the hippocampus. We conclude that the studied neocortical and allocortical areas showed a different pattern of FosB immunolocalization, which suggests a relative deficiency of transcriptional regulation in the Ammon's horn and may be responsible for distinct response to seizure-induced cellular insult.

Mitigation of nociception via transganglionic degenerative atrophy: possible mechanism of vinpocetine-induced blockade of retrograde axoplasmic transport.

Vinpocetine, a derivative of vincamine, widely used in the clinical pharmacotherapy of cerebral circulatory diseases, inhibits retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve, resulting in transganglionic degenerative atrophy (TDA) in the related ipsilateral superficial spinal dorsal horn, as shown in our previous publications. TDA induced by vinpocetine has been demonstrated to be followed by depletion of the marker enzyme fluoride-resistant acid phosphatase (FRAP) and its isoenzyme thiamine monophosphatase (TMP), and by the decrease in the pain-related neuropeptide substance P from laminae I-II-(III) from the segmentally related, ipsilateral substance of Rolando of the spinal cord. In the present paper, we report on the behavioral effects of perineurally administered vinpocetine. Nociception, induced by intraplantar injection of formalin, was mitigated by vinpocetine; increased expression of c-fos in the ipsilateral, segmentally related upper dorsal horn was also prevented. Since vinpocetine is not a microtubule inhibitor, and its chemical structure differs from that of vincristin and vinblastin (used formerly by us in the therapy of intractable, chronic neuropathic pain), its mode of action is enigmatic. We assume that the effect of vinpocetine in blocking retrograde axoplasmic transport of NGF might be related to its interaction with membrane trafficking proteins, such as signalling endosomes and the endocytosis-mediating "pincher" protein. Temporary, locally restricted decrease of nociception, induced by vinpocetine, might be useful in the clinical treatment of intractable, chronic neuropathic pain, since vinpocetine can successfully be applied by transcutaneous iontophoresis.

Blockade of AMPA-receptors attenuates 4-aminopyridine seizures, decreases the activation of inhibitory neurons but is ineffective against seizure-related astrocytic swelling.

The neurotransmitter glutamate plays a pivotal role in the development of the neuropathological sequelae following acute seizures. Our previous data proved the efficacy of the NMDA-receptor antagonists on the symptoms, survival and neuronal activation in the 4-aminopyridine- (4-AP) induced seizures. In this study, we examined the effects of two different doses of a non-competitive, selective, allosteric AMPA-receptor antagonist, GYKI 52466. GYKI 52466 was effective in prolonging the latency to generalised seizures and reduction of seizure mortality. However, the effects on neuronal c-fos expression and astrocyte swelling were complex. The 25mg/kg dose of GYKI 52466 was effective in reducing the c-fos immunoreactivity (IR) in the hippocampus only. In the neocortex the overall c-fos-IR cell counts were increased significantly. Investigation of the neocortical parvalbumin-containing interneuron population proved that GYKI 52466 decreased c-fos expression. The 50mg/kg dose of GYKI 52466 significantly reduced the c-fos-IR in the neo- and allocortex, not only in principal neurons, but also in the parvalbumin-positive interneurons. The GYKI 52466-pretreatment did not prevent the astrocyte swelling in the investigated cortical areas; thus we conclude that the AMPA-receptors have little if any involvement in the in the mediation of neuropathological alterations in acute convulsions.

Prevention of electrical stimulation-induced increase of c-fos immunoreaction in the caudal trigeminal nucleus by kynurenine combined with probenecid.

The systemic administration of nitroglycerine, regarded as a migraine model, was previously observed to result in an increased number of c-fos immunoreactive secondary sensory neurons in the caudal trigeminal nucleus, which forward nociceptive impulses to the thalamus. The present investigation tested the hypothesis of whether kynurenine in combination with systemically administered probenecid protects second-order trigeminal neurons against stimulation arriving via central processes of trigeminal ganglion cells. Electrical stimulation of the trigeminal ganglion, one of the experimental migraine models, is known to induce an increase in the number of c-fos immunoreactive second-order nerve cells projecting to the thalamus. Since the synapses between first- and second-order trigeminal neurons are presumed to be mediated by excitatory amino acids, postsynaptic NMDA receptors should be inhibited by kynurenic acid, an endogenous NMDA receptor antagonist. Kynurenic acid, however, does not cross the blood-brain barrier, and its use as a neuroprotective agent is therefore not feasible. In contrast, kynurenine, from which kynurenic acid is formed on the action of kynurenine aminotransferase, passes the blood-brain barrier without difficulty. After the i.p. injection of kynurenine combined with probenecid it was found that the stimulation-induced increase in the c-fos immunoreactivity of the secondary sensory neurons does not occur.

