Choline acetyltransferase, glutamic acid decarboxylase and somatostatin in the kainic acid model for chronic temporal lobe epilepsy

The aim of the study was to investigate neurochemical changes in a kainic acid (KA; 10 mg/kg, s.c.)-induced spontaneous recurrent seizure model of epilepsy, 6 months after the initial KA-induced seizures. The neuronal markers of cholinergic and gamma-aminobutyric acid (GABA)ergic systems, i.e. choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities, and a marker for neuropeptide, i.e. level of somatostatin, have been investigated. The brain regions investigated were the hippocampus, amygdala/piriform cortex, caudate nucleus, substantia nigra and the frontal, parietal, temporal and occipital cortices. Six months after KA injection, reduced ChAT activity was observed in the amygdala/piriform cortex (47% of control; p<0.001), increased ChAT activity in the hippocampus (119% of control; p<0.01) and normal ChAT activity in the other brain regions. The activity of GAD was significantly increased in all analysed cortical regions (between 146 and 171% of control), in the caudate nucleus (144% of control; p<0.01) and in the substantia nigra (126% of control; p<0.01), whereas in the amygdala/piriform cortex, the GAD activity was moderately lowered. The somatostatin level was significantly increased in all cortical regions (between 162 and 221% of control) as well as in the hippocampus (119% of control), but reduced in the amygdala/piriform cortex (45% of control; p<0.01). Six months after KA injection, the somatostatin:GAD ratio was lowered in the amygdala/piriform cortex (49% of control) and in the caudate nucleus (41% of control), whereas it was normal in the hippocampus and moderately increased in the cortical brain regions. A positive correlation was found between seizure severity and the reduction of both ChAT activities and somatostatin levels in the amygdala/piriform cortex. The results show a specific pattern of changes for cholinergic, GABAergic and somatostatinergic activities in the chronic KA model for epilepsy. The revealed data suggest a functional role for them in the new network that follows spontaneous repetitive seizures.     

Oxygen free radicals in rat limbic structures after kainate-induced seizures

Several indices of free radical generation were determined in limbic structures after kainate (KA)-induced seizure activity in adult and postnatal day (PND) 12 and 17 rats. Superoxide dismutase, catalase, and glutathione peroxidase activities were measured in piriform cortex and hippocampal subfields at 8, 16, 48 h, and 5 days after KA injection in adults and pups, and also at 3 weeks postinjection in adults. KA-induced seizure activity had no significant effect on enzyme activities in PND 12 and 17 rats. In adults, superoxide dismutase and catalase activities were significantly increased at 5 days after KA administration, and returned to preinjection levels by 3 weeks. Glutathione peroxidase activity was also increased significantly at 5 days postinjection, but remained elevated at 3 weeks. Lipid peroxidation, as indicated by malondialdehyde (MDA) concentration, exhibited an early significant increase at 8 and 16 h, followed at 48 h and 5 days by a significant decrease. At 3 weeks postinjection, MDA levels were still significantly decreased in CA3 and dentate gyrus. KA administration in PND 12 and 17 rats had no significant effect on MDA content. KA-induced seizure activity in adults also resulted in a large and sustained increase in protein oxidation in piriform cortex and hippocampus. The early increase in MDA and protein oxidation in adult rats strongly suggests the involvement of oxygen free radicals in the initial phases of KA-induced pathology, whereas the changes in scavenging enzyme activities and MDA content at 5 days and 3 weeks post KA injection possibly reflect glial proliferation subsequent to neuronal death.     

Alterations of taurine in the brain of chronic kainic acid epilepsy model

Summary. The aim of the study was to investigate the changes of taurine in the kainic acid (KA, 10 mg/kg, s.c.) chronic model of epilepsy, six months after KA application. The KA-rats used were divided into a group of animals showing weak behavioural response to KA (WDS, rare focal convulsion; rating scale <2 up to 3 h after KA injection) and a group of strong response to KA (WDS, seizures; rating >3 up to 3 h after KA injection). The brain regions investigated were caudate nucleus, substantia nigra, septum, hippocampus, amygdala/piriform cortex, and frontal, parietal, temporal and occipital cortices. KA-rats with rating <2 developed spontaneous WDS which occurred chronically and six months after KA injection increased taurine levels were found in the hippocampus (125.4% of control). KA-rats with rating >3 developed spontaneous recurrent seizures and six months after injection increased taurine levels were found in the caudate nucleus (162.5% of control) and hippocampus (126.6% of control), while reduced taurine levels were seen in the septum (78.2% of control). In summary, increased taurine levels in the hippocampus may involve processes for membrane stabilisation, thus favouring recovery after neuronal hyperactivity. The increased taurine levels in the caudate nucleus could be involved in the modulation of spontaneous recurrent seizure activity.     

