Lithium-pilocarpine-induced status epilepticus produces necrotic neurons with internucleosomal DNA fragmentation in adult rats

Prolonged and continuous epileptic seizures [status epilepticus (SE)] produce a widespread pattern of neuronal death, primarily in limbic brain regions. Because it has been suggested that seizure-induced neuronal death may be apoptotic in nature, we tested the hypothesis that lithium-pilocarpine-induced status epilepticus (LPCSE) produces apoptotic neurons. LPCSE lasting 3 h was induced in male Wistar rats which were allowed to recover for 24 or 72 h before perfusion-fixation. Neuronal death was assessed by light microscopy with the haematoxylin-and-eosin stain (H&E), with in situ DNA nick-end labelling (TUNEL stain), by electron microscopy, and by agarose gel electrophoresis of DNA extracted from vulnerable brain regions. Ultrastructurally, acidophilic neurons identified with H&E were dark, shrunken and necrotic in appearance, exhibiting pyknotic nuclei, irregular, dispersed chromatin clumps and cytoplasmic vacuolization. No cells with apoptotic features were seen. Acidophilic neurons were found in 21 out of 23 brain regions examined, and comprised 26-45% of the total number of neurons examined. A subset of these neurons (< 10% of the total number of neurons) were TUNEL-positive at 72 h, but not 24 h, after SE. Internucleosomal DNA cleavage (DNA 'laddering') was found in the six brain regions examined ultrastructurally 24 and 72 h after SE. These results indicate that, in adult rats, LPCSE produces neuronal injury with the appearance of necrosis rather than apoptosis. The necrotic neurons show nuclear pyknosis, chromatin condensation and internucleosomal DNA fragmentation, confirming the nonspecificity of these nuclear changes. Internucleosomal DNA cleavage and other programmed cell death mechanisms can be activated by SE in neurons which become necrotic.     

Neuropathology of limbic status epilepticus induced by electrical stimulation of naive rats.

Neuronal damage in relation to the duration of seizure was studied in limbic status epilepticus (SE) induced by electric stimulation of naive rats. Adult Sprague-Dawley rats were stimulated at the right amygdala to induce SE. To stop the seizures, diazepam was given to different groups of rats at 0.5 h (n = 4), 1 h (n = 6), 2 h (n = 6), and 3-4 h (n = 8) of SE. Eighteen hours after the end of SE, the rats were perfusion fixed. Naive (n = 6) and sham-operated (n = 4) rats served as controls. Horizontal paraffin sections were stained with acid fuchsin and cresyl violet. Neuronal damage was absent after 30 min of SE. Status epilepticus of 1 h or longer duration regularly caused neuronal damage to the cerebral cortex, thalamus, hippocampus, amygdala, and pars reticulata of the substantia nigra. Damage in the cerebral cortex predominated in the entorhinal, temporal, and pyriform regions. In the hippocampus, the dentate hilus was most severely affected, followed by CA3 and CA1. Damage to the dentate granule layer was mild. Further studies of the pathophysiology of excitotoxicity may help to protect patients from sequels of status epilepticus such as neuronal damage and epilepsy.     

Neuropathology of limbic status epilepticus induced by electrical stimulation of naive rats

Abstract

Neuronal damage in relation to the duration of seizure was studied in limbic status epilepticus (SE) induced by electric stimulation of naive rats. Adult Sprague-Dawley rats were stimulated at the right amygdala to induce SE. To stop the seizures, diazepam was given to different groups of rats at 0.5 h (n = 4), 7 h fn = 6), 2 h (n = 6), and 3-4 h (r\ = 8) of SE. Eighteen hours after the end of SE, the rats were perfusion fixed. Naive (n = 6) and sham-operated (n = 4) rats served as controls. Horizontal paraffin sections were stained with acid fuchsin and cresyl violet. Neuronal damage was absent after 30 min of SE. Status epilepticus of I h or longer duration regularly caused neuronal damage to the cerebral cortex, thalamus, hippocampus, amygdala, and pars reticulata of the substantia nigra. Damage in the cerebral cortex predominated in the entorhinal, temporal, and pyriform regions. In the hippocampus, the dentate hilus was most severely affected, followed by CA3 and CAT. Damage to the dentate granule layer was mild. Further studies of the pathophysiology of excitotoxicity may help to protect patients from sequels of status epilepticus such as neuronal damage and epilepsy. [Neurol Res 1999; 21: 399-403]     