Effect of vinpocetine on retrograde axoplasmic transport.

Vinpocetine, a derivate of vincamine, is widely used in the clinical pharmacotherapy of cerebral circulatory diseases. Herewith we report on a novel effect of vinpocetine: inhibition of retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve. Blockade of retrograde transport of NGF results in transganglionic degenerative atrophy (TDA) in the segmentally related ipsilateral superficial spinal dorsal horn, which is characterized by depletion of the marker enzymes fluoride-resistant acid phosphatase (FRAP) and thiamine monophosphatase (TMP). At the same time, pain-related neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), are depleted from lamina I-III from the segmentally related, ipsitateral Rolando substance of the spinal cord. On the basis of these experiments it is suggested that vinpocetine may result in a locally restricted decrease of nociception, that might be useful in clinical treatment of intractable pain. Pilot self-experiments support this assumption.

Alterations of seizure-induced c-fos immunolabelling and gene expression in the rat cerebral cortex following dexamethasone treatment.

We examined the effects of dexamethasone on the expression of the inducible transcription factor c-fos in 4-aminopyridine (4-AP) seizures. Induction of c-fos mRNA due to 4-AP-elicited convulsion was detected by means of the polymerase chain reaction (PCR) in samples from the neocortex. Adult male rats were pretreated with different doses of dexamethasone (0.5, 1, 3, 5mg/kg body weight); 1h later 5mg/kg 4-AP was injected intraperitoneally. Controls received the solvent of dexamethasone. Pretreatment with dexamethasone provided significant symptomatic protection against 4-AP-induced convulsions. Immunohistochemistry was used to evaluate the presence of the c-fos protein. The number of Fos-immunoreactive nuclei per section area was measured in the neocortex and hippocampus. Pretreatment with dexamethasone resulted in a dose-dependent, significant decrease of seizure-induced Fos-protein immunoreactivity in the neocortex, in the hilum of the dentate fascia, as well as in regions CA1-3 of the hippocampus, compared to control animals. Brains processed for mRNA isolation and PCR, displayed a significant increase of c-fos mRNA following the 4-AP treatment, while pretreatment with dexamethasone did not prevent or decrease this boosted c-fos mRNA expression. We conclude that seizure-induced c-fos expression and intracellular Fos-protein localization are mediated by transmitter and receptor systems, and dexamethasone significantly decreases Fos immunoreactivity, probably by regulating the intracellular traffic of the protein. We also conclude that dexamethasone does not interfere with the genomic regulation of c-fos mRNA synthesis.

Effect of 6-hydroxydopamine treatment on kynurenine aminotransferase-I (KAT-I) immunoreactivity of neurons and glial cells in the rat substantia nigra.

Parkinson's disease (PD), a progressive neurodegenerative disorder, is characterized by a preferential loss of dopaminergic neurons in the substantia nigra pars compacta (SNPC). Neurons in the SNPC are known to express tyrosine hydroxylase (TH); therefore, in a commonly used PD model, 6-hydroxydopamine (6-OHDA), a selective catecholamine neurotoxin, induces neuronal death in SNPC. We have shown with immunohistochemical techniques that kynurenine aminotransferase-I (KAT-I), the enzyme taking part in the formation of kynurenic acid (KYNA)--the only known endogenous selective NMDA receptor antagonist and a potent neuroprotective agent--is also expressed in the rat SNPC. We found that KAT-I and TH co-exist in the very same neurons of SNPC and that 6-OHDA injected into the lateral ventricle produced loss of the majority of nigral neurons. Densitometric analysis proved that, in consequence of 6-OHDA treatment, not only TH but also KAT-I immunoreactivity diminished considerably in the remaining SNPC neurons. Astrocytes in the substantia nigra were found to express KAT-I under normal conditions; the amount of this enzyme increased after administration of 6-OHDA, whereas microglial cells became KAT-I immunoreactive only after 6-OHDA treatment. Since intrinsic KYNA in SNPC neurons is perceptibly insufficient to protect them from the deleterious effect of 6-OHDA, it is hypothesized that biochemical approaches which increase KYNA content of the central nervous system might prevent the deleterious effect of 6-OHDA and, supposedly, also the neuronal degradation characterizing PD.

Calcium-binding proteins in GABAergic calyciform synapses of the reticular nucleus.

