| Abstrakt: |
Anterior Pericaudate White Matter Is Important for the Secondary Generalization of Kanic Acid-induced Amygdalar Seizures in Rats.Rationale:Previous studies indicated that the perirhinal cortex (PRC) is critical for the secondary generalization of amygdalar seizures (Yamada 1993, Imamura 1998). However, the exact pathway from the PRC to the sensorimotor cortex (SMC) remains obscure. McIntyre et al. suggested that a pathway from the PRC to the SMC exists in the white matter adjacent to the caudate nuclei, and we named this site the "anterior pericaudate white matter (APWM)." In this study, we investigated the role of the APWM in the secondary generalization of kanic acid (KA)-induced amygdalar seizures in rats.Methods:Twelve male Wistar rats were used in the experiment. All received stereotactic implantation of electrodes in the left amygdala (LA), left dorsal hippocampus (LH), and left sensorimotor cortex (LCx). A cannula was also inserted into the LA for the preparation of KA injection. The APWM was lesioned stereotactically in six rats (lesioned group) and the remaining six rats served as controls. All rats received KA injection (1.0μg) into the LA to provocate KA-induced amygdalar seizures, and electroclinical observation was made for 6 hours.Results:Stereotyped KA-induced amygdalar seizures appeared in all rats after 15 to 20 minutes. In the controls, secondary generalization was observed behaviorally and the apparent propagation to the LCx was observed with the electroencephalogram after 60 minutes. Seizure manifestations were less severe. In contrast, seizure propagation to the LCx was weaker in the lesioned group.Conclusions:The APWM may be an important area associated with the secondary generalization of KA-induced amygdalar seizures in rats. Vulnerability of the Hippocampal Redox Regulation in the Electrical Amygdaloid Kindled Rats.Purpose:Recent studies emphasize that the redox state in the brain plays an important pathophysiological role, contributing not only to neurotoxicity by free radical generation, but also to the function of transmembrane proteins. BothN-methyl-d-aspartate receptors (NMDA-R) and the excitatory amino acid transporters (EAATs), which are closely associated with glutamate neurotoxicity and epileptic activity, possess redox regulatory sites in their structures. The redox state in the brain is maintained at a constant balance between oxidative and reductive conditions. Under normal conditions, there is a steady-state, designated redox state, between the production of reactive oxygen species (ROS) and their destruction by the cellular antioxidant system. However, if ROS or free radicals are generated in excess of the amount that can be scavenged by the endogenous antioxidant system, the redox state in the brain would shift to an oxidative condition. Therefore, to explore the role of the redox state in epileptogenesis, it is important to investigate both the reductive, as well as oxidative, conditions simultaneously. To investigate the redox state in the hippocampus of the kindled rats, in vivo microdialysis was used with X-band electron paramagnetic resonance (EPR) spectroscopy.Methods:All animal experiments were reviewed and approved by the Animal Welfare Committee of Miyazaki Medical College (No. 1998-158-6). Twelve anesthetized male Wistar rats were operated upon to implant a single tripolar stainless steel electrode for kindling stimulation and 22-G guide cannulas coupled with dummy cannulas for in vivo microdialysis. The operated rats were divided into the kindled group (n= 6) and sham-operated (non-kindled) group (n= 6). Twenty-four hours after the last kindled seizure, 8 mM nitroxide radical (3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl; PCAM) was injected intraperitoneally (16μmol/kg), and the dynamics of PCAM was monitored by EPR spectrometer (FR 30, JEOL Ltd., Tokyo, Japan). Redox was estimated to be based on the principle that exogenous nitroxide radicals reaching the brain are reduced and lose their paramagnetism by reductants such as ascorbic acid,α-tocopherol, and GSH within the biological system. Therefore, the decay rate (i.e., the half-life) of the EPR signal intensity of the nitroxide radicals is assumed to reflect the antioxidant capability of the biological system.Results:The half-life was significantly prolonged in the kindled rats compared to the sham-operated group (p<0.01, Mann–Whitney U test).Discussion:The prolonged half-life of nitroxide radical elimination in the hippocampus of the amygdaloid-kindled rats signified