Status epilepticus triggers caspase-3 activation and necrosis in the immature rat brain

The mode and mechanism of neuronal death induced by status epilepticus (SE) in the immature brain have not been fully characterized. In this study, we analyzed the contribution of neuronal necrosis and caspase-3 activation to CA1 damage following lithium-pilocarpine SE in P14 rat pups. By electron microscopy, many CA1 neurons displayed evidence of early necrosis 6 hours following SE, and the full ultrastructural features of necrosis at 24-72 hours. Caspase-3 was activated in injured (acidophilic) neurons 24 hours following SE, raising the possibility that they died by caspase-dependent "programmed" necrosis.     

The pattern of 72-kDa heat shock protein-like immunoreactivity in the rat brain following flurothyl-induced status epilepticus

The inducible 72-kDa heat shock protein (HSP72) is a highly conserved stress protein that is expressed in CNS cells and may play a role in protection from neural injury. We used a monoclonal antibody to HSP72 and immunocytochemistry to localize HSP72 in the rat brain 24 h following either 30 or 60 min of flurothyl-induced status epilepticus. Sprague-Dawley rats were anesthetized with halothane, paralyzed, and ventilated, and remained normotensive and well oxygenated for the duration of the seizures. Seizure activity was quantified via analysis of the scalp EEG pattern. HSP72-like immunoreactivity (HSP72-LI) was induced in specific brain regions in a graded fashion that correlated, in part, with the duration and degree of seizure activity. Milder seizures produced HSP72-LI limited to layers 2 and 3 of frontoparietal cortex, dentate hilus cells, and CA3 pyramidal neurons. More extensive seizures led to HSP72-LI in layers 2, 3 and 5 of frontoparietal and visual cortex, dentate hilus cells, CA1 and CA3 pyramidal neurons, and certain thalamic and amygdaloid nuclei. These are similar to many, but not all, of the brain regions known to be injured with this model. No HSP72-LI was observed in sham-treated controls or flurothyl-treated animals whose seizures were controlled with pentobarbital. HSP72-LI thus localizes to certain regions of seizure-induced injury, and may provide a sensitive method of detecting neuronal 'stress' or injury relatively soon after status epilepticus. Whether or not HSP72 synthesis plays a protective role in the pathogenesis of seizures, or is only a marker for cell injury, remains to be determined.     

Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures.

Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 microM), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 microM) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 mM) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA.     

Epilepsy induced by extended amygdala-kindling in rats: lack of clear association between development of spontaneous seizures and neuronal damage

Most patients with temporal lobe epilepsy (TLE), the most common type of epilepsy, show pronounced loss of neurons in limbic brain regions, including the hippocampus, amygdala, and parahippocampal regions. Hippocampal damage in patients with TLE is characterized by extensive neuronal loss in the CA3 and CA1 sectors and the hilus of the dentate gyrus. There is a long and ongoing debate on whether this type of hippocampal damage, referred to as hippocampal sclerosis, is the cause or consequence of TLE. Furthermore, hippocampal damage may contribute to the progressive features of TLE. The present study was designed to determine whether development of spontaneous recurrent seizures (SRS) after extended kindling of the amygdala in rats is associated with neuronal damage. The kindling model of TLE was chosen because previous studies have shown that only part of the rats develop SRS after extended kindling, thus allowing to compare the brain pathology of rats that received the same number of amygdala stimulation but did or did not develop SRS. For extended kindling, rats were stimulated twice daily 3-5 days a week for up to about 280 stimulations. During long-term EEG/video monitoring, SRS were observed in 50% of the rats over the period of extended kindling. SRS often started with myoclonic jerks or focal seizures and subsequently progressed into secondarily generalized seizures, so that the development of SRS recapitulated the earlier kindling of elicited seizures. No obvious neurodegeneration was observed in the CA1 and CA3 sectors of the hippocampus, the amygdala, parahippocampal regions or thalamus. A significant bilateral reduction in neuronal density was determined in the dentate hilus after extended kindling, but this reduction in hilar cell density did not significantly differ between rats with and without observed SRS. Determination of the total number of hilar neurons and of hilar volume indicated that the reduced neuronal density in the dentate hilus was due to expansion of hilar area but not to neuronal damage. The data demonstrate that extended kindling does not cause any hippocampal damage resembling hippocampal sclerosis, but that SRS develop in the absence of such damage.     

