Pharmacology of Cortical Epileptic Afterdischarges in Rats

Summary: Afterdischarges (ADS) elicited by electrical stimulation of the sensorimotor cortical area are characterized by rhythmic spikes and spike-wave complexes in the EEG and by clonic face and forelimb seizures. We studied the sensitivity of such ADS to phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), primidone (PRM), and valproate (VPA) in 78 adult male Wistar rats with implanted electrodes. Neither PHT (30 and 60 mg/kg intraperitoneally, i.p.) nor CBZ (25 and 50 mg/kg i.p.) suppressed cortical ADS. Indeed, ADS were prolonged by higher doses of both drugs. PRM had a similar effect: A dose of 40 mg/kg transiently shortened ADS, but a dose of 80 mg/kg prolonged ADS. PB (20 and 40 mg/kg) and VPA (200 and 400 mg/kg) were effective in suppressing ADS. Higher doses of VPA and PB reduced the intensity of motor phenomena related to the stimuli but had no effect on the motor correlates of ADS. These findings suggest that cortically induced ADS are not a good model of secondarily generalized seizures. The response to VPA and PB suggests that cortical ADS may represent a model of myoclonic seizures.     

Repeated ECS induces GluR1 mRNA but not NMDAR1A-G mRNA in the rat hippocampus

The neural basis underlying the cognitive side effects of ECT is unknown. Recent studies suggest that the memory dysfunction may be caused by alterations in hippocampal synaptic efficacy [20]. In situ hybridization was used to examine the possible receptor mechanisms responsible for this effect. Repeated ECS markedly increased mRNA expression for the GluR1 subunit of the AMPA receptor, but not the NMDAR1A-G subtypes of the NMDA receptor, relative to control treatments. This effect was present 24 h after the last seizure and may be responsible for the expression of the ECS-induced increase in synaptic efficacy.     

托吡酯长期应用对癫痫幼鼠学习记忆及海马NMDAR1 mRNA表达的影响

目的:研究托吡酯(TPM)长期应用对癫癎幼鼠学习记忆功能的影响,并观察海马NMDAR1 mRNA表达的变化。方法:以锂-匹罗卡品制作癫癎模型,TPM灌胃,5周后使用Morris水迷宫评价大鼠的学习记忆功能,通过核酸原位杂交法观察海马各区NMDAR1mRNA表达的变化。结果:癫癎幼鼠的学习记忆功能较正常幼鼠明显下降;癫癎幼鼠学习记忆功能经中、小剂量TPM干预后未发生变化,而经大剂量TPM于预后则明显下降;大鼠海马各区NMDAR1 mRNA的表达在TPM干预后无明显变化。结论:癫癎持续状态(SE)后的慢性癫癎对幼鼠的视觉空间学习记忆功能造成损害,长期大剂量应用TPM可加重这一损害,该损害可能与海马CA1、CA3区NMDAR1 mRNA的表达无关。     

Asymmetrical reductions of hippocampal NMDAR1 glutamate receptor mRNA in the psychoses

The psychotomimetic properties of NMDA glutamate receptor antagonists suggest there may be disease related changes of this receptor in schizophrenia. Using in situ hybridisation histochemistry (ISHH), we measured mRNA for the obligatory NMDAR1 subunit of the NMDA glutamate receptor in post-mortem samples of hippocampus from schizophrenics, depressives, bipolar patients and normal controls. A significant main effect of diagnosis was observed in the dentate gyrus (ANOVA, p = 0.004) and a trend in the CA3 region (ANOVA, p = 0.06), with all psychiatric groups having reduced NMDAR1 mRNA levels compared to normal controls. In contrast to the affectively ill groups, the reductions in schizophrenics were more pronounced in the left side compared to the right. Expression of poly A mRNA also showed left-sided losses in the dentate gyrus in schizophrenia but reductions in NMDAR1 remained significant when expressed as a ratio of poly A. The findings confirm a recent report of reduced hippocampal NMDAR1 mRNA in schizophrenia. However, our new evidence suggests that this is a feature of both affective and schizophrenic disorders and that schizophrenia is distinguished from the others by left-sided reductions in hippocampal NMDAR1 gene expression.     

