Changes in mint1, a novel synaptic protein, after transient global ischemia in mouse hippocampus.

Mints (munc18-interacting proteins) are novel multimodular adapter proteins in membrane transport and organization. Mint1, a neuronal isoform, is involved in synaptic vesicle exocytosis. Its potential effects on development of ischemic damage to neurons have not yet been evaluated. The authors examined changes in mint1 and other synaptic proteins by immunohistochemistry after transient global ischemia in mouse hippocampus. In sham-ischemic mice, immunoreactivity for mint1 was rich in fibers projecting from the entorhinal cortex to the hippocampus and in the mossy fibers linking the granule cells of the dentate gyrus to CA3 pyramidal neurons. Munc18-1, a binding partner of mint1, was distributed uniformly throughout the hippocampus, and synaptophysin 2, a synaptic vesicle protein, was localized mainly in mossy fibers. After transient global ischemia, mint1 immunoreactivity in mossy fibers was dramatically decreased at 1 day of reperfusion but actually showed enhancement at 3 days. However, munc18-1 and synaptophysin 2 were substantially expressed in the same region throughout the reperfusion period. These findings suggest that mint1 participates in neuronal transmission along the excitatory pathway linking the entorhinal cortex to CA3 in the hippocampus. Because mint1 was transiently decreased in the mossy fiber projection after ischemia, functional impairment of neuronal transmission in the projection from the dentate gyrus to CA3 pyramidal neurons might be involved in delayed neuronal death.     

Chronological alterations of neurofilament 150 immunoreactivity in the gerbil hippocampus and dentate gyrus after transient forebrain ischemia

In this study, we observed the chronological alterations of neurofilament 150 (NF-150) immunoreactivity in the gerbil hippocampus and dentate gyrus after 5 min transient forebrain ischemia. NF-150 immunoreactivity in the sham-operated group was mainly detected in mossy fibers and in the hilar region of the dentate gyrus. NF-150 immunoreactivity and protein contents of NF-150 and RT 97 (polyphosphorylation epitopes of neurofilament) were significantly decreased at 15 min after ischemic insult. Between 30 min and 12 h after ischemic insult, NF-150 immunoreactivity and protein content were significantly increased as compared with the sham-operated group. Thereafter, NF-150 immunoreactivity and protein content started to decrease. At 12 h after ischemic insult, unlike dentate gyrus, NF-150 immunoreactivity increased in pyramidal cells of the CA1 region. Thereafter, NF-150 immunoreactivity in the CA1 region started to decrease, and 4 days after ischemic insult, NF-150 immunoreactivity nearly was similar to that of the sham-operated group. These biphasic patterns of NF-150 immunoreactivity in the hippocampus and dentate gyrus are reverse correlated with that of the intracellular calcium influx. For calcium detection in the CA1 region, we also conducted alizarin red staining. Alizarin red positive neurons were detected in some neurons at 15-30 min after ischemic insult. At 12 h after ischemia, alizarin red positive neurons were decreased. Thereafter, alizarin red positive neurons started to decrease, but alizarin positive neurons were significantly increased in dying neurons 4 days after ischemia. These results suggest that ischemia-related changes of NF-150 expression may be caused by the calcium following transient forebrain ischemia.     

Secretoneurin: A marker in rat hippocampal pathways

Secretoneurin is a 33-amino acid peptide, generated in brain by proteolytic processing of secretogranin II. The distribution of secretoneurin-like immunoreactivity and secretogranin II mRNA was investigated in the hippocampus of the rat. Secretogranin II mRNA was found in high concentrations throughtout the granule cell and pyramidal cell layers and in many local neurons, notably in the hilus of the dentate gyrus. The general distributional pattern of secretoneurin-like immunoreactivity was characterized by a prominent staining in the area of the terminal field of mossy fibers with an obvious staining in the infrapyramidal area of CA3 and a strongly immunopositive band in the inner third of the molecular layer of the dentate gyrus. Lesions of the granule cells by local injection of colchicine significantly reduced secretoneurin-like immunoreactivity in the terminal field of mossy fibers, but not in the inner molecular layer of the dentate gyrus. On the other hand, destruction of interneurons of the dentate gyrus (mossy cells and certain γ-aminobutyric acid-ergic interneurons) by kainic acid—induced seizures was associated with a reduction of secretoneurin-like immunoreactivity in the inner molecular layer of the dentate gyrus. However, 30 days after kainic acid—induced seizures, a strongly secretoneurin-immunoreactive band reappeared in this area, which at this late time point is due to sprouting of mossy fibers collaterals. Our experiments suggest a widespread distribution of secretoneurin-like immunoreactivity in neurons of the hippocampal formation with a preferential localization in excitatory pathways including associational/commissural fibers originating from secretoneurin-containing mossy cells. J. Comp. Neurol. 377:29-40, 1997. © 1997 Wiley-Liss, Inc.     