The microglial reaction in the rat dorsal hippocampus following transient forebrain ischemia.

We have examined the distribution and time course of the microglial reaction in the rat dorsal hippocampus after 25-min transient forebrain ischemia (four-vessel occlusion model). Microglial cells were visualized in brain sections using lectin staining with the Griffonia simplicifolia B4-isolectin following intervals of reperfusion ranging from 20 min to 4 weeks. Increased staining of microglial cells was detected in the dentate hilus and area CA1 as early as 20 min after reperfusion. These same regions demonstrated more intense microglial staining after 24 h. The strongest microglial reaction was observed 4-6 days after reperfusion when reactive microglia were abundant throughout all laminae of CA1 and the dentate hilus. Following longer reperfusion intervals, the microglial reaction became less intense; however, it remained above normal levels until the end of the fourth week. At all time points examined, microglial reactivity in the CA3 pyramidal and dentate granule cell layers was considerably lower than that observed in CA1 and dentate hilus. Our results are consistent with the known serial pathological changes associated with cerebral ischemia, but, in addition, show that the examination of the microglial reaction provides an extremely sensitive indicator of subtle and morphologically nonapparent neuronal damage during the early stages of injury.     

癫痫大鼠海马神经元和星形胶质细胞的病理演变

目的　探讨癫痫大鼠海马神经元和星形胶质细胞在点燃后各期的病理特点、时序及机制。方法　针对匹罗卡品癫痫大鼠模型,行Nissl、免疫组化和HE染色,观察海马神经元及星形胶质细胞的病理变化。结果　癫痫持续状态后超急性期（4h）,CA3区神经元呈嗜酸性变性、胞浆深染;急性期（24h）,嗜酸性变性最为显著,神经元固缩、核仁消失、突起断裂,星形胶质细胞水肿;缄默期（7d）,CA3、CA1区及门区神经元大量坏死、脱失,胶质增生肥大,海马构筑紊乱;慢性期（6w）,CA3、CA1区出现胶质瘢痕,遗有形态正常的神经元,且颗粒细胞层增厚。结论　癫痫时海马神经元先于星形胶质细胞发生病理改变,二者均参与癫痫发生。     

Age-dependent cyclooxygenase-2 induction and neuronal damage after status epilepticus in the postnatal rat hippocampus

PURPOSE: Epileptic seizures lead to age-dependent neuronal damage in the developing brain, particularly in the hippocampus, but the mechanisms involved have remained poorly elucidated. In this study, we investigated the contribution of apoptosis and inflammatory processes to neuronal damage after status epilepticus (SE) in postnatal rats. METHODS: SE was induced by an intraperitoneal injection of kainic acid (KA) in 21- and 9-day-old (P21 and P9) rats. The expression of Bax, Bcl-2 and caspase-3, markers for apoptosis, and cyclooxygenase-2 (COX-2), an indicator for activation of inflammatory processes, were studied from 6 h up to 1 week after SE by Western blotting and immunocytochemistry. Neuronal damage was verified by Fluoro-Jade B staining. RESULTS: In P21 rats, SE resulted in neuronal damage in the CA1 neurons of the hippocampus. COX-2 expression was extensively, but transiently, increased and its immunoreactivity pronouncedly enhanced in several hippocampal subregions, amygdala, and piriform cortex by 24 h after SE. The expression of Bax and caspase-3 remained unchanged, whereas the antiapoptotic factor Bcl-2 transiently decreased by 24 h. Single caspase-3 positive neurons appeared in the CA1 region of both control and KA-treated rats. In P9 rats, no neuronal death was detected, and COX-2 expression and immunoreactivity remained at the control level. DISCUSSION: Our results suggest that SE provokes age-specific effects on COX-2 expression. This together with the activation of putative inflammatory processes may contribute to neuronal cell death in the hippocampus of postnatal rats, whereas caspase-dependent apoptosis seems not to be involved in the death process.     

