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Notes: Abstract: [3H]Norepinephrine ([3H]NE) efflux from preloaded rat Hippocampal slices was increased in a dose-dependent manner by excitatory amino acids, with the following order of potencies: N-methyl-D-aspartate (NMDA) 〈 kainic acid (KA) 〈 L-glutamate ≥ D,L-homocysteate 〈 L-aspartate 〈 quinolinic acid 〈 quisqualic acid. The effect of the excitatory amino acids was blocked by physiological concentrations of Mg2+, with the exception of KA. D.,L-2-Amino-7-phosphonoheptanoic acid dose-dependently inhibited the NMDA effect (ID50= 69 μM), whereas at 1 mM it was ineffective versus KA. The release of [3H]-NE induced by quinolinic acid was blocked by 0.1 mM D,L-2-amino-7-phosphonoheptanoic acid. γ-D-Glutamyl-glycine dose-dependently inhibited the KA effect with an ID50 of 1.15 mM. Tetrodotoxin (2 μM) reduced by 40 and 20% the NMDA and KA effects, respectively. The data indicate that [3H]NE release from hippocampal slices can be used as a biochemical marker for pharmacological investigations of excitatory amino acid receptors and their putative agonists and antagonists.

Notes: Abstract: The technique of intracerebral dialysis in combination with a sensitive and specific radioenzymatic method was used for recovery and quantification of endogenous extracellular acetylcholine from the striata of freely moving rats. A thin dialysis tube was inserted transversally through the caudate nuclei, and the tube was perfused with Ringer solution, pH 6.1, at a constant rate of 2 μl min−1. The perfusates were collected at 10-min intervals. In the presence of 1 and 10 μM physostigmine, acetylcholine release was 4.5 ± 0.02 and 7.3 ± 0.3 pmol/10 min, respectively (not corrected for recovery). The latter concentration of the acetylcholinesterase inhibitor was used in all experiments. Under basal conditions, acetylcholine output was stable over at least 4 h. A depolarizing K+ concentration produced a sharp, reversible 87% increase in acetylcholine output. Both the basal and K+-stimulated release were Ca2+ dependent. The choline uptake inhibitor hemicholinium-3 (20 μg intracerebroventricularly) reduced striatal acetylcholine output to 35% of the basal value within 90 min. Scopolamine (0.34 mg/kg s.c.) provoked a sharp enhancement of acetylcholine release of ∼63% over basal values, whereas oxotremorine (0.53 mg/kg i.p.) transiently reduced acetylcholine release by 54%. These results indicate the physiological and pharmacological suitability of transstriatal dialysis for monitoring endogenous acetylcholine release.

Notes: Abstract: The extracellular content of taurine, glutamate, and glycine was measured by the novel method of brain dialysis in the acute phases following an intrahippocampal injection of the excitotoxic convulsant brain metabolite quinolinic acid (QUIN). Using bilaterally depth electrodes physically combined with hollow fibers for dialysis, it was possible to collect continuously brain perfusates while simultaneously monitoring brain activity in the unanesthetized rat. In separate animals, hippocampal amino acid tissue levels were measured 2 h after an intracerebral injection of a convulsant dose (156 nmol) of QUIN. When compared with those in animal receiving the nonconvulsant decarboxylation product of QUIN, nicotinic acid, no differences in tissue levels were detected. In contrast, the same dose of QUIN caused a selective increase (2.24-fold) in taurine levels in perfusates from the injected hippocampus. These changes were apparent prior to the onset of electrographic seizures and did not occur in the contralateral hippocampus where seizure activity was equally severe. Thus, increases in extracellular taurine, triggered by the presence of QUIN in the hippocampus, may reflect a selective tissue response to the neurotoxic (rather than the convulsant) effects of this excitotoxin.

Notes: Abstract: Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca2+ influx, we investigated whether SRIF counteracted NMDA-induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7–10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5–10 µM), simultaneously applied with excitatory amino acid, significantly reduced in a dose-dependent manner the neurotoxic effect of NMDA but not that of KA (0.25–0.5 mM). GABA (10 µM) partially protected neurons to a similar extent from NMDA- or KA-induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201-995; 10 µM) and vapreotide (RC 160; 10 µM), did not influence the NMDA-dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl-cyclic GMP (10 µM), a cyclic GMP analogue, whereas 8-(4-chlorphenylthio)-cyclic AMP (10 µM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose-dependent manner by SRIF (2.5–10 µM). Finally, both specific (Rp-8-bromoguanosine 3′,5′-monophosphate, 10 µM) and nonspecific [1-(5 isoquinolinylsulfonyl)-2-methylpiperazine (H7), 10 µM] cyclic GMP-dependent protein kinase (cGMP-PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA-induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP-PK presumably by regulation of the intracellular Ca2+ level.