a reduced in vivo antioxidant capability in the brain structure. These data confirmed an imbalance between free radical formation and the antioxidant capability, leading to an inclination of the redox state toward the oxidative condition, which may well explain the cell vulnerability and free radical neurotoxicity in the limbic system of kindled rats. These results were probably due to oxidative stress, such as ROS or free radicals, generated in the hippocampus of the kindled rats, and the persistent oxidized state of the hippocampus in this epilepsy model may be a result of the recurrent electrical stimulation. Cloned rat excitatory amino acids transporter (EAATs) subtypes distributed in the forebrain possess a sensing property, and undergo reciprocal functional changes in response to oxidation or reduction of reactive sulphydryls present in the structure. In particular, thiol oxidation and disulphide reduction result in reduced and increased uptake capacity, respectively. The thiol-based redox modulatory site in glutamate transporters is targeted by endogenous oxidants (such as ROS and free radicals) and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake. The oxidized redox state in the hippocampus of the kindled rats as shown in this study may be associated with the impaired regulation of the extracellular glutamate levels, causing increased glutamatergic excitatory synaptic transmission and glutamate neurotoxicity closely related to the pathophysiology of epilepsy. Apoptosis Induced by Bicuculline Involves the P/Q-Type Voltage-dependent Calcium Channel in Rat Hippocampal Slice Cultures.Purpose:The mechanism of epileptic cell damage has long been attributed to excitotocixity-induced necrosis. However, in recent years, evidence has accumulated to show that neurons also die by apoptosis following seizures. In this study, we subjected rat hippocampal slice cultures to chronic exposure to bicuculline (BiC), a GABAA receptor antagonist, to test the susceptibility of hippocampus neurons and determine whether the resulting neuronal cell damage involves apoptosis. We also investigated the type of voltage-dependent calcium channel (VDCC) involved in the neuronal cell death.Methods: Hippocampal slice cultures were prepared from 7–8-day-old Wistar rats and cultured by the interface method for 7 days. BiC was diluted to a final concentration of 6μM and applied for 12–48 hours. To determine the effects of the P/Q-type VDCC on neuronal cell toxicity, we used 200 nM ofω-agatoxin IVA, a P/Q- type VDCC antagonist, and roscovitine, a P/Q-type VDCC agonist. The viability marker PI was used to evaluate neuronal cell death in the slice cultures. In brief, 3 hours before treating the cultures with BiC and other drugs, 2μL of 2.5 mM PI was added and the PI uptake was recorded with a digital camera. The digital images were analyzed using a densitometry analysis program. To assess apoptosis, the cultivated slice was fixed with 4% paraformaldehyde (PFA) and immunocytochemistry was carried out with rabbit polyclonal antibodies specific to the activated form of caspase-3.Results: After exposure to 6μM BiC for 12 hours, the PI uptake in the CA2 sector (25.34± 5.60%, p<0.001) was significantly higher than that in the CA1 (12.46± 5.76%), CA3 (14.87± 2.86%), or DG (15.23± 4.70%) sector. After 24 hours, the PI uptake increased significantly in the CA2 (38.14± 6.93%, p<0.001) and CA3 (33.95± 9.45%, p<0.001) sectors compared with that of the CA1 (20.71± 5.79%) and DG (27.86± 10.32%) sectors. The PI uptake increased with time, and the area appeared to extend to the CA3 sector. The PI uptake was significantly inhibited after 12 hours in the presence of an antagonist (200 nMω-agatoxin; 16.25± 7.56, p<0.001), and was significantly enhanced with the addition of an agonist (20μM roscovitine; 30.25± 8.56, p<0.001). The effects of VDCC regulators were very clear after 24 or 48 hours. These results indicate that CA2 is the most vulnerable region in hippocampal slice cultures following chronic exposure to BiC, and that the neuronal cell damage process involves a CA2+ influx via P/Q-type VDCC. We also observed caspase-3 immunoreactivity (IR)-positive cells in the CA2 sector after exposure to BiC for 12 hours. The pattern of caspase-3 IR expression correlates well with the PI uptake pattern. However, there were fewer caspase-3 IR-positive cells than PI uptake cells. These findings indicate that the BiC induces neuronal death pathways involving necrosis and apoptosis, and almost all the neurons finally die via a common necrotic