Changes in protein kinase C isozymes in the rat hippocampal formation following hippocampal lesion

The cellular and regional distribution of the four protein kinase C (PKC) isoforms in the rat hippocampal formation and the response of PKC to lesions were determined by employing immunohistochemical and immunochemical techniques with antibodies specific to PKC (α), -(β I), -(β II), and -(γ). PKC (α) intensely stained the periphery of the pyramidal cell in the stratum pyramidale. The granule cells, glial cells, and mossy fibers were anti-PKC (α) negative. The cytoplasm, axons, and dendrites of basket cells and interneurons in the hilus were labeled with anti-PKC (α). Anti-PKC (β I) immunoreactivity was localized on the periphery of pyramidal cells and interneurons of the hilus, as well as the oriens, radiatum, and molecular layers of the CA regions. Anti-PKC (β II) immunoreactivity was mainly cytoplasmic, extending into the dendrites in the hippocampal pyramidal cells and the dentate granule cells, and also in some glial cells. In the stratum radiatum of the CA1, anti-PKC (γ) immunoreactivity localized to the pyramidal cell cytoplasm, extending into the dendrites. Following fimbria-fornix (FF) lesions, the anti-PKC (α) and -(β I) staining of the pyramidal cell periphery was markedly reduced. The anti-PKC (γ) staining of the pyramidal and granular cells of the dentate gyrus was reduced whereas the interneuron staining in the hilus was increased. In the FF-lesioned hippocampus, anti-PKC (α) and antiαPKC (β II) labeled reactive glial cells, whereas anti-PKC (β I) and -(γ) did not. Quantitative Western blot analysis revealed a dramatic increase in the particulate/total PKC for all isozyme forms, although the total levels of PKC, except PKC (γ), did not change following FF lesions. The PKC (γ) concentration doubled after FF lesions. Perforant path lesions resulted in a marked alteration in the neuronal staining in dentate gyrus with anti-PKC (α) and -(β I) and in increased numbers of anti-PKC (α)- and anti-PKC (β II)-positive glial cells. Anti-PKC (γ) staining did not change noticeably. The total PKC concentration did not change for isozymes α, β I, and γ, but PKC (βII) concentration increased by 48% following perforant path lesions as detected by Western blot analysis. The particulate/total PKC decreased for all four isozymes although the reduction in PKC (β I) concentration was not statistically significant. This change in PKC compartmentalization is in marked contrast to an increased level of particulate PKC following FF lesions. Thus, the effects of deefferentation and deafferentation for each PKC isoform were different.     

Transient increase of synapsin-I immunoreactivity in the mossy fiber layer of the hippocampus after transient forebrain ischemia in the mongolian gerbil

Synapsin-I is a vesicular phosphoprotein, which regulates neurotransmitter release, neurite development, and maturation of synaptic contacts during normal development and following various brain lesions in adulthood. In the present study, we have examined by immunohistochemistry possible modifications in the expression of synapsin-I in the hippocampus of Mongolian gerbils after transient forebrain ischemia. The animals were subjected to 5 min of transient forebrain ischemia through bilateral common carotid occlusion, and were examined at different time-points post-ischemia. Transient forebrain ischemia produces cell death of the majority of CA1 pyramidal neurons of the hippocampus and polymorphic hilar neurons of the dentate gyrus. This is followed by reactive changes, including synaptic reorganization and modifications in the expression of synaptic proteins, which provide the molecular bases of synaptic plasticity. Transient decrease of synapsin-I immunoreactivity was observed in the inner zone of the molecular layer of the dentate gyrus, thus suggesting denervation and posterior reinervation in this area. In addition, a strong increase in synapsin-I immunoreactivity was observed in the hilus of the dentate gyrus and in the mossy fiber layer of the hippocampus at 2, 4 and 7 days after ischemia. Parallel increases in synaptophysin immunoreactivity were not observed, thus suggesting a selective induction of synapsin-I after ischemia. The present results indicate that synapsin-I participates in the reactive response of granule cells to transient forebrain ischemia in the hippocampus of the gerbil, and suggest a role for this protein in the plastic adaptations of the hippocampus following injury.     

