Upregulation of Glutamate Receptors in Rat Cerebral Cortex with Neuronal Migration Disorders

Neuronal migration disorders (NMDs) constitute the main pathologic substrate of medically intractable epilepsy in human. This study is designed to investigate the changes in expression of glutamate receptor subtypes on radiation-induced NMD in rats. The lesion was produced by intrauterine irradiation (240 cGy) on E17 rats, and then 10 weeks old rats were used for the study. The pathologic and immunohistochemical findings for glutamate receptor subunit proteins on NMD cortex were correlated with development of behavioral seizures and EEG abnormality. Spontaneous seizures uncommonly occurred in NMD rats (5%); however, clinical stages of seizures were significantly increased in NMD rats by an administration of kainic acid. Brains taken from irradiated rats revealed gross and histopathologic features of NMD. Focal cortical dysplasia was identified by histopathology and immunohistochemistry with neurofilament protein (NF-M/H). Significantly strong NR1 and NR2A/B immunoreactivities were demonstrated in cytomegalic and heterotopic neurons of NMD rats. The results of the present study indicate that epileptogenesis of NMD might be caused by upregulation of glutamate receptor expression in dysplastic neurons of the rat cerebral cortex with NMDs.

Glutamate Receptor Subunits Gene Expression in Kainate-induced Temporal Lobe Epilpsy Model

BACKGROUND: There is considerable controversy about the exact molecular mechanisms of excitatory amino acid receptors in epileptogenesis. METHODS: We used in situ hybridization to examine the hybridization density (HD) of n-methyl- D-aspartic acid receptor type 1 (NMDAR-1) and alpha-amino-3-hydroxy -5-methyl-4-isoxazole-propionate (AMPA) receptor type 2 (GluR-2) mRNA, in the hippocampus obtained from the kainic acid (KA)-induced status epilep-ticus (SE) model. Some Sprague-Dawley rats were injected with KA (10 mg/Kg; I.p.), and others with MK-801 (4 mg/kg) 20 minutes prior to KA. The rats were allowed to have 4-hour SE and were killed at 8 hours or 4 weeks after KA or MK-801/KA injection. HD of NMDAR-1 and GluR-2 mRNA in subfields of the hippocampus was measured by an image analysis system. RESULTS: A typical neuropathological finding of hippocampal sclerosis and spontaneous repetitive seizures (SRS) were observed in the KA injected rats, but not in the MK-801 pretreated rats, killed at 4 weeks. Compared with controls, the rats killed at 8 hours after KA showed increased CA1, CA2, and CA3 NMDAR-1 HD, and stratum granulosum (SG) GluR-2 HD. The increase of NMDAR-1, not GluR-2, HD was blocked effectively by MK-801. The increase of SG GluR-2 HD remained until 4 weeks after the KA injection. CONCLUSIONS: Not only the NMDAR-1activa-tionbut also the GluR-2 activation is an important factor in delaying hippocampal neuronal loss and epileptogenesis. (J Korean Neurol Assoc 19(1):36~44, 2001

c-JUN Expression and Apoptotic Cell Death in Kainate-Induced Temporal Lobe Epilepsy

Following kainate (KA)-induced epilepsy, rat hippocampal neurons strongly ex-press immediate early gene (IEG) products, i.e., c-FOS and c-JUN, and neural stress protein, HSP72. Prolonged expression of c-JUN and c-FOS 48 hr after cerebral ischemia has been underwent delayed neuronal death. However, it is not yet clear whether IEGs actually assume the essential roles in the cell death process or simply as a by-product due to external stimuli because of the prolonged expression of c-FOS, more than one week, on intact CA2 neurons of the hippocampus in a KA-induced epilepsy model. This study investigated the relationships between prolonged expression of c-JUN and hippocampal neuronal apoptosis in a KA-induced epilepsy model. Epileptic seizure was induced in rats by a single microinjection of KA (1g/l) into the left amygdala. Characteristic seizures and hippocampal neuronal injury were developed. The expression of c-JUN was evaluated by immunohistochemistry, and neuronal apoptosis by in situ end labeling. The seizures were associated with c-JUN expression in the hippocampal neurons, of which the level showed a positive correlation with that of apoptosis. Losses of hippocampal neurons, especially in the CA3 region, were partly caused by apoptotic cell death via a c-JUN-mediated signaling pathway. This is thought to be an important component in the pathogenesis of hippocampal neuronal injury via KA-induced epilepsy.

