Cellular expression of connexins in the rat brain: neuronal localization, effects of kainate-induced seizures and expression in apoptotic neuronal cells

The identification of connexins (Cxs) expressed in neuronal cells represents a crucial step for understanding the direct communication between neurons and between neuron and glia. In the present work, using a double-labelling method combining in situ hybridization for Cx mRNAs with immunohistochemical detection for neuronal markers, we provide evidence that, among cerebral connexins (Cx26, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45 and Cx47), only Cx45 and Cx36 mRNAs are localized in neuronal cells in both developing and adult rat brain. In order to establish whether connexin expression is influenced in vivo by abnormal neuronal activity, we examined the short-term effects of kainate-induced seizures. The results revealed an unexpected expression of Cx26 and Cx45 mRNA in neuronal cells undergoing apoptotic cell death in the CA3-CA4, in the hilus of the hippocampus and in other brain regions involved in seizure-induced lesion. However, the expression of Cx26 and Cx45 mRNAs was not associated with detectable expression of corresponding proteins as evaluated by immunohistochemistry with specific antibodies. Moreover, in the same brain regions Cx32 and Cx43 were up-regulated in non-neruronal cells whereas the neuronal Cx36 was down-regulated. Taken together the present results provide novel information regarding the specific subpopulation of neurons expressing Cx45 and raise the question of the meaning of connexin mRNA expression in the neuronal apoptotic process.     

Expression of the proto-oncogene bcl-2 is increased in the rat brain following kainate-induced seizures

The proto-oncogene bcl-2 is an important suppressor of apoptotic cell death in development and of both apoptotic and necrotic cell death in mature neurons. We studied expression of bcl-2 and the related gene, bax, which may promote cell death, after seizures induced by systemic kainate injection in rats. Expression of bcl-2 mRNA was studied by in situ hybridization. Bax and bcl-2 protein expression was studied by immunocytochemistry. Histologic analysis of cresyl violet-stained paraffin sections was performed at 72 h. bcl-2 protein was expressed in CA1 neurons, a region that is injured, yet survives after seizures. Bcl-2 mRNA was expressed in CA3, a region where there is extensive neuronal death at 72 h, but the bcl-2 protein was not translated. However, bax protein expression in CA3 was increased at 24 h. These results support a possible role for bcl-2 in promoting survival of CA3 after seizures.     

Temporal changes in expression of neuronal nitric oxide synthase mRNA in the rat hippocampus associated with kainate-induced seizures

We studied the temporal changes in expression of neuronal nitric oxide (NO) synthase (nNOS) mRNA in the hippocampus of rats treated with kainic acid by use of in situ hybridization technique. Intraperitoneal injection of 10 mg kg-1 kainic acid decreased expression of nNOS mRNAs in the dentate gyrus and CA3 region of the hippocampus at 3 h and 8 h and increased it in the dentate gyrus and CA1 at one week after treatment. Although our previous study indicated that administration of kainic acid increased NO generation in the rat hippocampus, present results suggest that the injection of kainic acid results in differential regulation of nNOS mRNA and NO formation in the rat hippocampus.     

Cycloheximide prevents kainate-induced neuronal death and c-fos expression in adult rat brain

The present study was directed at evaluating the possible involvement of protein synthesis in excitotoxin-induced neuronal damage and prolonged expression of the proto-oncogene, c-fos. Kainic acid-induced seizure activity elicited varying degrees of neuronal damage and cell loss in selectively vulnerable regions of the adult rat limbic system. Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter behavioral seizure characteristics, but markedly attenuated damage to susceptible neuronal populations. A prolonged increase in c-fos mRNA was observed byin situ hybridization up to 16 h after the onset of seizures in regions exhibiting neuronal death. Pretreatment with cycloheximide did not affect the transient induction of c-fos observed in numerous structures, but significantly reduced the prolonged expression of c-fos mRNA in kainatevulnerable regions. Despite producing massive seizure activity, systemic kainic acid administration during the early postnatal period did not induce any neuronal death, and did not result in prolonged c-fos expression in any brain structures. The developmental onset of selective neuronal vulnerability coincided with that of prolonged c-fos expression in susceptible neuronal populations. In adult rats, seizure activity induced by pentylenetetrazole did not produce neuronal damage nor did it produce prolonged c-fos expression. These results not only demonstrate that kainate-induced neurotoxicity and the prolonged expression of c-fos are both prevented by cycloheximide, but also strengthen the idea that prolonged c-fos expression is a marker of neuronal death.     