Circadian rhythms in neurotransmitter receptors in discrete rat brain regions

Circadian rhythms were measured in alpha 1-, alpha 2- and beta-adrenergic, acetylcholine muscarinic (ACh), and benzodiazepine (BDZ) receptor binding in small regions of rat brain. Rhythms in alpha 1-receptor binding were measured in olfactory bulb, frontal, cingulate, piriform, parietal, temporal and occipital cortex, hypothalamus, hippocampus, pons-medulla, caudate-putamen and thalamus-septum. No rhythm was found in cerebellum. Rhythms in alpha 2-receptor binding were measured in frontal, parietal and temporal cortex, and pons-medulla. No rhythm was found in cingulate, piriform or occipital cortex, or hypothalamus. Rhythms in binding to beta-receptors were measured in olfactory bulb, piriform, insular, parietal and temporal cortex, hypothalamus and cerebellum. No rhythms were found in frontal, entorhinal, cingulate, or occipital cortex, hippocampus, caudate-putamen, or pons-medulla. Rhythms in ACh receptor binding were measured in olfactory bulb, parietal cortex and caudate-putamen. No rhythms were found in frontal or occipital cortex, nucleus accumbens, hippocampus, thalamus-septum, pons-medulla or cerebellum. Rhythms in BDZ receptor binding were measured in olfactory bulb, olfactory and occipital cortex, olfactory tubercle, nucleus accumbens, amygdala, caudate-putamen, hippocampus and cerebellum. No rhythms were found in parietal cortex, pons-medulla or thalamus-septum. The 24-hr mean binding to receptors varied between 3- and 10-fold, the highest in cortex and the lowest, usually, in cerebellum. The piriform cortex was particularly high in alpha 1- and alpha 2-adrenergic receptors; the nucleus accumbens and caudate, in ACh receptors; and the amygdala, in BDZ receptors. Most adrenergic and ACh receptor rhythms peaked in subjective night (the period when lights were off under L:D conditions), whereas most BDZ receptor rhythms peaked in subjective day (the time lights were on in L:D). Perhaps in the rat, a nocturnal animal, the adrenergic and ACh receptors mediate activity and the functions that accompany it, and the BDZ receptors mediate rest, and with it, sleep.     

Responses of Heat Shock Proteins hsp27, αB-Crystallin, and hsp70 in Rat Brain After Kainic Acid-Induced Seizure Activity

We determined the changes in the levels of the mammalian small heat shock protein of 25-28 kDa (hsp27) and the hsp alphaB-crystallin in various regions of rat brain after kainic acid-induced seizure activity by means of specific immunoassays. The levels of hsp27 in the hippocampus and entorhinal cortex were markedly increased and reached a maximum (1.5-2 microg/mg of protein) 2-4 days after the seizure. The levels of hsp27 in these regions were considerably high even 10 days after the seizure. A marked increase in levels of mRNA for hsp27 was also observed in the hippocampus of rats 1-2 days after the seizure. A severalfold increase in the levels of alphaB-crystallin was observed in the hippocampus and entorhinal cortex of rats 2 days after the seizure. However, the maximum levels were <50 ng/mg of protein. The levels of protein sulfhydryl group and glutathione were significantly reduced in the hippocampus of rats at 24 h after the seizure, which might have enhanced the expressions of hsp27 and alphaB-crystallin. The expression of inducible mammalian hsp of 70 kDa (hsp70) was also enhanced in the hippocampus of rats after the seizure, as detected by western and northern blotting analyses. Immunohistochemically, an intensive staining of hsp27 was observed in both glial cells and neurons in the hippocampus, piriform cortex, and entorhinal cortex of rats with kainic acid-induced seizure. However, in the cerebellum, where the receptors for kainic acid are also rich, hsp27 was barely induced in the same rats. This might be due to high levels of the cerebellar calcium-binding proteins parvalbumin and 28-kDa calbindin-D, which might have a protective effect against the kainic acid-inducible damage.     

Formation of the base modification 8-hydroxyl-2'-deoxyguanosine and DNA fragmentation following seizures induced by systemic kainic acid in the rat

The formation of oxidative DNA damage as a consequence of seizures remains little explored. We therefore investigated the regional and temporal profile of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) formation, a hallmark of oxidative DNA damage and DNA fragmentation in rat brain following seizures induced by systemic kainic acid (KA). Formation of 8-OHdG was determined via HPLC with electrochemical detection, and single- and double-stranded DNA breaks were detected using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated nick end-labeling (TUNEL), respectively. Systemic KA (11 mg/kg) significantly increased levels of 8-OHdG within the thalamus after 2 h, within the amygdala/piriform cortex after 4 h, and within the hippocampus after 8 h. Levels remained elevated up to sevenfold within these areas for 72 h. Smaller increases in 8-OHdG levels were also detected within the parietal cortex and striatum. PANT-positive cells were detected within the thalamus, amygdala/piriform cortex, and hippocampus 24-72 h following KA injection. TUNEL-positive cells appeared within the same brain regions and over a similar time course (24-72 h) but were generally lower in number. The present data suggest oxidative damage to DNA may be an early consequence of epileptic seizures and a possible initiation event in the progression of seizure-induced injury to DNA fragmentation and cell death.     