Effects of norepinephrine and neuropeptide Y on the contractility of small mesenteric artery from 2K1C and DOCA-salt hypertensive rats

The present study was conducted to investigate the possible role of the sympathetic nervous system in two-kidney, one clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt hypertension. 2K1C and DOCA-salt hypertension were made in Sprague-Dawley rats. Four weeks after induction of hypertension, systolic blood pressure measured in conscious state was significantly higher in 2K1C (216+/-18 mmHg) and DOCA-salt (205+/-29 mmHg) groups than that in control (128+/-4 mmHg). The third branches (<300 micrometer in outer diameter) of the mesenteric artery were isolated and cut into ring segments of 2apprx3 mm in length. Each ring segment was mounted in tissue bath and connected to a force displacement transducer for measurement of isometric tension. The arterial rings were contracted by application of norepinephrine (NE) in a dose-dependent manner. The amplitude of the NE-induced contraction of the vessels was significantly larger in hypertension than in control. The NE-induced contraction was significantly enhanced by neuropeptide Y (NPY) in hypertension. Reciprocally, NPY-elicited vasocontraction was increased by NE in hypertension. These results suggest that the sympathetic nervous system contributes to the development of 2K1C and DOCA-salt hypertension.

Altered Calcium Current of the Vascular Smooth Muscle in Renal Hypertension

The present study was aimed at investigating whether the calcium current in the vascular smooth muscle (VSM) cells is altered in renal hypertension. Two-kidney, one clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt hypertension were made in Sprague-Dawley rats. Rats without clipping the renal artery or implanting DOCA were used as control for 2K1C and DOCA-salt hypertension, respectively. Four weeks after clipping, systolic blood pressure was significantly higher in 2K1C rats than in control (192+/-24 and 119+/-4 mmHg, respectively, n=16 each). DOCA-salt rats also showed a higher blood pressure (180+/-15 mmHg, n=18) compared with control (121+/-6 mmHg, n=14). VSM cells were enzymatically and mechanically isolated from basilar arteries. Single relaxed VSM cells measured 5 ~ 10 mum in width and 70 ~ 150 mum in length were obtained. VSM cells could not be differentiated in size and shape between hypertensive and normotensive rats under light microscopy. High-threshold (L-type) calciumcurrents were recorded using whole-cell patch clamp technique. The amplitude of the current recorded from VSM cells was larger in 2K1C hypertension than in control. Neither the voltage-dependence of the calcium current nor the cell capacitance was significantly affected by 2K1C hypertension. By contrast, the amplitude of the calcium current was not altered in DOCA-salt hypertension. These results suggest that high-threshold calcium current of the VSM cells is altered in 2K1C hypertension, and that calcium channel may not be involved in calcium recruitment of VSM in DOCA-salt hypertension.

Augmented expression of cardiac atrial natriuretic peptide system in hypertensive rats

The present study was aimed at investigating the regulation of atrial natriuretic peptide (ANP) system in association with either enhanced or attenuated activity of the renin-angiotensin system (RAS). The cardiac tissue mRNA and peptide levels of ANP were measured in rats with two-kidney, one clip (2K1C) or deoxycorticosterone acetate (DOCA)-salt hypertension. Plasma renin concentration was increased in 2K1C hypertension along with increases of renin mRNA and protein contents in the clipped kidney. On the contrary, it was suppressed in DOCA-salt hypertension along with decreases of renin mRNA and protein contents in the remaining kidney. The plasma ANP concentration was similarly increased in both models of hypertension. The cardiac tissue ANP contents were not significantly changed, but the tissue ANP mRNA levels were upregulated in the hypertrophied heart in these two models of hypertension. It is suggested that the cardiac ANP system is transcriptionally enhanced by cardiac hypertrophy associated with hypertension, independent of the systemic RAS.