pathway in the rat hipppocampal slice cultures.Conclusions: We demonstrated that the neuronal death induced by BiC in hippocampal slice cultures was initiated in the CA2 region and the area seemed to extend to the CA3 sector. Neuronal death was mediated by pathways involving necrosis and apoptosis, and the P/Q-type VDCC played an important role in the two neuronal cell death pathways. Impairment of Monoaminergic Transmission inμ3B Knockout Mice.Purpose:Recently, we have generatedμ3B gene knockout (μ3BKO) mice resulting in the loss of neuron-specific adaptor protein complex type 3B (AP-3B) and spontaneous epileptic seizures. Our previous studies have demonstrated impaired GABA release and abnormal propagation of neuronal excitability via the temporoammonic pathway in (μ3BKO mice. However, whether other inhibitory neurotransmissions such as monoaminergic transmission are involved in the abnormal neuronal excitability of (μ3BKO mice has not been clarified. Thus, to explore the possible mechanisms of spontaneous seizure inμ3BKO mice, the present study determined the hippocampal dopamine (DA) and serotonin (5-HT) releases inμ3BKO mice and the background C57BL/6J (wild-type) mice, using in vitro minislices and in vivo microdialysis methods.Methods: All experiments were carried out in accordance with the specifications of the Animal Research Committee of Hirosaki University and met the Guidelines for Animal Experimentation of Hirosaki University. The (μ3BKO mice were generated essentially as described by Nakatsu et al. (2004), and maintained with a C57BL/6J background. Two- to 16-week-oldμ3BKO and wild-type mice were used. Determination of the neurotransmitter release from the hippocampal minislice (350μm× 350μm) and microdialysis experiments were performed according to the methods of Okada et al. (2001) and Nakatsu et al. (2004). Basal and 50 mM K+-evoked releases of DA and 5-HT were determined by high-performance liquid chromatography (HPLC) equipped with electrochemical detection (Eicom, Kyoto, Japan) and a graphite carbon electrode set at+450 mV (vs. An Ag/AgCl reference electrode).Results: In minislice experiments, there were no significant differences in hippocampal basal releases of DA and 5-HT betweenμ3BKO mice and wild-type mice during the whole study period. However, the 50 mM K+-evoked DA and 5-HT releases were impaired inμ3BKO mice aged 6 weeks or older (p<0.01). In order to investigate under a more physiological condition, an in vivo microdialysis experiment was further carried out in 8-week-old mice. Remarkably, the K+-evoked DA and 5-HT releases were significantly impaired in (μ3BKO mice (p<0.01). Perfusion of 10 (μM GABAA receptor antagonist, bicuculline, through the microdialysis probe increased DA and 5-HT releases significantly (p<0.01). However, the increased DA and 5-HT stimulated by bicuculline in (μ3BKO mice did not reach the levels in wild-type mice (p<0.05).Discussion: The present study demonstrated the age-dependent impairment of monoaminergic transmission in (μ3BKO mice. Previously, we have established that impaired GABA release in (μ3BKO mice is caused, at least in part, by a reduction in vesicular GABA transporter, although there is no change in glutamate release. Considering also these previous efforts, the present study suggests that AP-3B plays an important role in the formation and function of synaptic vesicles, particularly the inhibitory type. The impairment of inhibitory neurotransmissions might be involved in, at least partially, the increased seizure susceptibility in (μ3BKO mice. (μ3B deficiency causes an impairment of monoamine release possibly because of the reduction of vesicular monoamine transporters. Further studies are required to elucidate the possible mechanisms. The (μ3BKO mice may serve as a novel animal model for the study of epilepsy in the future. Role of Glutamic Acid Receptors in the Inferior Colliculus in Audiogenic Epileptic Rats.Purpose:Glutamic acid receptors andγ-aminobutyric acid and (GABA) in the inferior colliculus (IC) have been reported to play important roles in audiogenic seizures. These receptors have been strongly implicated in the seizure mechanisms in many epilepsy models. The present study investigates the effects of glutamic acid receptors in the IC using a rat model of audiogenic seizures.Methods:Sprague-Dawley (SD) rats neonatally exposed to 0.02% propylthiouracil (PTU) through mother's milk showed a high incidence of audiogenic seizures after maturation. The SD rats (300–450 g) were anesthetized with pentobarbital (60 mg/kg