Biochemical consequences of kainic acid injection into the lateral brain ventricle in rat

Effects of a single intraventricular injection of kainic acid (KA) in a dose of 0.1 microgram per rat on the activity of different brain neurotransmitter systems were investigated. A decreased level of norepinephrine at 3 and 24 h and acceleration of its utilization at 3 h after application of KA were observed. These changes were also accompanied by a decreased level of dopamine at 24 h, increased utilization of dopamine at 3 h, increased levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid at 3 and 24 h, as well as by shortened time of the turnover of 5-hydroxytryptamine. No disturbances in the function of the aminergic systems were noted at 120 h after injection of KA. Lowered activity of glutamic acid decarboxylase in the striatum, hippocampus, hypothalamus and cerebellum was observed at 24 h after administration of KA. At 480 h following application of KA, this lowering persisted in the hippocampus only. The most prominent changes in the level of gamma-aminobutyric were observed at 120 h in the striatum, hippocampus and cerebellum. A decreased level of gamma-aminobutyric acid was found in the striatum and cerebellum at 480 h following injection of KA. The observed changes in the dynamic equilibrium between various neurotransmitter systems may be a consequence of the direct or indirect influence of KA.     

Dantrolene protects neurons against kainic acid induced apoptosis in vitro and in vivo

Apoptotic cell death induced by kainic acid (KA) in cultures of rat cerebellar granule cells (CGC) and in different brain regions of Wistar rat pups on postnatal day 21 (P21) was studied. In vitro , KA (100–500 μM) induced a concentration-dependent loss of cell viability in MTT assay and cell death had apoptotic morphology as studied by chromatin staining with propidium iodide (PI). In vivo , twenty-four hours after induction of status epilepticus (SE) by an intraperitoneal KA injection (5 mg/kg) we quantified apoptotic cells in hippocampus (CA1 and CA3), parietal cortex and cerebellum using PI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. We report that dantrolene, a specific ryanodine receptor antagonist, was able to significantly reduce the apoptotic cell death in CGC cultures and in hyppocampal CA1 and parietal cortex regions. Our finding can be valuable for neuroprotective therapy strategies in patients with repeated generalized seizures or status epilepticus.     

Biochemical markers of apoptosis in different brain regions after training

Activity of caspase-3 and content of DNA fragments with sizes 0.2-0.6 kbp and more than 4.0 kbp in the worm of the cerebellum, hippocampus and prefrontal cortex of the adult rat brain were estimated in 4 and 24 hours after the procedure of acoustic startle habituation and fear conditioning. Heterochronic changes in apoptotic markers in the examined brain structures were observed after training. Caspase-3 activity was decreased in the worm of the cerebellum and hippocampus, and DNA fragmentation was suppressed in the hippocampus and brain cortex. At the same time, both caspase activity and DNA fragmentation were increased in the hypothalamus. These results provide evidence for the involvement of apoptosis in the mechanisms of learning and memory in adult brain.     

Changes in Cholinergic but Not in GABAergic Markers in Amygdala, Piriform Cortex, and Nucleus Basalis of the Rat Brain Following Systemic Administration of Kainic Acid

Three days after systemic administration of kainic acid (15 mg/kg, s.c.), selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, and high-affinity choline uptake) and GABAergic parameters [benzodiazepine and gamma-aminobutyric acid (GABA) receptors] were studied in the frontal and piriform cortex, dorsal hippocampus, amygdaloid complex, and nucleus basalis. Kainic acid treatment resulted in a significant reduction of choline acetyltransferase activity in the piriform cortex (by 20%), amygdala (by 19%), and nucleus basalis (by 31%) in comparison with vehicle-injected control rats. A lower activity of acetylcholinesterase was also determined in the piriform cortex following parenteral kainic acid administration. [3H]Quinuclidinyl benzilate binding to muscarinic acetylcholine receptors was significantly decreased in the piriform cortex (by 33%), amygdala (by 39%), and nucleus basalis (by 33%) in the group treated with kainic acid, whereas such binding in the hippocampus and frontal cortex was not affected by kainic acid. Sodium-dependent high-affinity choline uptake into cholinergic nerve terminals was decreased in the piriform cortex (by 25%) and amygdala (by 24%) after kainic acid treatment. In contrast, [3H]flunitrazepam binding to benzodiazepine receptors and [3H]muscimol binding to GABA receptors were not affected 3 days after parenteral kainic acid application in any of the brain regions studied. The data indicate that kainic acid-induced limbic seizures result in a loss of cholinergic cells in the nucleus basalis that is paralleled by degeneration of cholinergic fibers and cholinoceptive structures in the piriform cortex and amygdala, a finding emphasizing the important role of cholinergic mechanisms in generating and/or maintaining seizure activity.     