Notes: Electrical stimulation of the parafascicular but not the ventrolateral or dorsomedial thalamic nucleus (ten 0.5 ms, 10 V pulses, 140 pA) of freely moving rats induced a frequency-dependent (2.5, 5,10 and 20 Hz) increase in the extracellular acetylcholine (ACh) content of the dorsal striatum, assessed by trans-striatal microdialysis. The time-dependent effect of 10 Hz stimulation was studied. The peak increase, 39% above baseline, was attained during 4 min of stimulation. This was blocked by coperfusion with 5 pM tetrodotoxin, indicating that the release we measured represents a physiological process. The facilitatory effect of parafascicular nucleus stimulation does not appear to be associated with indirect action through the cerebral frontal cortex because acute lesion of the excitatory corticostriatal afferents, which by itself reduced basal ACh release by 40%, did not modify the effect of 10 Hz stimulation. The possible involvement of the fasciculus retroflexus in the facilitation of ACh release was also ruled out. The non-competitive NMDA-type receptor antagonist MK-801, applied by reversed dialysis (30 pM) or systemically injected (0.2 mg/kg), significantly reduced the basal ACh output and prevented the tetanus-evoked increase in ACh release. The results provide in vivo evidence that the activity of the cholinergic neurons in the dorsal striatum is trans-synaptically modulated by parafascicular nucleus excitatory afferents through activation of the NMDA subtype of glutamate receptors that is probably located in the striatum.

Notes: Several lines of evidence suggest that N-methyl-D-aspartate (NMDA) receptors significantly contribute to the development of kindling. In addition, a lasting enhancement of the NMDA receptor function has been suggested to play a significant role in the chronic hyperexcitability occurring in the hippocampus after kindling epileptogenesis. We have investigated whether hippocampal kindling induces changes in the NMDA receptor at the molecular level by assessing the expression of mRNAs of the different spliced variants at the N-terminal (exon 5) and C-terminal (exon 21) position of the NMDA receptor 1 (NR1) gene by means of the reverse transcription-polymerase chain reaction. Alternative splicing at exon 5 confers different sensitivity of the NMDA receptor to polyamines while exon 21 encodes a 37-amino acid insert containing the major phosphorylation sites for protein kinase C. One week after the acquisition of stage 5 of kindling in rats (generalized tonic-clonic seizures), the relative abundance of the two alternatively spliced forms at the C-terminal domain, respectively containing (+) or lacking (−) exon 21, was reversed compared to controls (implanted with electrodes but not stimulated) in the dorsal hippocampus ipsilateral and contralateral to the electrical stimulation. The exon 21+/exon 21− mRNA ratio for controls was 1.3 ± 0.04 (mean ± SE); for ipsilaterally kindled rats it was 0.64 ± 0.05 (P 〈 0.05), and for contralaterally kindled rats it was 0.48 ± 0.07 (P 〈 0.01). Similar bilateral effects were observed in the ventral hippocampus (temporal pole). No changes were found 4 weeks after stage 5 seizures and 1 week after the induction of a single afterdischarge. No significant alterations were induced by kindling in the relative abundance of the spliced variants containing or lacking exon 5. Our findings show selective changes in alternative splicing of the NR1 gene after repeated application of an epileptogenic stimulus. This may generate receptors with different functional properties, which may contribute to the increased sensitivity for the induction of generalized seizures during kindling.

