GAP-43 mRNA localization in the rat hippocampus CA3 field.

Gene expression of the axonal growth-associated protein, GAP-43, has been studied in the adult rat brain by in situ hybridization histochemistry. This protein is synthesized at high levels in neuronal somata in immature and regenerating neurons, but after establishment of mature synaptic relations its synthesis generally declines sharply, thus providing a marker denoting propensity for exhibiting synaptic plasticity. Detailed examination of the distribution of mRNA for GAP-43 in rat hippocampus is selectively and robustly expressed in the pyramidal neurons of field CA3 and, to a lesser extent, the polymorph neurons of the hilus of the dentate gyrus. Additional hippocampal regions of moderate expression include the tenia tecta and the subicular and entorhinal fields, but CA1 and CA2 are strikingly lower in signal. The significance of this pattern of localization is considered in the context of the phosphorylation of GAP-43 and its role in influencing synaptic events underlying the establishment and maintenance of long-term potentiation and plasticity in the hippocampus.     

Gene expression of growth-associated proteins,GAP-43 and SCG10, in the hippocampal formation of the Macaque monkey: Nonradioactive in situ hybridization study

We performed nonradioactive in situ hybridization histochemistry in the monkey hippocampal formation that includes the hippocampus, the subicular complex, and the entorhinal cortex to detect the expression of mRNA for two growth-associated proteins: GAP-43 and SCG10. Overall, the distribution patterns overlapped but were partially distinct. In the hippocampus, the intense hybridization signals for both GAP-43 and SCG10 mRNAs were observed in the pyramidal cell layer of Ammon's horn, especially in CA3 subfields. The intense hybridization signals were also observed in the stratum oriens of Ammon's horn and the polymorphic layer of the dentate gyrus. In the granule cell layer of the dentate gyrus, many GAP-43 mRNA-positive cells were observed, whereas a few positive cells with weak signals were observed for SCG10 mRNA. Throughout the subicular complex, the hybridization signals for both mRNAs were weak. In the entorhinal cortex, both mRNAs were abundant in the caudal field. These subregion-specific expression of the growth-associated proteins may reflect the functional specialization regarding plasticity in each region of the monkey hippocampal formation. Hippocampus 1998;8:533–547. © 1998 Wiley-Liss, Inc.     

The gene for heparin-binding epidermal growth factor-like growth factor is stress-inducible: its role in cerebral ischemia.

The functions of the epidermal growth factor (EGF) family members in the adult brain are not known. This study investigated the changes in the expression of members of the EGF family following global ischemia employing in situ hybridization and immunohistochemical techniques to elucidate their roles in pathological conditions. EGF mRNA was not detected in either the control or the postischemic rat brain. Although transforming growth factor-alpha (TGF-alpha) mRNA was widely expressed in the normal brain, its expression did not change appreciably following ischemia. By contrast, heparin-binding EGF-like growth factor (HB-EGF) mRNA expression was rapidly increased in the CA3 sector and the dentate gyrus of the hippocampus, cortex, thalamus, and cerebellar granule and Purkinje cell layers. EGF receptor mRNA, which was widely expressed, also showed an increase in the CA3 sector and dentate gyrus. Conversely, HB-EGF mRNA did not show any increase prior to ischemic neuronal injury in the CA1 sector, the region most vulnerable to ischemia. Immunohistochemical detection of HB-EGF in the postischemic brain suggested a slight increase of immunostaining in the dentate gyrus of the hippocampus and the cortex. These findings showed that the gene encoding HB-EGF is stress-inducible, indicating the likelihood that HB-EGF is a neuroprotective factor in cerebral ischemia.     

Central 5,7-Dihydroxytryptamine Lesions Decrease Hippocampal Glucocorticoid and Mineralocorticoid Receptor Messenger Ribonucleic Acid Expression

Corticosteroids exert effects on the hippocampus by binding to intracellular glucocorticoid and/or mineralocorticoid receptors, but the relative importance of each receptor type in mediating corticosteroid effects is poorly understood. There is an extensive serotoninergic (5-HT) innervation of the hippocampus which interacts with corticosteroid-sensitive cells. We have investigated the effect of intracerebroventricular 5,7-dihydroxytryptamine lesions of 5-HT neurons on glucocorticoid and mineralocorticoid receptor messenger ribonucleic acid (mRNA) expression in the rat hippocampus using in situ hybridization histochemistry. In controls, glucocorticoid receptor mRNA was highly expressed in dentate gyrus granule cell neurons, and in pyramidal cells of CA1 and CA2, but levels in CAS and CA4 were significantly lower. 5,7-dihydroxytryptamine-lesioned animals showed significantly less glucocorticoid receptor mRNA in the dentate gyrus (76% decrease), CA1 (42% decrease) and CA2 (52% decrease; all P<0.05 compared with controls). Mineralocorticoid receptor mRNA was expressed at a similar level in all hippocampal subregions in control rats. 5,7-dihydroxytryptamine lesioning led to a significant decrease in mineralocorticoid receptor mRNA expression in CA3 (56% fall) and CA4 (45% fall; both P<0.05), but not in the other subregions. Thus the 5-HT innervation regulates hippocampal corticosteroid receptor mRNA expression.     