Tissue-Specific Regulation of Angiotensinogen and Angiotensin II Receptor Genes in Two-Kidney, One Clip Hypertensive Rats / 대한신장학회잡지

The present study was aimed at investigating the molecular regulation of the renin- angiotensin system (RAS) in two-kidney, one clip (2K1C) hypertension. The expression of renin, angiotensinogen and angiotensin II receptor genes was determined by Northern blot analysis in rats made 2K1C hypertensive for 2 or 4 weeks. The expression of renin gene was increased in the clipped kidney and decreased in the contralateral non-clipped kidney at weeks 2 and 4. The expression of angiotensinogen gene was not significantly altered at week 2, but increased at week 4 in the clipped kidney. However, it was not significantly altered in the contralateral kidney either at week 2 or 4. Nor was the expression of angiotensinogen gene significantly altered in the liver either at week 2 or 4. On the other hand, the expression of angiotensin II receptor gene was decreased at week 2, and increased at week 4 in the clipped kidney, whereas it was not significantly changed in the contralateral kidney either at week 2 or 4. In the liver, the expression of angiotensin II receptor gene was not significantly altered at week 2, but decreased at week 4. These results suggest that the components of RAS are transcriptionally regulated in 2K1C hypertension in a manner dependent on tissues and duration of hypertension.

Role of endogenous nitric oxide in the control salivary secretion and blood flow

The present study was designed to investigate whether endogenous nitric oxide (EDNO) is involved in submandibular vasodilation and salivation induced by parasympathetic nerve stimulation. Effects of Nw-nitro-L-arginine-methyl ester (L-NAME) which blocks the synthesis of EDNO from L-arginine on the submandibular vasodilation and salivation induced by chorda stimulation or administration of various vasodilators were examined in anesthetized cats. Effect of L-NAME on K+ efflux induced by carbachol was also examined using the excised submandibular slice in vitro. In the submandibular slices, acetylcholine (10(-5) mol/L) or vasoactive intestinal polypeptide (VIP, 10(-5) mol/L) increased NO2 contents, which was prevented by pretreatment with L-NAME. Salivary secretion in response to the chorda stimulation (3 V, 1 msec, 10 ~ 20 Hz) was completely blocked by treatment with atropine (1 mg/kg). Increased blood flow response to the low frequency (1, 2, 5 Hz) stimulation was significantly reduced, whereas the blood flow induced by the higher frequency (10, 20 Hz) stimulation was not affected. Lingual-arterial infusion of L-NAME (100 mg/kg) significantly diminished the vasodilatory and salivary responses to the chorda stimulation at all stimuli frequencies used. Intra-arterial infusion of L-NAME (100 mg/kg) markedly diminished the vasodilatory responses to acetylcholine (5 mug/kg), VIP (5 mug/kg) or bradykinin (5 mug/kg). In the excised submandibular slice, K+ efflux in response to carbachol (10(-5) mol/L) was significantly decrease by pretreatment with L-NAME (10(-5) mol/L). In the isolated submandibular artery precontracted with phenylephrine (10(-5) mol/L), the vasorelaxation induced by ACh (10-7 mol/L) was reversed into a contraction by methylene blue (10(-4) mol/L). These results suggest that EDNO may play an important role in vasodilation and secretion of the submandibular gland.