AMP-activated protein kinase is highly expressed in neurons in the developing rat brain and promotes neuronal survival following glucose deprivation

Adenosine monophosphate-activated protein kinase (AMPK) is a member of metabolite-sensing kinase family that plays important roles in responses of muscle cells to metabolic stress. AMPK is a heterotrimer of a catalytic α subunit (α1 or α2), and β (β1 or β2) and γ (γ1 or γ2) subunits. Because the brain has a high metabolic rate and is sensitive to changes in the supply of glucose and oxygen, we investigated the expression of AMPK in rat embryonic and adult brain and its role in modifying neuronal survival under conditions of cellular stress. We report that catalytic (α1 and α2) and noncatalytic (β2 and γ1) subunits of AMPK are present at high levels in embryonic hippocampal neurons in vivo and in cell culture. In the adult rat brain, the catalytic subunits α1 and α2 are present in neurons throughout the brain. The AMPK-activating agent AICAR protected hippocampal neurons against death induced by glucose deprivation, chemical hypoxia, and exposure to glutamate and amyloid β-peptide. Suppression of levels of the AMPK α1 and α2 subunits using antisense technology resulted in enhanced neuronal death following glucose deprivation, and abolished the neuroprotective effect of AICAR. These findings suggest that AMPK can protect neurons against metabolic and excitotoxic insults relevant to the pathogenesis of several different neurodegenerative conditions.     

Effects of pilocarpine- and kainate-induced seizures on thyrotropin-releasing hormone biosynthesis and receptors in the rat brain

Summary. The expression of mRNA coding for prepro-thyrotropin releasing hormone (preproTRH) was estimated in the rat brain in two animal models of limbic seizures, evoked by systemic administration of pilocarpine (400 mg/kg ip) or kainate (12 mg/kg ip). As shown by an in situ hybridization study, after 24 h both pilocarpine- and kainate-induced seizures profoundly increased the preproTRH mRNA level in the dentate gyrus. After 72 h, the preproTRH mRNA level was back to control values. Kainate-treated rats showed an elevated level of TRH in the hippocampus, septum, frontal and occipital cortex after 24 and 72 h, whereas in the striatum and amygdala the TRH level was raised after 72 h only. In the hypothalamus, TRH levels was lowered after 3 and 24 h, and returned to the control after 72 h. Pilocarpine-induced seizures also elevated the TRH level after 72 h in the majority of the above structures, except for the hypothalamus and amygdala where no changes were found at any time point. A radioreceptor assay showed that kainate decreased the B_max value of TRH receptors in the striatum and hippocampus after 3 and 24 h, respectively, and had no effect on the K_d values. In contrast, pilocarpine-induced seizures lowered the B_max of TRH receptors in the striatum, hippocampus and piriform cortex after 72 h only, and decreased K_d values in the striatum, amygdala and frontal cortex. These data showed that pilocarpine- and kainate-induced seizures enhanced likewise preproTRH mRNA in the dentate gyrus; on the other hand, they differed with respect to time- and structure-related changes in TRH tissue levels and TRH receptors. These differences may have functional significance in TRH-dependent control mechanism of the seizure activity in these two models of limbic epilepsy. Received February 2, 1998; accepted November 25, 1998     

Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity

The expression of excitatory amino acid transporters (EAATs) in rat hippocampus was studied following kainic acid-induced seizure activity in vivo and in hippocampal slice cultures. Protein and mRNA levels of the glial (EAAT2) and neuronal (EAAT3) transporters were determined with affinity-purified antibodies and oligonucleotide probes, respectively. Kainate treatment decreased EAAT3 immunoreactivity in stratum lacunosum moleculare within 4 h of seizure onset. Upon pyramidal cell death (5 days after kainate treatment), EAAT3 immunoreactivity in stratum pyramidale of CA1 and in stratum lacunosum moleculare was almost completely eliminated. The rapid effect of kainate on EAAT3 expression was confirmed by in situ hybridization; EAAT3 mRNA levels were decreased in CA1 and CA3 regions within 4-8 h of seizure onset. Kainate treatment had an opposite effect on levels and expression of EAAT2. Developmental studies indicated that the rapid regulation of transporter expression was not observed in rats younger than 21 days, an observation congruent with previous reports regarding the resistance of young rats to kainate. In hippocampal organotypic cultures, which lack a major excitatory input from the entorhinal cortex, kainate produced a slow decrease in [3H]d-aspartate uptake. This study indicates that an early effect of kainate treatment consists of down-regulation of the neuronal transporter EAAT3 in restricted hippocampal regions, together with a modest increase in the expression of the glial transporter EAAT2. Differential regulation of neuronal and glial glutamate transporters may thus play a role in kainate-induced seizure, neurotoxicity and neuronal plasticity.     

The cardiac sodium channel mRNA is expressed in the developing and adult rat and human brain

Expression of the rat (RH-I/SkM2) and human (hH1/SCN5A) tetrodotoxin-resistant (TTX-R), voltage-sensitive sodium channels is thought to be specific to cardiac tissue. We detected RH-I/SkM2 mRNA in newborn rat brain using both RNase protection assay analysis and in situ hybridization and in adult rat brain using RNase protection assay analysis. This expression was observed primarily in developing limbic structures of the cerebrum and diencephalon, and in the medulla of the brain stem. Using RT-PCR analysis, we detected hH1/SCN5A mRNA in both fetal and adult human brain. Interestingly, mutations in the human cardiac sodium channel are known to lead to cardiac abnormalities, which result in arrhythmias and frequently in sudden cardiac death. If these mutant channels were also expressed in limbic regions of the brain, alterations in channel function could have drastic effects on the brain’s signaling ability, possibly promoting seizure activity.     

L-4-chlorokynurenine attenuates kainate-induced seizures and lesions in the rat

Blockade of the strychnine-insensitive glycine site of the NMDA receptor is considered an attractive strategy for the development of novel neuroprotective and anticonvulsive agents. 7-Cl-kynurenic acid (7-Cl-KYNA) is a potent, selective antagonist of the NMDA/glycine receptor but penetrates poorly through the blood-brain barrier. Its prodrug, L-4-Cl-kynurenine (4-Cl-KYN), readily enters the brain from the circulation and provides antiexcitotoxic neuroprotection after systemic application. We now examined the effect of 4-Cl-KYN on seizures and neuronal loss caused by the systemic administration of the chemoconvulsant kainate (KA). 4-Cl-KYN (50 mg/kg, ip) was given 10 min before and 30, 120, and 360 min after KA (10 mg/kg, sc). Microdialysis and tissue level measurements in 4-Cl-KYN-treated rats showed increases in the concentration of 7-Cl-KYNA in several limbic brain regions of KA-injected animals. Continuous EEG recording for 24 h revealed that 4-Cl-KYN significantly delayed seizure onset and reduced the total time spent in seizures. Repeated 4-Cl-KYN administration also prevented KA-induced lesions in the piriform cortex and provided protection of hippocampal pyramidal cells in area CA1. In contrast, neurons in the hilus and in layer III of the entorhinal cortex were not protected. Consistent with the in vivo results, in vitro application of 7-Cl-KYNA to brain slices containing hippocampus and entorhinal cortex preferentially blocked low Mg(2+)-induced seizure activity in hippocampal pyramidal cells. Taken together, these data suggest that a prodrug approach using 4-Cl-KYN might offer advantages in the treatment of temporal lobe epilepsy.     