Development and Pharmacological Suppression of Secondary Afterdischarges in the Hippocampus of Amygdala-kindled Rats

The development and spread of afterdischarges in the ipsilateral limbic system during amygdala kindling, a model of complex partial seizures, was studied in male and female rats. Kindling stimulation was performed in the basolateral amygdala, and afterdischarges were recorded from the stimulation electrode and electrodes in the nucleus accumbens, the posterior piriform cortex and the ventral hippocampus, all implanted on the right side of the brain. All structures showed primary afterdischarges already after the first stimulation, indicating a close anatomical and physiological connection to the epileptogenic focus. The development of robust secondary afterdischarges, which occurred after the end of the primary afterdischarges in the amygdala and which always originated in the hippocampus but also spread to one or more of the other recording sites, is described. The secondary afterdischarges initially occurred after about nine kindling stimulations in both male and female rats, and were associated with an increase in primary afterdischarge duration and a progression from focal to motor seizures. In order to test the effect of common antiepileptic drugs on the secondary afterdischarges, a group of female rats were treated with valproate, carbamazepine or phenytoin. All drugs suppressed the secondary afterdischarges, although they had a different anticonvulsant efficacy on motor seizures and afterdischarge duration after amygdala stimulation. While valproate and carbamazepine dose-dependently reduced all parameters of the kindled seizure, including the secondary afterdischarges in the hippocampus, phenytoin suppressed the secondary afterdischarges also in the absence of any anticonvulsant effect, suggesting that recurrent hippocampal activation is not crucial for the kindled state. Recording of secondary afterdischarges in the hippocampus may offer the possibility of studying the conditions for development and pharmacological suppression of recurrent hippocampal activation in amygdala-kindled rats.     

Interleukin-1 Receptor Type I mRNA Levels in Brain Regions From Male and Female Rats

Interleukin-1 (IL-1) induces a variety of neurological manifestations by direct action in the central nervous system (CNS). The IL-1 receptor type I (IL-1RI) mediates IL-1 signalling. In the present study, the steady-state content of IL-1RI mRNA was determined by a sensitive RNase protection assay in brain regions obtained from normal male and nonestrous female Wistar rats. The results show that brain regions differ in IL-1RI mRNA content. Highest levels of IL-1RI mRNA were detected in the male and female cerebral cortex. High levels of IL-1RI mRNA were also observed in the brain stem and its structures, and the cerebellum. Male and female rats exhibited similar differential profile of IL-1RI mRNA levels in the frontal cortex, hippocampus, hypothalamus, and cerebellum. The present data on brain distribution of IL-1RI mRNA levels suggest that distinct brain regions may depend differentially on the IL-1 system.     

Accurate quantification of the mRNA of NMDAR1 splice variants measured by competitive RT-PCR

N-methyl-D-aspartate receptors (NMDAR) belong to the subclass of ionotropic glutamate receptors and are widely distributed in the vertebrate brain. Molecular cloning has revealed the existence of six NMDAR subunits: one NMDAR1 (NR1), four different NMDAR2 (NR2A-D) and one NMDAR3A (NR3A). Alternative splicing of the single NR1 gene generates eight isoforms with distinct functional properties [M. Hollmann, J. Boulter, C. Maron, L. Beasley, J. Sullivan, G. Pecht, S. Heinemann, Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor, Neuron 10 (1993) 943-954 [8]; R.S. Zukin, M.V.L. Bennett, Alternatively spliced isoforms of the NMDAR1 receptor subunit, TiNS 18 (1995) 306-313 [20]]. Despite the progress made in the functional analysis of NMDARs the molecular architecture of this receptor remains to be elucidated. In situ hybridization studies have already indicated that splicing of the NR1 gene is regionally regulated in the rodent brain, which may contribute to functional diversity of NMDARs in distinct brain areas [D.J. Laurie, P.H. Seeburg, Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA, J. Neurosci. 14 (1994) 3180-3194 [10]; D.G. Standaert, C.M. Testa, A.B. Young, J.B. Penney Jr., Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat, J. Comp. Neurology 343 (1994) 1-16 [18]; M. Hollmann, S. Heinemann, Cloned glutamate receptors, Ann. Rev. Neurosci. 17 (1994) 31-108 [9]]. Since in situ hybridization techniques do not allow accurate quantification of distinct NR1 splice variants and are also very time-consuming, an accurate and sensitive competitive RT-PCR assay was developed. This method was then used to study the distribution of three NR1 splice variants in the rat brain, and the results are compared with former in situ hybridization studies.     