i.p.). Microdialysis probes were implanted stereotaxically into the right IC (A; 8.8 mm, L; 1.6 mm, D; 6.25 mm). On the following day, artificial CSF was perfused at a flow rate of 2μl/min and the sound of a ringing bell applied as a stimulus. Microdialysates were collected every 30 minutes for 6 hours and then analyzed for glutamate, aspartate, and GABA by HPLC using a reverse-phase column. The microdialysis probe implantation site was examined histologically.Results:All rats exposed to PTU (PTU rat) showed running fits and generalized tonic–clonic convulsions induced by the sound stimuli. PTU rats did not show convulsions when treated with MK801 (1 mg/kg i.p.) (a NMDA receptor-associated channel antagonist) before sound stimulation. The glutamate concentration of the microdialysate was significantly higher in PTU rats than in control rats (no PTU exposure) in response to sound stimulation. With MK801 treatment before sound stimulation, the glutamate concentration in PTU rats was suppressed, showing no significant increase compared to control rats. The microdialysate concentrations of aspartate and GABA in PTU rats and control rats did not differ significantly after sound stimulation only or after and MK801 plus sound stimulation.Conclusions:The present results demonstrate that postnatal exposure to PTU induces susceptibility to audiogenic seizures in rats. PTU rats showed running fits and seizures similar to rats genetically prone to epilepsy stimulated by sound. The IC is an important site for the initiation of audiogenic seizures. As to the roles of the IC, GABA and glutamate have been strongly implicated as the major inhibitory and excitatory neurotransmitters, respectively. In our study, the GABA concentration did not differ significantly between PTU rats and control rats, whereas the IC glutamate concentration showed a significant and prolonged increase in PTU rats in response to sound stimulation. MK801 treatment prior to sound stimulation obliterated this significant glutamate increase. These results suggest that the mechanism of audiogenic seizures involves the glutamic acid receptors. Electrical Stimulation Induced After–discharges in the Rat Neocortex: Effect of Stimulus Patterns.Purpose: There has been increased interest in the use of electrical stimulation of selected brain regions as a treatment modality in some patients with pharmacoresistant epilepsy. However, little is known about the effect of these various stimulation patterns and frequencies in the induction or suppression of seizure activity. In order to explain this relationship, we created an acute focal seizure rat model using focal electrical stimulation and examined the effect of two different stimulation patterns (regular versus irregular) on electrically induced neocortical afterdischarges (ADs).Methods: Five Sprague-Dawley rats were used in this study. Epidural stainless steel screw electrodes were implanted on bilateral frontal cortices (anterior: 3 mm, lateral:±3 mm from Bregma, Paxinos), right motor area (A: -1 mm, L: right 2.5 mm), bilateral sensory areas (A:–1 mm. L:±5 mm), and occipital area (A:–5 mm, L:±3 mm). These electrodes were connected to a socket, and were fixed on the rats’ scalps using dental cement. Both stimulation and recordings were carried out using a switching device. Epidural electrodes of the right motor area and right sensory area were stimulated using a bipolar mode. Biphasic irregular 50 Hz stimulation with 0.3 ms pulse width was used to the study random stimulus effect. Irregular stimuli were created using LabVIEW version 6.1. Stimulus impulses were randomly given within 1 second and repeated five times during 5 seconds stimulation. Biphasic 50 Hz regular stimulation with 5-second duration was used as a regular stimulation pattern. Regular and irregular stimuli were tested randomly five times for each rat. Threshold intensities that produce behavioral changes and for AD induction were compared against irregular and regular stimuli using two-way ANOVA.Results: The thresholds obtained in producing behavioral changes were 1.66± 0.53 mA and 1.73± 0.47 mA for regular and irregular stimuli, respectively (not significant). The thresholds for inducing ADs were 2.48± 0.88 mA and 2.51± 0.85 mA for regular and irregular stimuli, respectively (not significant).Conclusions: Random or irregular electrical stimulation of the rat neocortex did not show any significant differences in the induction of behavioral changes or afterdischarges as compared to regular stimuli. [ABSTRACT FROM AUTHOR] |
Plný text