Regional distribution and time-course of calpain activation following kainate-induced seizure activity in adult rat brain

Systemic injection of kainic acid (KA) in adult rat elicits a pattern of neuronal pathology which exhibits several features of human temporal lobe epilepsy. KA-induced seizure activity is accompanied by the activation of the calcium-dependent protease calpain in limbic structures. In the present study, we evaluated the spatio-temporal activation of calpain after the onset of seizure activity by immunohistochemistry using an antibody for the spectrin breakdown product (sbdp) generated by calpain-mediated spectrin proteolysis. In addition, we compared the changes in sbdp immunoreactivity with those in immunoreactivity to subunits of the Glu/AMPA receptors (GluR₁ andGluR₂₃). One hour after seizure onset, sbdp accumulation was observed in selected interneurons in stratum oriens and in the hilus of the dentate gyrus. By 4 h, sbdp immunoreactivity was prominent in dendritic fields of the hippocampus as well as in neurons in thalamus and piriform cortex. By 8 h, sbdp immunoreactivity had disappeared from interneurons but was localized in pyramidal cell bodies in hippocampus. Intense labeling of cell bodies and dendritic fields persisted until 5 days following KA treatment. Changes in GluR subunit immunoreactivity were mirror images of those seen for sbdp. In general, increased sbdp immunoreactivity in dendritic fields was associated with decreased (GluR₁ immunoreactivity. However, increased sbdp immunoreactivity in neuronal perikarya was also associated with increased GluR immunoreactivity. These results indicate that calpain activation following seizure onset exhibits a specific spatio-temporal pattern, with activation in restricted interneurons preceding widespread activation in pyramidal neurons. Calpain activation also precedes neuronal pathology and could thus represent an initial trigger for neuronal pathology. Finally, the results suggest that calpain activation produces rapid alterations in GluR subunit properties which could be involved in the hyperexcitability observed following seizure activity.     

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.     

Induction of brain derived neurotrophic factor mRNA by seizures in neonatal and juvenile rat brain

Seizures have been shown to regulate neurotrophin expression in adult mammalian brain. However, there has been some controversy as to whether seizures affect neurotrophin expression in very immature brain. In the present study, we have examined the effects of seizures induced by pilocarpine following lithium pretreatment or by kainic acid on the expression of brain derived neurotrophic factor (BDNF) mRNA in developing rat brain by in situ hybridization. In adult brain, lithium/pilocarpine treatment resulted in dramatic elevations of hybridization to BDNF cRNA in neocortical and limbic brain structures. In developing brain, lithium/pilocarpine induced elevations of BDNF mRNA in the hippocampus, piriform and entorhinal cortex as early as postnatal day 7 (P7). By P12, the pattern of enhanced expression was similar to that of the adult. Maximal elevations of hybridization were present 2 to 4 h following pilocarpine injection. Electrophysiological recording demonstrated that lithium/pilocarpine treatment resulted in electrographic seizures. Pretreatment with diazepam blocked the seizures as well as the elevation of BDNF mRNA. Kainic acid induced elevations of BDNF mRNA in the CA3 subfield of the hippocampal pyramidal cell layer, but not in other brain areas in pups as young as P7. These data indicate that seizures during the neonatal and early juvenile period of brain development induce elevated BDNF mRNA expression, and that different methods of seizure induction yield different patterns of elevations in hybridization. Furthermore, BDNF may be capable of playing a role in the development of seizure susceptibility in the immature brain.     

Immunocytochemically detectable metallothionein is expressed by astrocytes in the ischaemic human brain

Immunocytochemically detectable metallothionein is expressed by astrocytes in the ischaemic human brain
The metallothioneins in vitro are both effective free radical and transitional metal ion scavengers. For this reason we investigated the expression of this protein in the normal CNS and after ischaemia using a monoclonal anti-metallothionein antibody to metallothionein isoforms I and II. Using immunohistochemistry and confocal laser scanning microscopy, we found anti-GFAP and anti-metallothionein were co-localized in astrocytes at the edges of infarcts. Energy dispersive X-ray micro-analysis demonstrated high levels of copper and iron in the metallothionein positive reactive astrocytes. On the basis of the in vitro properties of these proteins, the functional importance of metallothionein in reactive astrocytes could be as a free radical scavenger and metal ion chelator.     