Expression and changes of Ca2+-ATPase in neurons and astrocytes in the gerbil hippocampus after transient forebrain ischemia

Ca2+-ATPase is one of the most powerful modulators of intracellular calcium levels. In this study, we focused on chronological changes in the immunoreactivity and protein levels of Ca2+-ATPase in the hippocampus after 5 min of transient forebrain ischemia. Ca2+-ATPase immunoreactivity was significantly altered in the hippocampal CA1 region and in the dentate gyrus, but not in the CA2/3 region after ischemic insult. In the sham-operated group, Ca2+-ATPase immunoreactivity was detected in the hippocampus. Ca2+-ATPase immunoreactivity in the CA1 region and in the dentate gyrus, and its protein levels peaked 3 h after ischemic insult. At this time, CA1 pyramidal cells and dentate polymorphic cells showed strong Ca2+-ATPase immunoreactivity. Thereafter, Ca2+-ATPase immunoreactivity reduced in the CA1 region and in the dentate gyrus. One day after ischemic insult, Ca2+-ATPase immunoreactivity was observed in some CA1 non-pyramidal cells, and 4 days after ischemic insult, Ca2+-ATPase immunoreactivity was detected in astrocytes throughout the CA1 region, but Ca2+-ATPase immunoreactivity in the dentate gyrus had nearly disappeared. Our results suggest that Ca2+-ATPase changes may be associated with a response to ischemic damage in hippocampal CA1 pyramidal cells, and that increased Ca2+-ATPase immunoreactivity in the reactive astrocytes may be associated with the maintenance of intracellular calcium levels.     

Induction of Calcitonin Gene-Related Peptide-like Immunoreactivity in Hippocampal Neurons Following Ischemia: A Putative Regional Modulator of the CNS Injury/Immune Response

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and immune cell modulator. In two studies within the hippocampal formation (HF), CGRP-like immunoreactivity (CGRP-LI) was increased in the inner molecular layer of the dentate gyrus after adrenalectomy and in mossy cells after colchicine-induced destruction of granule neurons. Given the increase in CGRP-LI following damage to the granule cell region of the HF, we investigated another trauma model, ischemia, that targeted different areas of the HF, CA1 region, and subiculum to ascertain the regional expression of this peptide after insult. Following ischemia, light microscopic evaluation showed CGRP-LI in basket cell-like neuronal perikarya within the dorsal subiculum and CA1 region of the hippocampus and in varicose fibers within the CA2 region of the hippocampus. Control rats rarely expressed CGRP-LI within neurons in these regions. In ischemic brains, double-labeled immunocytochemistry with antibodies to various neural markers demonstrated co-localization of CGRP-LI primarily within surviving subicular and CA1 cells resembling interneurons containing parvalbumin-LI or calbindin-LI. Electron microscopic analysis of the CA1 region from ischemic brains showed that CGRP-LI was contained in terminals with numerous small synaptic vesicles that formed symmetric synapses with perikarya and large dendrites of pyramidal cells, some of which were degenerating. Collectively, the data from this study and our previous study indicate that damage induces CGRP-LI expression in interneurons and nonprincipal cells in the area of damage, and we hypothesize that CGRP expression in surviving neurons within damage-related regions of the hippocampus is likely to be an important, and possibly a protective, component of the response of the nervous system to injury.     