Notes: Galanin, a 29- or 30-amino acid neuropeptide, has been implicated in the modulation of seizures. In this study, we constructed a recombinant adeno-associated viral (AAV) vector to constitutively over-express galanin (AAV-GAL). The vector mediated efficient transduction of HEK 293 cells in vitro and robust galanin expression in vivo when injected into the rat dorsal hippocampus. Rats were administered kainic acid intrahippocampally 2.5 months following AAV-GAL or empty vector (AAV-Empty) injection to study the effect of vector-mediated galanin over-expression on seizures. AAV-GAL-injected rats showed a decreased number of seizure episodes and total time spent in seizures compared to AAV-Empty rats, despite similar latencies to development of the first EEG seizure and similar levels of neuronal damage in the CA3 region for both groups. These data show that recombinant AAV mediates strong and stable over-expression of galanin when injected into the rat hippocampus resulting in a significant anticonvulsive effect. The seizure suppression effect of galanin expression in the hippocampus by viral vectors may lead to novel therapeutic strategies for the treatment and management of intractable seizures with focal onset such as temporal lobe epilepsy.

Notes: Recent evidence shows that neuropeptide expression in the CNS is markedly affected by seizure activity, particularly in the limbic system. Changes in neuropeptides in specific neuronal populations depend on the type and intensity of seizures and on their chronic sequelae (i.e. neurodegeneration and spontaneous convulsions). This paper reviews the effects of seizures on somatostatin-containing neurons, somatostatin mRNA and immunoreactivity, the release of this peptide and its receptor subtypes in the CNS. Differences between kindling and status epilepticus in rats are emphasized and discussed in the light of an inhibitory role of somatostatin on hippocampal excitability. Pharmacological studies show that somatostatin affects electrophysiological properties of neurons, modulates classical neurotransmission and has anticonvulsant properties in experimental models of seizures. This peptidergic system may be an interesting target for pharmacological attempts to control pathological hyperactivity in neurons, thus providing new directions for the development of novel anticonvulsant treatments.

Notes: Limbic status epilepticus was induced in rats by unilateral 60-min electrical stimulation of the CA3 region of the ventral hippocampus. As assessed by RT-PCR followed by Southern blot analysis, transcripts of interleukin-1β, interleukin-6, interleukin-1 receptor antagonist and inducible nitric oxide synthase were significantly increased 2 h after status epilepticus in the stimulated hippocampus. Induction was maximal at 6 h for interleukin-1β (445%), interleukin-6 (405%) and tumour necrosis factor-α (264%) and at 24 h for interleukin-1 receptor antagonist (494%) and inducible nitric oxide synthase (432%). In rats with spontaneous seizures (60 days after status epilepticus), interleukin-1β mRNA was still higher than controls (241%). Immunocytochemical staining of interleukin-1β, interleukin-6 and tumour necrosis factor-α was enhanced in glia with a time-course similar to that of the respective transcripts. Sixty days after status epilepticus, interleukin-1β immunoreactivity was increased exclusively in neurons in one third of the animals. Multiple intracerebroventricular injections of interleukin-1 receptor antagonist (0.5 μg/3 μL) significantly decreased the severity of behavioural convulsions during electrical stimulation and selectively reduced tumour necrosis factor-α content in the hippocampus measured 18 h after status epilepticus. Thus, the induction of spontaneously recurring seizures in rats involves the activation of inflammatory cytokines and related pro- and anti-inflammatory genes in the hippocampus. These changes may play an active role in hyperexcitability of the epileptic tissue.

Notes: Several lines of evidence indicate that neuropeptide Y (NPY)-mediated neurotransmission in the hippocampus is altered by limbic seizures. The functional consequences of this change are still unresolved and clearly depend on the type of NPY receptors involved. We have investigated the role of NPY Y1 receptor subtypes, which are enriched in the dentate area of the hippocampus, on EEG seizures induced by a local injection of 0.04 μg kainic acid in rats.Intrahippocampal administration of 10 μg BIBP3226 (N2- (diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]d-arginamide), a non-peptide selective antagonist at the NPY Y1 receptors, increased threefold on average (P 〈 0.01) the time to onset of seizures and reduced the number of seizures and the total time in seizures three- and fourfold, respectively (P 〈 0.01). Its inactive S-enantiomer BIBP3435 was ineffective on seizure activity. One microgram [Leu31,Pro34]NPY, an agonist at Y1 receptors, did not modify per se the EEG sequelae induced by kainic acid but it antagonized the anticonvulsant effect of BIBP3226.These results indicate that NPY Y1 receptors in the hippocampus are involved in epileptic phenomena and suggest that selective Y1 receptor antagonists may be of value for attenuating limbic seizures.