Large calyciform synapses in the rat reticular thalamic nucleus are characterized by the presence of gamma-aminobutyric acid. Presynaptic terminals are also loaded with calcium-binding proteins such as parvalbumin, calbindin, calretinin and calcineurin. The number of calyciform terminals containing gamma-aminobutyric acid and parvalbumin is 2005 in young adult rats; calbindin is present in 1,500, calretinin in 850 and calcineurin in 560 calyciform terminals. Developmental studies revealed that gamma-aminobutyric acid and calcium-binding proteins are virtually absent from calyciform terminals at birth but their occurrence increased considerably during postnatal life, suggesting increasing regulation of presynaptic calcium signaling during postnatal life. It is concluded that synaptic activity of large calyciform gamma-aminobutyric acid-containing synapses of the reticular thalamic nucleus is mediated, regulated or accompanied by calcium ions.

Lateral entorhinal cortex lesions rearrange afferents, glutamate receptors, increase seizure latency and suppress seizure-induced c-fos expression in the hippocampus of adult rat.

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurones in chronic epilepsy. Here we analysed the effects of one-sided lateral EC (LEC) and temporoammonic (alvear) path lesion on the development and properties of 4-aminopyridine-induced seizures. Electroencephalography (EEG) analysis of freely moving rats identified that the lesion increased the latency of the hippocampal seizure significantly and decreased the number of brief convulsions. Seizure-induced neuronal c-fos expression was reduced in every hippocampal area following LEC lesion. Immunocytochemical analysis 40 days after the ablation of the LEC identified sprouting of cholinergic and calretinin-containing axons into the dentate molecular layer. Region and subunit specific changes in the expression of ionotropic glutamate receptors (iGluRs) were identified. Although the total amount of AMPA receptor subunits remained unchanged, GluR1(flop) displayed a significant decrease in the CA1 region. An increase in NR1 and NR2B N-methyl-d-aspartate (NMDA) receptor subunits and KA-2 kainate receptor subunit was identified in the deafferented layers of the hippocampus. These results further emphasize the importance of the lateral entorhinal area in the spread and regulation of hippocampal seizures and highlight the potential role of the rewiring of afferents and rearrangement of iGluRs in the dentate gyrus in hippocampal convulsive activity.

Effect of electrical stimulation of the reticular nucleus of the rat thalamus upon c-fos immunoreactivity in the retrosplenial cortex.

Electrical stimulation of the reticular nucleus of the rat thalamus results in activation of c-fos immunoreactivity in nerve cells of the ipsilateral retrosplenial cortex. The c-fos immunoreactive neurons are mainly concentrated in lamina IV of the retrosplenial cortex. Conversely, electrical stimulation of the retrosplenial cortex induced c-fos immunoreactivity in the ipsilateral reticular nucleus of the thalamus. The results of the electrical stimulation suggest a direct synaptic connection between the cerebral cortex and the ipsilateral reticular thalamic nucleus. Simultaneous immunohistochemical staining proves that the majority of nerve cells and dendro-dendritic terminals in the reticular thalamic nucleus contain parvalbumine and, at the same time, also GABA. The role of GABA-ergic parvalbumine immunoreactive terminals in the reticular thalamic nucleus seems to be related to integration and processing of impulses and attentional gating, distinguishing between noxious and innocuous inputs.

GABAergic parvalbumin-immunoreactive large calyciform presynaptic complexes in the reticular nucleus of the rat thalamus.

In the reticular thalamic nucleus of the rat, nearly all neurons are parvalbumin-immunoreactive. We found that in addition, though superficially similar to large parvalbumin-immunoreactive neurons, also numerous peculiar parvalbumin-immunoreactive complexes are present in the reticular thalamic nucleus which are not identical with parvalbumin-immunoreactive perikarya, as shown by nuclear variation curves. Light and electron microscopic immunocytochemical studies revealed that these parvalbumin-immunoreactive complexes are brought about by parvalbumin-immunoreactive calyciform terminals which establish synapses with large, parvalbumin-immunonegative dendritic profiles. Transection of thalamo-reticular connections did not cause any alteration of calyciform terminals in the reticular thalamic nucleus. Nuclear counterstaining revealed that parvalbumin-immunoreactive calyciform terminals originated from local parvalbumin-immunoreactive interneuronal perikarya, which, depending of the length of the "neck" protruding from the perikaryon, establish somato-dendritic, axo-dendritic or dendro-dendritic synapses. Light and electron microscopic immunocytochemical investigations prove that the parvalbumin-immunoreactive calyciform complexes contain also GABA, that are likely to be inhibitory. In accordance with literature data, our results suggest that parvalbumin-immunoreactive GABAergic calyciform terminals in the reticular thalamic nucleus may be instrumental in intrinsic cell-to-cell communications and, as such, may be involved in synchronisation of thalamo-cortical oscillations, in the production of sleep spindles and in attentional processes.

Neocortical c-fos mRNA transcription in repeated, brief, acute seizures: is c-fos a coincidence detector?