Poor correlation between delayed neuronal death induced by transient forebrain ischemia,and immunoreactivity for parvalbumin and calbindin D-28k in developing gerbil hippocampus

In the normal developing hippocampus of the gerbil, parvalbumin-immunoreactive neurons first appear in the stratum pyramidale of CA3 at postnatal day 15 (P15), and in CA2 and hilus of the dentate gyrus from P21 onwards. Immunoreactive terminals also follow the same sequence from CA3 to CA1 to reach adult patterns by the end of the 1st month. Calbindin D-28k immunoreactivity is seen in the external part of the upper blade of the dentate gyrus at P5, and progresses to the granule cell and molecular layers of the whole gyrus by P15, except for a thin band of immature cells located at the base of the granule cell layer which are calbindin negative. Calbindin immunoreactivity in mossy fibers progresses from the external to the hilar region of CA3 during the same period. A few immunoreactive cells are also found in the stratum radiatum/lacunare of the CA3, but no calbindin-immunoreactive cells are observed in the CA1 and CA2 subfields. The adult pattern of calbindin immunoreactivity is reached at P21. Vulnerability following transient forebrain ischemia for 20 min was examined in the hippocampal formation of gerbils during postnatal development. No cellular damage was seen in animals aged 7 days. Dying cells were observed at the base of the granule cell layer of the dentate gyrus in animals aged 15, 21 and 30 days. Pyramidal cells in the CA3 subfield were also sensitive to ischemia in gerbils aged 15 days, and less frequently in animals aged 21 days. The adult pattern of cellular damage, characterized by selective vulnerability of the CA1 subfield, was seen from day 30 onwards. These findings show that the pattern of selective vulnerability following transient forebrain ischemia is different in young and adult gerbils, and suggest that little, if any, correlation exists between resistance to delayed cellular damage and parvalbumin and calbindin D-28k content in the hippocampus of young gerbils.Supported in part by grant FIS 93-131 and a grant from the Fundacio Pi i Synyer (to A.T.)     

The distribution of chromogranin A-like immunoreactivity in the human hippocampus coincides with the pattern of resistance to epilepsy-induced neuronal damage

The distribution of chromogranin A-like immunoreactivity in the hippocampus of adult humans who were free of neurological disease was examined by immunohistochemical methods. Immunoreactivity was restricted to the cytoplasm of certain neuronal populations, most notably the mossy fibers of denate granule cells (and a subset of their perikarya), and the perikarya of pyramidal cells of the cornu Ammonis 2 (CA2) sector. Additionally, staining was observed in neurons in the stratum oriens, a population of neurons at the periphery of the CA4 sector, scattered, probably short-axon perikarya in the CA1 sector, and fibers in the perforant path and the molecular layer of the dentate gyrus. Pyramidal neurons in the CA1 and CA3 sectors were not immunoreactive. The two prominently immunoreactive neuronal populations, CA2 pyramids and dentate granule cells, are those spared in human and experimental epileptic brain damage, whereas CA1 and CA3 pyramids, lacking chromogranin, are characteristically destroyed in this condition. The known activities of chromogranin in the periphery as a calcium-binding protein and as a precursor of active peptides (autocrine inhibitory modulators) suggest that its distribution in the hippocampus may help to explain the observed pattern of resistance to epileptic brain damage.     

TRANSIENT ISCHEMIA-INDUCED EXPRESSION AND CHANGES OF TYROSINE KINASE A IN THE HIPPOCAMPAL DENTATE GYRUS OF THE GERBIL

The present study examined ischemia-related changes in tyrosine kinase A (trkA) immunoreactivity and its protein content in the dentate gyrus after 5 min of transient forebrain ischemia in gerbils. One day after ischemic insult, cresyl violet-positive polymorphic cells showed ischemic degeneration. The ischemia-induced changes in trkA immunoreactivity were found in the polymorphic layer (PL) and granule cell layer (GCL) of the dentate gyrus. In the sham-operated group, trkA immunoreactivity in the dentate gyrus was very weak. From 30 min after ischemia, trkA immunoreactivity was increased in the dentate gyrus and peaked in the dentate gyrus at 12 h after ischemia-reperfusion. Thereafter, trkA immunoreactivity was decreased time-dependently after ischemia-reperfusion. Four days after ischemic insult, trkA immunoreactivity was similar to that of the sham-operated group. In addition, it was found that ischemia-related changes in trkA protein content were similar to the immunohistochemical changes. These results suggest that the chronological changes of trkA in the dentate gyrus after transient forebrain ischemia may be associated with ischemic damage in polymorphic cells of the dentate gyrus.     