Hypothermia protects somatostatinergic neurons in rat dentate hilus from zinc accumulation and cell death after cerebral ischemia

We have previously shown that somatostatin (SS) immunoreactive (-i) neurons, located in the rat dentate hilus, are vulnerable to cerebral ischemia (Johansen et al., 1987). Within 40 h after ischemia, the cells show clear signs of cell death. At the same time, we observed that dying cells, located in the projection field of the mossy fibers (dentate hilus and CA3 mossy fiber layer), accumulate free zinc. We now demonstrate that the hilar cells, accumulating zinc after ischemia, are SS-i cells. Since it is known that hypothermia can ameliorate ischemic brain damage, we furthermore studied whether hypothermia (29°C) protects the vulnerable SS-i neurons in hilus from zinc accumulation and ischemic cell death. We found that hypothermia both prevented ischemia-induced neuronal zinc accumulation and cell death. We speculate that hilar SS-i cells are highly vulnerable to ischemia, and develop rapid ischemic cell death, because they accumulate zinc shortly after ischemia.     

Glutamate receptor 1-immunopositive neurons in the gliotic CA1 area of the mouse hippocampus after pilocarpine-induced status epilepticus

Significant reduction in glutamate receptor 1 (GluR1)- and GluR2/3-immunopositive neurons was demonstrated in the hilus of the dentate gyrus in mice killed on days 1, 7 and 60 after pilocarpine-induced status epilepticus (PISE). In addition, GluR1 and GluR2/3 immunostaining in the strata oriens, radiatum and lacunosum moleculare of areas CA1-3 decreased drastically on days 7 and 60 after PISE. Neuronal loss observed in the above regions may account, at least in part, for a decrease in GluR immunoreactivity. By contrast, many GluR1-immunopositive neurons were observed in the gliotic area of CA1. Of these, about 42.8% were immunopositive for markers for hippocampal interneurons, namely calretinin (7.6%), calbindin (12.8%) and parvalbumin (22.4%). GluR1 or GluR2/3 and BrdU double-labelling showed that the GluR1- and GluR2/3-immunopositive neurons at 60 days after PISE were neurons that had survived rather than newly generated neurons. Furthermore, anterograde tracer and double-labelling studies performed on animals at 60 days after PISE indicated a projection from the hilus of the dentate gyrus to gliotic areas in both CA3 and CA1, where the projecting fibres apparently established connections with GluR1-immunopositive neurons. The projection to CA1 was unexpected. These novel findings suggest that the intrinsic hippocampal neuronal network is altered after PISE. We speculate that GluR1-immunopositive neurons in gliotic CA1 act as a bridge between dentate gyrus and subiculum contributing towards epileptogenesis.     

The Protein Phosphatase Inhibitor Okadaic Acid Induces Heat Shock Protein Expression and Neurodegeneration in Rat Hippocampusin Vivo

The tumor promoter okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A and therefore it is a useful tool for studying the participation of protein phosphorylation in cellular processes. Since it has been shown that in cultured neurons OKA behaves as a potent neurotoxin, in the present work we have administered different doses of this compound into the dorsal rat hippocampus, in order to assess its neurotoxicityin vivo.Cresyl violet staining of hippocampal sections revealed that as early as 3 h after injection of 300 ng OKA a notable neurodegeneration occurred in the CA1 subfield, the dentate gyrus, and the hilus, particularly in the former. Neuronal death was more evident at 24 h and at this time the extent of damage was dose-dependent. The process of neuronal death was accompanied by a loss of the microtubule-associated protein MAP2, as assessed by immunocytochemistry. Moreover, OKA treatment resulted in a notable expression of the inducible heat shock protein 72 in the surviving neurons of the injected hippocampus and in the corresponding CA1 and hilus of the apparently normal contralateral hippocampus. The expression of the heat shock protein was partially prevented in the injected hippocampus and completely blocked in the contralateral CA1 region, by the systemic previous administration of the NMDA receptor antagonist MK-801. These results suggest that protein hyperphosphorylation due to inhibition of phosphatasesin vivoinduces neuronal stress and subsequent neurodegeneration.     

Temporospatial expression of HSP72 and c-JUN, and DNA fragmentation in goat hippocampus after global cerebral ischemia