Localization of type I insulin-like growth factor receptor messenger RNA in the adult rat brain by in situ hybridization.

Using multiple 35S-labeled oligonucleotide probes concurrently, the type I insulin-like growth factor receptor (IGF-I-R) mRNA was demonstrated by Northern blot hybridization in newborn and adult rat brain as a single species of approximately 11 kilobases. The probes were used to localize IGF-I-R mRNA by in situ hybridization in slices of adult rat brain. The highest levels of IGF-I-R mRNA expression were found in the glomerular and mitral cell body layers of the olfactory bulb, the granule cell body layers of the dentate gyrus and cerebellum, the pyramidal cell body layers of the piriform cortex and Ammon's horn, and the choroid plexus. The lowest levels of IGF-I-R mRNA expression were found in white matter. At the cellular level, IGF-I-R mRNA was expressed by a variety of neurons, by epithelial cells of the choroid plexus, and by ependymal cells of the third ventricle. Of the neuron types studied, the highest levels of IGF-I-R mRNA were consistently found in perikarya of mitral and tufted cells in the olfactory bulb, in pyramidal cells of the piriform cortex and Ammon's horn, and in granule cells of the dentate gyrus. There was a close congruency between the distribution of IGF-I binding and IGF-I-R mRNA at the regional level. Neuropil layers in the cerebral cortex, olfactory bulb, hippocampus, and cerebellum contained a high level of IGF-I binding, whereas the adjacent cell body layers contained a high level of the IGF-I-R mRNA. We conclude that in these regions, IGF-I-R mRNA is synthesized in neuronal cell bodies, and the receptors are transported to axons and dendrites in adjacent synapse-rich layers, where appropriate IGF effects are achieved.     

Cloning and localization of rgpr85 encoding rat G-protein-coupled receptor

In an attempt to isolate genes involved in the brain development using ordered differential display PCR, we cloned rgpr85 which encodes rat G-protein-coupled receptor with high degree of identity to the amine-like neurotransmitter receptors. This gene was found to be localized at rat chromosome 4q21. In situ hybridization demonstrated that rgpr85 was predominantly expressed in the developing brain and spinal cord. Hybridization signal was especially abundant within the embryonic cortical plates where postmitotic cortical neurons are localized. In the cerebral cortex, the expression of rgpr85 was gradually decreased postnatally and became undetectable by P18. However, weak but significant expression of rgpr85 was maintained in the adult hippocampal formation, olfactory bulb, and cerebellum. Interestingly, rgpr85 expression was transiently induced in the adult hippocampal formation, piriform cortex, and amygdaloid complex by kainic acid (KA) treatment. Thus, dynamic regulation of rgpr85 expression suggests an importance of rgpr85-mediated signaling in the development of cerebral cortex and in the KA-induced responses in the adult brain.     

Distribution and Developmental Regulation of Metabotropic Glutamate Receptor 7a in Rat Brain

Abstract: To determine the regional and cellular distribution of the metabotropic glutamate receptor mGluR7a, we used rabbit anti-peptide polyclonal-targeted antibodies against the C-terminal domain of mGluR7a. Here we report that immunocytochemistry at the light-microscopic level revealed that mGluR7a is widely distributed throughout the adult rat brain, with a high level of expression in sensory areas, such as piriform cortex, superior colliculus, and dorsal cochlear nucleus. In most brain structures, mGluR7a immunoreactivity is characterized by staining of puncta and fibers. However, in some regions, including the locus ceruleus, cerebellum, and thalamic nuclei, both cell bodies and fibers are immunopositive. The changes in levels of mGluR7a during development were investigated with immunoblotting and immunocytochemical analysis. Immunoblot analysis revealed that the levels of mGluR7a are differentially regulated across brain regions during postnatal development. In cortical regions (hippocampus, neocortex, and olfactory cortex), mGluR7a levels were highest at postnatal day 7 (P7) and P14, then declined in older rats. In contrast, mGluR7a levels were highest at P7 in pons/medulla and cerebellum and decreased markedly between P7 and P14. In these regions, mGluR7a immunoreactivity was at similar low levels at P14 and P21 and in adults. Immunocytochemical analysis revealed that staining for mGluR7a was exceptionally high in fiber tracts in P7 animals relative to adults. Furthermore, the pattern of mGluR7a immunoreactivity in certain brain structures, including cerebellum, piriform cortex, and hippocampus, was significantly different in P7 and adult animals. In summary, these data suggest that mGluR7a is widely distributed throughout the rat brain and that this receptor undergoes a dynamic, regionally specific regulation during postnatal development.