Transient ischemia-induced expression and changes of tyrosine kinase A in the hippocampal dentate gyrus of the gerbil

The present study examined ischemia-related changes in tyrosine kinase A (trkA) immunoreactivity and its protein content in the dentate gyrus after 5 min of transient forebrain ischemia in gerbils. One day after ischemic insult, cresyl violet-positive polymorphic cells showed ischemic degeneration. The ischemia-induced changes in trkA immunoreactivity were found in the polymorphic layer (PL) and granule cell layer (GCL) of the dentate gyrus. In the sham-operated group, trkA immunoreactivity in the dentate gyrus was very weak. From 30 min after ischemia, trkA immunoreactivity was increased in the dentate gyrus and peaked in the dentate gyrus at 12 h after ischemia-reperfusion. Thereafter, trkA immunoreactivity was decreased time-dependently after ischemia-reperfusion. Four days after ischemic insult, trkA immunoreactivity was similar to that of the sham-operated group. In addition, it was found that ischemia-related changes in trkA protein content were similar to the immunohistochemical changes. These results suggest that the chronological changes of trkA in the dentate gyrus after transient forebrain ischemia may be associated with ischemic damage in polymorphic cells of the dentate gyrus.     

Glucose metabolism and neurogenesis in the gerbil hippocampus after transient forebrain ischemia

Recent evidence exists that glucose transporter 3 (GLUT3) plays an important role in the energy metabo-lism in the brain. Most previous studies have been conducted using focal or hypoxic ischemia models and have focused on changes in GLUT3 expression based on protein and mRNA levels rather than tissue levels. In the present study, we observed change in GLUT3 immunoreactivity in the adult gerbil hippocampus at various time points after 5 minutes of transient forebrain ischemia. In the sham-operated group, GLUT3 immunoreactivity in the hippocampal CA1 region was weak, in the pyramidal cells of the CA1 region in-creased in a time-dependent fashion 24 hours after ischemia, and in the hippocampal CA1 region decreased signiifcantly between 2 and 5 days after ischemia, with high level of GLUT3 immunoreactivity observed in the CA1 region 10 days after ischemia. In a double immunolfuorescence study using GLUT3 and gli-al-ifbrillary acidic protein (GFAP), we observed strong GLUT3 immunoreactivity in the astrocytes. GLUT3 immunoreactivity increased after ischemia and peaked 7 days in the dentate gyrus after ischemia/reperfu-sion. In a double immunolfuorescence study using GLUT3 and doublecortin (DCX), we observed low level of GLUT3 immunoreactivity in the differentiated neuroblasts of the subgranular zone of the dentate gyrus after ischemia. GLUT3 immunoreactivity in the sham-operated group was mainly detected in the subgran-ular zone of the dentate gyrus. These results suggest that the increase in GLUT3 immunoreactivity may be a compensatory mechanism to modulate glucose level in the hippocampal CA1 region and to promote adult neurogenesis in the dentate gyrus.     

Spatial and temporal distribution of N-type Ca(2+) channels in gerbil global cerebral ischemia

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (alpha(1A)), N-type (alpha(1B)), and L-type (alpha(1C) and alpha(1D)) Ca(2+) channels were observed in the hippocampus at days 1-5 after ischemic injury. However, increased expression of N-type Ca(2+) channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca(2+) channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca(2+) concentration ([Ca(2+)](i)) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca(2+) channels may prove useful for future investigations.     