The role of gene induction (expression of HSP72 and c-JUN proteins) and delayed ischemic cell death (in situ labeling of DNA fragmentation) have been investigated in the goat hippocampus after transient global cerebral ischemia. The animals were subjected to 20-min ischemia (bilateral occlusion of the external carotid arteries plus bilateral jugular vein compression) and allowed to reperfuse for 2 h, and then 1, 3, and 7 days. Histological signs of cell loss were not found in the hippocampus at 2 h, 1 day, or 3 days of reperfusion. However, such an ischemic insult produced extensive, selective, and delayed degeneration in the hippocampus, as 68% of the neurons in CA1 had died at 7 days, but cell loss was not detected in CA3 and dentate gyrus fields. Concomitantly, a high percentage of TUNEL-positive CA1 neurons (60+/-9%, mean +/- SEM) was seen at 7 days, but not at the earlier time points. Mild induction of HSP72 was detected in the goat hippocampus after ischemia. The maximum percentage of HSP72-positive neurons (10-15%) was shown at 3 days of reperfusion and was concentrated mainly in the CA3 field, subiculum, and hilus, rather than in the CA1 field, whereas HSP72 expression was hardly detected at 7 days. At this later time point, scattered induction of nuclear c-JUN was found in a few neurons. The results show that: 1) postischemic delayed neuronal death selectively affects the CA1 field in the goat hippocampus, a phenomenon which seems to take longer to develop than in previously reported rodent models; and 2) postischemic expression of c-JUN does not appear to be related to cell death or survival, while the inability of most CA1 neurons to express HSP72 could contribute to neuronal death.     

Regional and time‐dependent neuroprotective effect of hypothermia following oxygen‐glucose deprivation

The neuroprotective effect of hypothermia has been demonstrated in in vivo and in vitro models of cerebral ischemia. In regard to the hippocampus, previous studies have mainly focused on CA1 pyramidal neurons, which are very vulnerable to ischemia. But the dentate gyrus (DG), in which neuronal proliferation occurs, can also be damaged by ischemia. In this study, we explored the neuroprotective effect of postischemic hypothermia in different areas of the hippocampus after mild or severe ischemia. Organotypic hippocampal slice cultures were prepared from 6‐ to 8‐day‐old rats and maintained for 12 days. Cultures were exposed to 25 or 35 min of oxygen and glucose deprivation (OGD). Neuronal damage was quantified after 6, 24, 48, and 72 h by propidium iodide fluorescence. Mild hypothermia (33°C) was induced 1 h after the end of OGD and was maintained for a period of 24 h. Short OGD produced delayed neuronal damage in the CA1 area and in the DG and to a lesser extend in the CA3 area. Damage in CA1 pyramidal cells was totally prevented by hypothermia whereas neuroprotection was limited in the DG. Thirty‐five‐minute OGD induced more rapid and more severe cell death in the three regions. In this case, hypothermia induced 1 h after OGD was unable to protect CA1 pyramidal cells whereas hypothermia induced during OGD was able to prevent cell loss. This study provides evidence that neuroprotection by hypothermia is limited to specific areas and depends on the severity of the ischemia. © 2014 Wiley Periodicals, Inc.     

Kainic acid dose affects delayed cell death mechanism after status epilepticus

Kainic acid (KA)-induced status epilepticus (SE) produces hippocampal neuronal death, which varies from necrosis to apoptosis or programmed cell death (PCD). We examined whether the type of neuronal death was dependent on KA dose. Adult rats were induced SE by intraperitoneal injection of KA at 9 mg/kg (K9) or 12 mg/kg (K12). Hippocampal neuronal death was assessed by TUNEL staining, electron microscopy, and Western blotting of caspase-3 on days 1, 3 and 7 after SE induction. K12 rats showed higher a mortality rate and shorter latency to the onset of SE when compared with K9 rats. In both groups, acidophilic and pyknotic neurons were evident in CA1 at 24h after SE and neuronal loss developed from day 3. The degenerated neurons became TUNEL-positive on days 3 and 7 in K9 rats but not in K12 rats. Caspase-3 activation was detected on days 3 and 7 in K9 rats but was undetectable in K12 rats. Ultrastructural study revealed shrunken neurons exhibiting pyknotic nuclei containing small and dispersed chromatin clumps 24h after SE in CA1. No cells exhibited apoptosis. On days 3 and 7, the degenerated neurons were necrotic with high electron density and small chromatin clumps. There were no ultrastructural differences between the K9 and K12 groups. These results revealed that differences in KA dose affected the delayed cell death (3 and 7 days after SE); however, no effect was seen on the early cell death (24h after SE). Moderate-dose KA induced necrosis, while low-dose KA induced PCD.     