Flavin-Containing Monooxygenase mRNA Levels are Up-Regulated in ALS Brain Areas in SOD1-Mutant Mice

Flavin-containing monooxygenases (FMOs) are a family of microsomal enzymes involved in the oxygenation of a variety of nucleophilic heteroatom-containing xenobiotics. Recent results have pointed to a relation between Amyotrophic Lateral Sclerosis (ALS) and FMO genes. ALS is an adult-onset, progressive, and fatal neurodegenerative disease. We have compared FMO mRNA expression in the control mouse strain C57BL/6J and in a SOD1-mutated (G93A) ALS mouse model. Fmo expression was examined in total brain, and in subregions including cerebellum, cerebral hemisphere, brainstem, and spinal cord of control and SOD1-mutated mice. We have also considered expression in male and female mice because FMO regulation is gender-related. Real-Time TaqMan PCR was used for FMO expression analysis. Normalization was done using hypoxanthine-guanine phosphoribosyl transferase (Hprt) as a control housekeeping gene. Fmo genes, except Fmo3, were detectably expressed in the central nervous system of both control and ALS model mice. FMO expression was generally greater in the ALS mouse model than in control mice, with the highest increase in Fmo1 expression in spinal cord and brainstem. In addition, we showed greater Fmo expression in males than in female mice in the ALS model. The expression of Fmo1 mRNA correlated with Sod1 mRNA expression in pathologic brain areas. We hypothesize that alteration of FMO gene expression is a consequence of the pathological environment linked to oxidative stress related to mutated SOD1.     

Cellular and Subcellular Distribution of NMDAR1 Splice Variant mRNA in the Rat Lumbar Spinal Cord

The regional distribution of alternatively spliced messenger RNA encoding the N -methyl-D-aspartate (NMDA) receptor R1 subunit (NMDAR1) variants was examined by in situ hybridization in the rat lumbar spinal cord. Splice-specific oligonucleotide probes [recognizing full-length mRNA (NMDAR1-1), deletion exon 21 (NMDAR1-2), deletion exon 22 (NMDAR1-3), combined deletion exons 21 and 22 (NMDAR1-4) and mRNA which lacks (NMDAR1-a) or contains exon 5 (NMDAR1-b)] detected marked differences in abundance and distribution of N- and C-terminal spliced variants. The NMDAR1-a, NMDAR1-2 and NMDAR1-4 mRNAs were evenly distributed throughout all laminae of the dorsal and ventral horns. In the superficial dorsal horn NMDAR1-b mRNA was preferentially detected in laminae II inner and III, while NMDAR1-1 mRNA was restricted to laminae I to III. Large neurons in laminae IV and V contained mainly NMDAR1-a, NMDAR1-2 and NMDAR1-4 mRNAs and occasionally NMDAR1-b. The NMDAR1-3 variant was only detected in very low abundance, being restricted to occasional cells in lamina I and II. In the ventral horn, motor neurons showed strong signals for NMDAR1-a, NMDAR1-b, NMDAR1-2 and NMDAR1-4 mRNAs. Serial sectioning through large motor neurons permitted the detection of multiple splice variants in single neurons. Analysis of the subcellular distribution of the mRNAs revealed that the NMDAR1-1 mRNA was almost exclusively found in the cell nucleus, NMDAR1-a mRNA was largely in the cytoplasm, while all other splice variants showed a homogeneous distribution between nucleus and cytoplasm. Comparison of the in situ hybridization images with functional analyses of heteromeric recombinant receptors will be necessary to ascertain whether splice variants of the NMDAR1 receptor subunit can account for some of the known electrophysiological properties of spinal cord neurons under physiological and pathophysiological conditions.     

Increases in mRNA levels for Talpha1-tubulin in the rat kindling model of epilepsy

The expression of mRNA for Talpha1-tubulin, a cytoskeletal protein, was studied in the rat kindling model of epilepsy. The Talpha1-tubulin mRNA level increased significantly in the dentate gyrus (DG) and CA3 of hippocampus ipsilateral to stimulation from 8 h to 4 weeks after amygdaloid kindled seizures. The peak increase was observed at 1 week after the last seizures both in the DG and CA3. These results suggest that the microtubule formation contributes to synaptic remodeling and reorganization of neural networks, which may be based on the kindling-inducing epileptogenesity.     