Expression of GAP-43 in the Granule Cells of Rat Hippocampus After Seizure-induced Sprouting of Mossy Fibres: In Situ Hybridization and Immunocytochemical Studies

The axonal growth-associated protein GAP-43 is believed to play some role in the synaptic remodelling that takes place in the hippocampus of adult rats after certain experimental lesions. GAP-43 mRNA is highly expressed in adult CA3 pyramidal cells but almost absent in the dentate granule cells. We analysed whether the sprouting of granule cell axons, the mossy fibres of the hippocampus, caused by kainic acid-induced seizures in adult rats was associated with any induction of GAP-43 mRNA in granule cells and with any changes in the immunostaining pattern of GAP-43 in the hippocampus. Increased GAP-43 mRNA expression was found to be induced in granule cells 18, 24 and 30 h after a systemic injection of kainic acid which induced generalized seizures in adult rats, and returned to control levels by 48 h post-treatment. No effect was observed in other regions of the hippocampus. However, when kainic acid was injected into 15-day-old rats, which responded with generalized seizures but no sprouting of mossy fibres, there was no induction of GAP-43 mRNA in the granule cells, suggesting a close relation between GAP-43 expression and sprouting of these cells. Seven days after kainic acid injections, GAP-43 immunostaining was decreased in the inner molecular layer of the dentate gyrus except for a thin supragranular band, whereas 30 days after treatment all animals showed increased GAP-43 immunoreactivity in the whole inner molecular layer. Since collaterals of mossy fibres grow in the inner molecular layer after kainic acid-induced seizures, these results support the theory that GAP-43 plays a role in synaptic remodelling in the adult central nervous system.     

Vesicular GABA transporter mRNA expression in the dentate gyrus and in mossy fiber synaptosomes

In the normal granule cells of the dentate gyrus, glutamate and both gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) coexist. GAD expression is increased after seizures, and simultaneous glutamatergic and GABAergic neurotransmission from the mossy fibers to CA3 appears, supporting the hypothesis that GABA can be released from the mossy fibers. To sustain GABAergic neurotransmission, the amino acid must be transported into synaptic vesicles. To address this, using RT-PCR we looked for the presence and regulation of expression of the vesicular GABA transporter (VGAT) mRNA in the dentate gyrus and in mossy fiber synaptosomes of control and kindled rats. We found trace amounts of VGAT mRNA in the dentate gyrus and mossy fiber synaptosomes of control rats. In the dentate gyrus of kindled rats with several seizures and of control rats subject to one acute seizure, no changes were apparent either 1 or 24 h after the seizures. However, repetitive synaptic or antidromic activation of the granule cells in slices of control rats in vitro induces an activity-dependent enhancement of VGAT mRNA expression in the dentate. Surprisingly, in the mossy fiber synaptosomes of seizing rats, the levels of VGAT mRNA were significantly higher than in controls. These data show that the granule cells and their mossy fibers, besides containing machinery for the synthesis of GABA, also contain the elements that support its vesiculation. This further supports the notion that local synaptic molecular changes enable mossy fibers to release GABA in response to enhanced excitability.     

Differential expression of S100beta and glial fibrillary acidic protein in the hippocampus after kainic acid-induced lesions and mossy fiber sprouting in adult rat

The expression of S100beta and glial fibrillary acidic protein (GFAP) was analyzed following bilateral injection of kainic acid (KA), a glutamate derivative, into the CA3 region of the adult rat hippocampus. This treatment produces a progressive degeneration of the pyramidal neurons of the hippocampus while sparing the granule cells of the dentate gyrus which undergo sprouting of their axons in the supragranular layer. Messenger RNA and protein levels were measured, by Northern blot and ELISA, in the hippocampus of lesioned and sham-operated rats 1, 7, and 30 days after KA injection. A significant increase of GFAP and its mRNA was demonstrated at each time point, whereas S100beta mRNA levels were significantly enhanced only 30 days after the KA injection and the levels of S100beta protein remained unchanged at all time points. However, when analyzed by immunohistochemistry the S100beta showed clear changes in its expression and distribution depending on the region considered. One month after KA injection, S100beta immunoreactivity was considerably reduced in the stratum radiatum of CA3 region, but there was increased S100beta immunoreactivity in the stratum moleculare. In particular, a notable band of S100beta positive, hypertrophic astrocytes appeared in the supragranular layer of the dentate gyrus where the sprouting of mossy fiber collaterals was detected by Timm's staining. These data show for the first time that an increase in S100beta expression in subpopulations of reactive astrocytes may be involved in the structural reorganization of the hippocampus following KA-induced neurodegeneration.     