Neuronal damage following non-lethal but repeated cerebral ischemia in the gerbil

Summary Brief, non-lethal transient forebrain ischemia in the gerbil can injure selectively vulnerable neurons when such ischemia is induced repeatedly. The influence of the number and interval of the ischemic insults on neuronal damage, as well as the time course of damage, following repeated 2-min forebrain ischemia were examined. A single 2-min forebrain ischemia were examined. A single 2-min ischemic insult caused no morphological neuronal damage. A moderate number of hippocampal CA1 neurons were destroyed following two ischemic insults with a 1-h interval, and destruction of almost all CA1 neurons resulted from three or five insults at 1-h intervals. Three and five insults also resulted in moderate to severe damage to the striatum and thalamus, depending on the number of episodes. Although three ischemic insults at 1-h intervals caused severe neuronal damage, this number of insults at 5-min and 4-h intervals caused destruction of relatively few neurons, and non neurons were destroyed at 12-h intervals. Following three ischemic insults at 1-h intervals, damage to the striatum, neocortex, hippocampal CA4 subfield and thalamus was observed at 6–24 h of survival, whereas damage to the hippocampal CA1 subfield appeared at 2–4 days. The results indicate that even a brief non-lethal ischemic insult can produce severe neuronal damage in selectively vulnerable regions when it is induced repeatedly at a certain interval. The severity of neuronal damage was dependent on the number and interval of ischemic episodes.     

Methamphetamine-induced neuronal necrosis: the role of electrographic seizure discharges

We have evidence that methamphetamine (METH)-induced neuronal death is morphologically necrotic, not apoptotic, as is currently believed, and that electrographic seizures may be responsible. We administered 40 mg/kg i.p. to 12 male C57BL/6 mice and monitored EEGs continuously and rectal temperatures every 15 min, keeping rectal temperatures <41.0 °C. Seven of the 12 mice had repetitive electrographic seizure discharges (RESDs) and 5 did not. The RESDs were often not accompanied by behavioral signs of seizures—i.e., they were often not accompanied by clonic forelimb movements. The 7 mice with RESDs had acidophilic neurons (the H&E light-microscopic equivalent of necrotic neurons by ultrastructural examination) in all of 7 brain regions (hippocampal CA1, CA2, CA3 and hilus, amygdala, piriform cortex and entorhinal cortex), the same brain regions damaged following generalized seizures, 24 h after METH administration. The 5 mice without RESDs had a few acidophilic neurons in 4 of the 7 brain regions, but those with RESDs had significantly more in 6 of the 7 brain regions. Maximum rectal temperatures were comparable in mice with and without RESDs, so that cannot explain the difference between the two groups with respect to METH-induced neuronal death. Our data show that METH-induced neuronal death is morphologically necrotic, that EEGs must be recorded to detect electrographic seizure activity in rodents without behavioral evidence of seizures, and that RESDs may be responsible for METH-induced neuronal death.     

GDNF family ligands and receptors are differentially regulated after brain insults in the rat

Expression of mRNAs for glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and their receptors was studied in adult rat brain using in situ hybridization after 40 kindling-evoked, rapidly recurring seizures or 10 min of global forebrain ischaemia. Following seizures, GDNF and NTN mRNAs were elevated in dentate granule cells, and c-Ret mRNA in hilar neurons and non-pyramidal cells in CA1 and CA3 regions. GFRalpha-1 mRNA levels showed more widespread increases in the dentate granule cell layer and hilus, CA1 and CA3 pyramidal layers, basolateral amygdala and parietal cortex. The expression of GFRalpha-2 mRNA increased in the piriform cortex and decreased in the CA1 region and basolateral amygdala. Forebrain ischaemia induced elevated expression of GDNF mRNA in dentate granule cells, GFRalpha-1 mRNA in the dentate granule cell layer, hilus and CA3 pyramidal layer, and GFRalpha-2 mRNA in the parietal cortex. The gene expression patterns observed here suggest that GDNF and NTN may act as target-derived factors, but also in an autocrine or paracrine manner. GFRalpha-1 can be coexpressed with GFRalpha-2 and c-Ret mRNAs in the same hippocampal or thalamic neurons, but other neurons contain GFRalpha-1 alone or together with c-Ret mRNA. The gene expression changes for the ligands, and the receptor components are region-, cell- and insult-specific, and occur independently of each other, mainly within 24 h after seizures or ischaemia. This dynamic regulation of GDNF and NTN circuits primarily at the receptor level may be important for the effectiveness of neuroprotective responses but could also trigger plastic changes, e.g. those underlying the development of epileptic syndromes.