Autoantibodies against the NMDAR subunit NR1 are associated with neuropsychiatric outcome after ischemic stroke

Background and PurposePreexisting autoantibodies against N-methyl-D-aspartate-receptor subunit NR1 (NMDAR1-AB) in acute ischemic stroke patients with previously intact blood-brain-barrier were associated with smaller evolution of lesion size. Effects of chronic exposure to NMDAR1-AB long after stroke, however, have remained unclear. We investigated in a prospective follow-up study whether long-term neuropsychiatric outcome after stroke differs depending on NMDAR1-AB status.MethodsBlood samples for NMDAR1-AB analysis were collected within 24 h after ischemic stroke from n = 114 patients. Outcome was assessed 1–3 years later using NIHSS, modified Rankin-scale, Barthel-Index, RBANS (Repeatable Battery for the Assessment of Neuropsychological Status) subcategories (immediate/delayed memory, attention, visuoconstruction), anamnesis evaluating neuropsychiatric symptoms (e.g. hallucinations, psychomotor slowing, reduced alertness, depressiveness, fatigue) and questionnaires (Beck's Depression Inventory-BDI, Fatigue Impact Scale-FIS). Scores were generated to cover RBANS plus neuropsychiatric symptoms (Score A; n = 96) or only neuropsychiatric symptoms (Score B; n = 114, including patients unable to conduct RBANS). Depression/fatigue were measured in patients, capable to perform questionnaires (n = 86).ResultsNMDAR1-AB (IgM, IgA, IgG) were detected in n = 27 patients (23.7%). NMDAR1-AB seropositive patients showed inferior results in Score A (p = 0.006), Score B (p = 0.004), BDI (p = 0.013) and FIS (p = 0.018), compared to seronegative patients. Multiple regression analysis including covariates age, NIHSS at day 7 post-stroke, and days from stroke to follow-up, showed NMDAR1-AB seropositivity associated with worse outcome in Scores A (b: 1.517, 95%CI: 0.505–2.529, p = 0.004) and B (b: 0.803, 95%CI: 0.233–1.373; p = 0.006). Also FIS was unfavorably associated with NMDAR1-AB seropositivity (binary logistic regression: OR: 3.904, 95%CI: 1.200–12.695; p = 0.024).ConclusionsEven though the numbers of included patients are low, our data apparently indicate that NMDAR1-AB seropositivity at the time point of acute ischemic stroke is associated with neuropsychiatric symptoms including cognitive dysfunction and fatigue years after stroke. Preclinical proof of a causal relation provided, targeted immunosuppression may be a future prophylactic option to be clinically evaluated.     

Lasting changes in NMDAR1 mRNA level in various regions of cerebral cortex in epileptogenesis of amygdaloid-kindled rat

The involvement of NMDA receptor subunit, NR1, with kindling phenomenon has been reported, but the role of NR1 in epileptogenesis is still unknown. We have examined the expression levels of NR1 mRNA in the cerebral cortices of amygdaloid-kindled rats. Northern blot analysis showed a significant increase in NR1 mRNA expression level in the ipsilateral frontal and temporal cortices at 4 weeks after the last generalized seizure. At the same time, NR1 mRNA decreased in the bilateral piriform cortices. These data suggest that NR1-mediated transmission may have an impact in the neurobiological basis of enduring epileptogenesis.     

L-type Calcium Channels are Involved in Iron-induced Neurotoxicity in Primary Cultured Ventral Mesencephalon Neurons of Rats

In the present study,we investigated the mechanisms underlying the mediation of iron transport by Ltype Ca^2+ channels(LTCCs)in primary cultured ventral mesencephalon(VM)neurons from rats.We found that cotreatment with 100 lmol/L FeSO4 and MPP^+(1-methyl-4-phenylpyridinium)significantly increased the production of intracellular reactive oxygen species,decreased the mitochondrial transmembrane potential and increased the caspase-3 activation compared to MPP^+ treatment alone.Co-treatment with 500 lmol/L CaCl2 further aggravated the FeSO4-induced neurotoxicity in MPP^+-treated VM neurons.Co-treatment with 10 lmol/L isradipine,an LTCC blocker,alleviated the neurotoxicity induced by co-application of FeSO4 and FeSO4/CaCl2.Further studies indicated that MPP^+treatment accelerated the iron influx into VM neurons.In addition,FeSO4 treatment significantly increased the intracellular Ca^2+ concentration.These effects were blocked by isradipine.These results suggest that elevated extracellular Ca^2+ aggravates ironinduced neurotoxicity.LTCCs mediate iron transport in dopaminergic neurons and this,in turn,results in elevated intracellular Ca^2+ and further aggravates iron-induced neurotoxicity.     