Immunohistochemical distribution of beta-protein kinase C in rat hippocampus determined with an antibody against a synthetic peptide sequence

An antibody directed against a synthetic peptide sequence specific for the beta-subtype of protein kinase C (PKC) was used to determine the distribution of beta-PKC in rat hippocampus by immunocytochemistry. PKC was distributed primarily in the stratum oriens and radiatum of the CA1 region. Positive staining cell bodies were only observed after colchicine treatment in pyramidal cells (CA2-CA4) and granule cells of the dentate gyrus. The discrete localization of various subtypes of PKC should provide clues to their functions.     

脑缺血选择性海马CA1区神经元损害的实验研究

采用Ｐｕｌｓｉｎｅｌｉ－Ｂｒｉｅｒｌｅｙ４血管阻塞脑缺血模型观察了大鼠全脑缺血２０ｍｉｎ再灌流８ｈ，ｃ－ｆｏｓ基因表达及再灌流７ｄ海马ＣＡ１区迟发性神经元损害。在缺血再灌流早期（８ｈ）海马ＣＡ１区极少ｃ－ｆｏｓ表达，而齿状回、海马ＣＡ３区、杏仁核大量ｃ－ｆｏｓ表达。缺血再灌流晚期（７ｄ）镀银染色显示海马ＣＡ１区神经元及其突触终末带呈黑色溃变相，而齿状回、海马ＣＡ３区、杏仁核呈金黄色正常相。相邻切片ＨＥ染色示缺血组海马ＣＡ１区核完整的锥体细胞数（５±２．６个／２００μｍ）与对照组（４０±２．９个／μｍ）比较差异有显著意义（Ｐ＜０．０１）。脑缺血诱导的ｃ－ｆｏｓ基因表达对于缺血易损海马ＣＡ１区迟发性神经元坏死可能起直接的调控作用。     

Ischemic preconditioning upregulates expression of phospholipase D2 in the rat hippocampus

To investigate the possible involvement of phospholipase D2 (PLD2) in the induction of ischemic tolerance, we analyzed the distribution and time course of PLD2 expression in the rat hippocampus after a sublethal period of ischemia. Forebrain ischemia was induced by four-vessel occlusion for 3 min. Increased PLD2 immunoreactivity after this sublethal ischemia was observed in CA1 pyramidal neurons of the rat hippocampus. In tolerance-acquired CA1 neurons, PLD2 immunoreactivity was upregulated as early as 12 h post-ischemia and was most prominent at 1–3 days, with expression sustained for at least 7 days, as shown by a time course of immunoblotting and measurement of the enzymatic activity of PLD. PLD2 expression was also increased in ischemia-resistant CA3 neurons and dentate granule cells, although weaker staining intensity was noted. Further, we showed that, in cultured SK-N-BE(2)C human neuroblastoma cells, overexpression of PLD2 inhibited cell death by chemical hypoxia induced with potassium cyanide and deoxyglucose. These data suggest that upregulation of PLD2 might be involved in the neuroprotective mechanism of ischemic tolerance in the rat hippocampus. This research was supported by a grant (M103KV010010-06K2201-01010) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, the Republic of Korea.     

The comparative immunocytochemical distribution of 28 kDa cholecalcin (CaBP) in the hippocampus of rat, guinea pig and hedgehog

The distribution of 28 kDa cholecalcin (calcium-binding protein, CaBP) in the hippocampal formation of the rat, guinea pig and European hedgehog was examined by immunocytochemistry. The extension of the mossy fibers (the axons of the granule cells of the dentate gyrus) was also studied using the Timm's sulfide-silver method. Cholecalcin was present in all mossy fibers. In the rat, only those pyramidal cells not reached by the labeled mossy fibers displayed cholecalcin immunoreactivity. Immunocytochemical staining of the hedgehog hippocampus showed that contacts between cholecalcin-containing mossy fibers and cholecalcin-containing pyramidal cells are possible. Consequently, the protein is probably not involved in the control of mossy fiber extension. Strikingly, no guinea pig pyramidal cells showed cholecalcin immunoreactivity. The possible involvement of cholecalcin in the differential excitability of pyramidal cells in the CA3 and CA1 areas of the hippocampus could therefore be tested in a comparative study of rat, guinea pig and hedgehog.