The effect of acute brief seizures on neocortical c-fos expression was investigated in rats injected with 5 mg/kg 4-aminopyridine. Electroencephalography in freely moving animals with implanted neocortical electrodes detected an average of 2.67 tonic-clonic convulsions within 1 h following the 4-AP treatment. Tissue samples of the somatosensory neocortex were collected at 30 min, 1 h, 3 h, 5 h and 8 h following the treatment for PCR and immunohistochemistry. The c-fos mRNA displayed the first significant rise at 1 h, and remained significantly higher through 3 h. The number of c-fos protein immunoreactive cells was significantly elevated already at 30 min, peaked at 1 h, and declined by 5 h. We conclude that in repetitive, brief seizures, the first convulsion does not increase c-fos RNA transcription, whilst the second causes a long-lasting gene expression and a large increase of c-fos protein synthesis. The phenomenon may have implications in the pathogenesis of human and animal epilepsies.

Repeated 4-aminopyridine seizures reduce parvalbumin content in the medial mammillary nucleus of the rat brain.

Parvalbumin (Pv) containing fast spiking neurons play a crucial role in synchronizing the activity of excitatory neuronal circuits in the brain. Alterations of parvalbumin content in these neurons can affect their spike characteristics and, ultimately, may increase the susceptibility of neuronal circuits to epileptic seizures. In the present study, we examined whether repeated 4-aminopyridine (4-AP)-induced seizures modify the regional parvalbumin contents in the rat brain. 4-Aminopyridine was injected intraperitoneally in adult rats, controls received the solvent. Animals were sacrificed at 3 h after a single acute treatment, or following repeated, daily treatments of 12 days. In situ hybridization (ISH) indicated significantly decreased parvalbumin mRNA level in the medial mammillary nucleus (MM) at 12 days. Western blotting revealed 20.1% significant decrease of parvalbumin content in the medial mammillary area, while parvalbumin immunohistochemistry indicated no change of the number of immunoreactive cells in the medial mammillary nucleus. The results reveal the downregulation of the transcription of the parvalbumin gene and the decrease of parvalbumin synthesis in medial mammillary nucleus neurons in response to experimental seizures.

Dynamics and regional distribution of c-fos protein expression in rat brain after a closed head injury.

The objective of this study was to define the time- and brain-area-related distribution of c-fos expression in the brain during the first 24 h following a closed head injury in rats. In the control groups (n = 32), only a few c-fos positive nuclei were observed in the brain and the c-fos staining did not change during the next 24 h. In the closed head injury group c-fos-positive cells were rare in the brain regions during the first 30 min. During the next 2 h, the number of c-fos-positive cells increased rapidly in the basal ganglions, the ventricular ependyma cells the corticospinal tract, the area postrema, the cerebral neocortex, and the corpus callosum. The increase was highest in the corpus callosum (317 +/- 44.5 mm(-2)), in the thalamic reticular nucleus (474.8 +/- 49.2 mm(-2)), in the dentate hilus (1090 +/- 187 mm(-2)) and in the cerebral neocortex (992 +/- 93 mm(-2)). Thereafter, the elevated c-fos expression gradually decreased and at 6 h post-closed head injury no significant differences were observed between the controls and the trauma group. We conclude that a closed head injury induces a large, transient increase of c-fos expression in the brain. Since the observed time course and regional differences in c-fos expression are in good agreement with the cognitive and memory deficits observed after human TBI it can be utilized in further investigations, especially to test the effects of various forms of pharmacological or cellular therapy.

Kynurenine aminotransferase in the supratentorial dura mater of the rat: effect of stimulation of the trigeminal ganglion.

Electrical stimulation of the trigeminal ganglion has been widely used as a model of nociception, characterizing migraine. This treatment is known to evoke release of neuropeptides and neurotransmitters from nerve fibers of the dura mater. On the basis of immunocytochemical investigations, we found that under normal conditions, surface membranes of Schwann cells surrounding nerve fibers in the supratentorial dura mater display kynurenine aminotransferase-immunoreaction (KAT-IR); also KAT-IR are the granules of mast cells and the cytoplasms of macrophages (histiocytes). In consequence of stimulation of the trigeminal ganglion, Schwann cells in the dura mater became conspicuously swollen while their KAT-IR decreased considerably; also KAT-IR of mast cells and macrophages decreased significantly. At the same time, nitric oxide synthase (NOS)-IR of nerve fibers in the dura mater increased, suggesting release of nitric oxide (NO), this is known to be involved in NMDA receptor activation leading to vasodilation followed by neurogenic inflammation. Because kynurenic acid (KYNA) is an antagonist of NMDA receptors, we hypothesize that KYNA and its synthesizing enzyme, KAT, may play a role in the prevention of migraine attacks.