SC1, a brain extracellular matrix glycoprotein related to SPARC and follistatin, is expressed by rat cerebellar astrocytes following injury and during development

In the nervous system, extracellular matrix components are believed to influence cell shape, proliferation and migration during development and following injury. SC1 is a secreted glycoprotein expressed during neural development and in the adult brain. The molecule shows partial sequence homology to the anti-adhesive extracellular matrix molecule SPARC/osteonectin and to follistatin. We have made a surgical lesion in the adult rat cerebellum and examined changes in SC1 expression at 1 to 14 days after injury. Dual in situ hybridization/immunohistochemistry demonstrated that SC1 mRNA was induced in astrocytes surrounding the wound, reaching maximal levels at 10 days post-lesion. Immunohistochemistry revealed changes in the deposition of SC1 protein in radial fibres of Bergmann glia. SC1 protein was also detected at the border of the lesion, suggesting an association with the glial scar. Double immunohistochemistry with the astrocytic marker GFAP demonstrated that astrocytes also express SC1 during postnatal development.     

病毒感染性脑病反应性星形细胞内皮素一l的表达

目的：对8例急性单纯疱疹病毒性脑炎、2例进行性多灶性白质脑病（PML）、2例亚急性硬化性全脑炎（SSPE）尸检材料进行研究,以探讨内皮素一1在这些疾病中的作用。方法：采用免疫组织化学（SP法）技术。结果：脑反应性星形细胞存在内皮索－1的表达,而对照组均明性。结论：提示内皮素－1可能参与病毒感染性脑病的病理过程。     

GFAP mRNA levels following stab wounds in rat brain

We previously reported that glial fibrillary acidic protein (GFAP) levels increased significantly at 3 days after stab wounds, relative to sham-operated controls, reaching a maximum of 200% of control value at 5-7 days. They then fell to near-normal values by 21 days. To determine whether these protein changes correlated with changes in GFAP mRNA we performed Northern blot analyses. Total RNA, isolated from lesioned, sham-operated and intact rat forebrains, was hybridized with 32P-labeled mouse GFAP cDNA and quantified by densitometry. The maximum increase in total RNA content in lesioned animals was only 20% over controls at 12 h. GFAP mRNA levels increased to 2-fold control values at 6 h and reached 5-fold at 12 h. Thereafter they remained at 3.5- to 6-fold until 5 days and then declined to 1.5-fold by 21 days. The rapid increase of GFAP message at 12 h preceded a significant increase in GFAP by 2 days and the decrease of message after 5 days was more precipitate than the slow decrease in GFAP content. Sham-operated animals showed no significant changes in GFAP mRNA, compared to intact controls, during the period 3 h to 14 days postoperation. GFAP mRNA and GFAP in the stab-wound model reached levels similar to those found in the experimental autoimmune encephalomyelitis (EAE) model, but returned to normal much more rapidly.     

alpha-synuclein is developmentally expressed in cultured rat brain oligodendrocytes

Although a neuronal protein, alpha-synuclein is a major component of glial cytoplasmic inclusions (GCIs) in oligodendrocytes of multiple system atrophy (MSA) brains. Because alpha-synuclein has not been identified in oligodendrocytes of normal brains, we examined cultured rat brain oligodendrocytes during in vitro development and showed that alpha-synuclein mRNA and protein are present in cultured oligodendrocytes. The expression of alpha-synuclein was developmentally regulated; it increased to peak levels at 2 or 3 days in culture but declined thereafter. Indirect immunofluorescence further shows that alpha-synuclein was localized predominantly in cell bodies and primary processes of oligodendroglia. Thus, GCIs may be a consequence of altered rather than de novo expression of alpha-synuclein in MSA oligodendrocytes.