Increases in mRNA levels for Tα1-tubulin in the rat kindling model of epilepsy

The expression of mRNA for Tα1-tubulin, a cytoskeletal protein, was studied in the rat kindling model of epilepsy. The Tα1-tubulin mRNA level increased significantly in the dentate gyrus (DG) and CA3 of hippocampus ipsilateral to stimulation from 8 h to 4 weeks after amygdaloid kindled seizures. The peak increase was observed at 1 week after the last seizures both in the DG and CA3. These results suggest that the microtubule formation contributes to synaptic remodeling and reorganization of neural networks, which may be based on the kindling-inducing epileptogenesity.     

Alterations of NMDAR1 and NMDAR2A/B immunoreactivity in the hippocampus after perforant pathway lesion

Immunohistochemical techniques were employed to examine the changes in immunolabeling of the N‐methyl‐d ‐aspartate (NMDA) receptor subunits NMDAR1 and NMDAR2A/B within the hippocampus 1, 3, 7, 14 and 30 days after a unilateral perforant pathway lesion was made in a rat brain. At 1 day post‐lesion, we observed a decrease in NMDAR1 immunolabeling in the granule cells in the dentate gyrus as well as in the mossy cells in the polymorphic region ipsilateral to the lesion, while an increase in diffuse neuropil labeling was observed. At 3 days post‐lesion, we observed a marked increase in NMDAR1 immunolabeling in the outer molecular layer of the dentate gyrus as well as in the stratum moleculare in the CA fields ipsilateral to the lesion. Although this increase was less marked at 7 and 14 days post‐lesion, an increase in NMDAR1 immunolabeling was evident at 30 days post‐lesion. In contrast, although a transient increase in NMDAR2A/B immunolabeling was observed in the outer molecular layer at 3 days post‐lesion, no other changes were detectable at any of the time points examined. Our study suggests that each subunit of the NMDA receptor displays a different response to deafferentation of the perforant pathway. We have previously observed that changes in the immunoreactivity of the receptor subunits of another class of glutamate receptor, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoaxolepropionate (AMPA), occur at 30 days post‐lesion but not after a relatively short survival time. NMDA receptor subunits demonstrate an earlier response to the loss of the perforant pathway fibers than do the AMPA receptor subunits.     

大鼠局灶性脑缺血皮质白细胞介素1β mRNA的表达

目的:观察大鼠局灶性脑缺血皮质白细胞介素1β(IL-1β)mRNA及其受体和受体拮抗剂表达的动态变化。方法:采用尼龙线栓法大鼠脑缺血模型,逆转录PCR方法观察缺血后其相对表达量。结果:缺血侧IL-1βmRNA在各缺血时间点皆有表达,12～24 h达高峰;IL-1RA在缺血48 h表达最强;IL-RI在缺血侧和缺血对侧均稳定表达(包括假手术组);IL-RⅡ仅在缺血侧有表达,24～48 h表达较高。结论:局灶性脑缺血时IL-1βmRNA表达增高,其受体及受体拮抗剂mRNA表达亦增高;表达程度与时相的差别说明它们在脑缺血中可能起着不同的作用。     

Effects of Pentobarbital on Entorhinal Tetanic Responses and the Progression of Afterdischarges During the Early Course of Amygdala Kindling in Rats

Summary: We investigated the relationship between the progress of afterdischarges (AD) and the development of facilitated entorhinal tetanic responses by amygdala kindling stimulations in conscious and pentobarbital (PB)-treated rats. The entorhinal responses consisted of deep negative components and the following shallow positive components. The negative potential (mean±SE) reflecting excitatory synaptic activation in the test response evoked by a single stimulation (600 μA) before kindling stimulations was greater in PB-treated rats (1.3 ± 0.21 mV, n = 6) than in conscious rats (0.5 ± 0.08 mV, n = 9).The positive potential reflecting inhibitory synaptic activation in the test response was also greater in PB-treated rats (0.6 ±0.14 mV, n = 6) than in conscious rats (0.2 ±0.04 mV, n = 9).The magnitude of the tetanic response was estimated as the area between the excitatory negative potential and the baseline in the averaged tetanic response during each kindling stimulation (10 Hz, 100 pulses).The magnitude of the tetanic response was significantly enhanced in association with the prolongation of AD duration in the conscious rats. In the PB-treated (50 mg/kg intraperitoneally, i.p.) rats, enhancement of tetanic response was very slight and the progress of AD duration was prevented. There was a linear correlation >( r = 0.9) between the magnitude of tetanic response and AD duration. These findings indicate that PB suppresses kindling-induced enhancement of excitatory synaptic activation in tetanic responses and that this enhancement is intimately related to the development of AD.