Altered vascular calcium regulation in hypertension

The present study was aimed at investigating whether the vascular calcium regulation is altered in hypertension. Two-kidney, one clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt hypertension were made in rats, and their thoracic aortae were taken 4 weeks later. The isometric contractile response and calcium uptake of the endothelium-denuded aortic preparations were determined. Caffeine (0.1-35 mmol/L) induced a greater contraction in 2K1C and DOCA-salt hypertension than in normotensive control. When the vascular calcium store was functionally-depleted by a repeated exposure to caffeine, it took longer to reload the store and to resume the initial contraction force in response to caffeine in both 2K1C and DOCA-salt hypertension. The vascular 45Ca uptake following the functional depletion of the cellular store was also greater in both models of hypertension than in control. Ryanodine, calcium channel activator of the sarcoplasmic reticulum, attenuated the restoration of caffeine-induced vascular contraction, which was not affected by either 2K1C or DOCA-salt hypertension. Nifedipine, an L-type Ca2+ channel blocker, attenuated the restoration of caffeine-induced contraction, which was not affected by DOCA-salt hypertension, but was more pronounced in 2K1C hypertension. Nifedipine also diminished the vascular 45Ca uptake, which was not affected by DOCA-salt hypertension, but was more pronounced in 2K1C hypertension. Ouabain, a Na+, K+/-ATPase inhibitor, increased the caffeine-induced contraction by a similar magnitude in control and 2K1C hypertension, which was, however, markedly attenuated in DOCA-salt hypertension. Ouabain enhanced the vascular 45Ca uptake, the degree of which was not affected by 2K1C hypertension, but was markedly attenuated in DOCA-salt hypertension compared with that in control. Cyclopiazonic acid, a selective inhibitor of Ca2+/-ATPase of the sarcoplasmic reticulum, attenuated the restoration of caffeine-induced contraction, which was not affected by 2K1C hypertension, but was more marked in DOCA-salt hypertension. These results suggest that the increased vascular calcium storage may be attributed to an enhanced calcium influx in 2K1C hypertension, and to an impaired Na+/-K+ pump activity of the, cell membrane and subsequently increased calcium pump activity of the cellular store in DOCA-salt hypertension.

Electrophysiological properties of the neurons dissociated from the nucleus raphe magnus in postnatal rats

Neurons in the nucleus raphe magnus are involved in descending modulation of nociceptive transmission. In this study, we attempted to investigate electrophysiological properties of the NRM neurons dissociated from the postnatal rat medulla. The NRM neurons in the coronal slices of and the dissociated neurons from the postnatal rat medullae were immunohistochemically identified using antibody against serotonin. Relatively small number of neurons were positively stained in both preparations. The positively stained neurons displayed large cell body with double or multiple neurites. Using whole-cell patch clamp configuration ionic currents were recorded from the dissociated NRM-like neurons selected by criteria such as size and shape of cell body and cell population. Two types, high- and low-threshold, of voltage-dependent calcium currents were recorded from the dissociated NRM-like neurons. Some neurons displayed both types of calcium currents, whereas others displayed only high-threshold calcium current. Voltage-dependent potassium currents were also recorded from the dissociated NRM neurons. Some neurons displayed both transient outward and delayed rectifier currents but others showed only delayed rectifier current. These results suggest that there are at least two types of calcium currents and two types of potassium currents in the dissociated NRM neurons.

Spontaneous electrical activity in cerebellar Purkinje neurons of postnatal rats

Although cerebellar Purkinje cells display spontaneous electrical activity in vivo and in slice experiments, the mechanism of the spontaneous activity generation has not been clearly understood. The aim of this study was to investigate whether cerebellar Purkinje cells of postnatal rats generate spontaneous electrical activity without synaptic inputs. Dissociated cerebellar Purkinje cells were used for reducing synaptic inputs in the present study. Cerebellar Purkinje cells with dendrites were dissociated from postnatal rats using enzymatic treatment followed by mechanical trituration. Spontaneous electrical activities were recorded from dissociated cells without any stimulus using whole-cell patch clamp configuration. Two types, spontaneously firing or quiescent, of dissociated Purkinje cells were observed in postnatal rats. Both types of cells were identified as Purkinje cells using immunocytochemical staining technique with anti-calbindin after recording. Spontaneously active cells displayed two patterns of firing, repetitive and burst firings. Two thirds of dissociated Purkinje cells displayed repetitive firing and the rest of them did burst firing under same recording condition. Repetitive firing activities were maintained even after further isolation using either physical or pharmacological techniques. Neither high magnesium solution nor excitatory synaptic blockers, AP-5 and DNQX, block the spontaneous activity. These results demonstrate that spontaneous electrical activity of isolated cerebellar Purkinje cells in postnatal rats is generated by intrinsic membrane properties rather than synaptic inputs.