aFGF, bFGF and flg mRNAs Show Distinct Patterns of Induction in the Hippocampus Following Kainate-induced Seizures

We report that kainic acid-induced seizures lead to marked increases in mRNAs encoding basic and acidic fibroblast growth factors (bFGF and aFGF, respectively) and fig, one of their receptors, in the rat hippocampus. Anticonvulsant pretreatment inhibits the up-regulation of these mRNAs. The observed increase in fig mRNA levels involves the pyramidal cells of all hippocampal subfields and the granular ceils of the dentate gyrus. The increased expression of aFGF and bFGF mRNAs is limited to neuron populations that are resistant to seizure-induced injury, the granular cells of dentate gyrus and pyramidal cells of CA1 region, respectively. The results suggest that the increase in the FGFs and fig may play pivotal roles in neuron survival and in long-term changes occurring in the hippocampus following seizure activity.     

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.     

Time-course study of SCG10 mRNA levels associated with LTP induction and maintenance in the rat Schaffer-CA1 pathway in vivo

The maintenance of long-term potentiation (LTP) depends on altered gene expression. Previously, we found the expression of neuronal growth associated protein SCG10, which is involved in neurite outgrowth and neural regeneration, was up-regulated by LTP induction in the rat hippocampal Schaffer-collateral CA1 pathway. Here we studied the temporal expression pattern of SCG10 mRNA after LTP induction using permanently implanted electrodes in the same CA1 pathway. The real-time RT-PCR showed that both SCG10 mRNA 1 and 2 kb forms were increased at the 3 h, but not at 1 or 24 h. In situ hybridization revealed an increase of SCG10 2 kb mRNA level in ipsilateral CA3 and CA1 areas, but not their contralateral counterparts or either side of dentate gyrus. These results suggest that SCG10 may play a role in the maintenance of synaptic plasticity through a transient regulation of microtubule dynamics, which facilitates the structural remodeling of the presynaptic element during the consolidation period.     

Increased cyclin D1 in vulnerable neurons in the hippocampus after ischaemia and epilepsy: a modulator of in vivo programmed cell death?

Several observations suggest that delayed neuronal death in ischaemia, epilepsy and other brain disorders includes an apoptotic component, involving programmed cell death (PCD). PCD is hypothesized to result, in part, from aberrant control of the cell cycle. Because they are instrumental in mitosis, cyclins D are key markers to evaluate whether neurons indeed progress into the cell cycle in situations of pathology. Therefore, we investigated in rat brains, the expression of cyclins D in the delayed neuronal death that occurs following transient global ischaemia and kainate-induced seizures. Following a four-vessel occlusion insult, quantitative in situ hybridization revealed a highly significant and persistent 100% increase of cyclin D1 mRNA in the vulnerable pyramidal neurons of the CA1 hippocampal region. Ischaemia also induced a smaller and transient cyclin D1 mRNA increase in the resistant CA3 area and dentate gyrus. In contrast, the cyclin D2 and D3 mRNAs, expressed constitutively in the adult rat hippocampus, were not upregulated. Following kainate-induced seizures, cyclin D1 mRNA was induced in the vulnerable CA3 region, and to a lesser extent, in non-vulnerable regions. Cyclin D1 immunohistochemistry revealed increased protein levels in the cytoplasm and nucleus of neurons commited to die after ischaemia. Double labelling experiments indicate that cyclin D1 is also expressed in reactive astrocytes but not in microglial cells. Finally, we report that in neurons, cyclin D1 expression peaks before nuclear condensation and the appearance of DNA fragmentation. We propose that cyclin D1, when expressed at high levels in lesioned neurons, may act as a modulator of apoptosis.     

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.     

Alpha1-adrenoreceptor in human hippocampus: binding and receptor subtype mRNA expression

Alpha1-adrenoreceptors (AR), of which three subtypes exist (alpha1A-, alpha1B- and alpha1D-AR) are G-protein-coupled receptors that mediate the actions of norepinephrine and epinephrine both peripherally and centrally. In the CNS, alpha1-ARs are found in the hippocampus where animal studies have shown the ability of alpha1-AR agents to modulate long-term potentiation and memory; however, the precise distribution of alpha1-AR expression and its subtypes in the human brain is unknown making functional comparisons difficult. In the human hippocampus, 3H-prazosin (alpha1-AR antagonist) labels only the dentate gyrus (molecular, granule and polymorphic layers) and the stratum lucidum of the CA3 homogeneously. Human alpha1A-AR mRNA in the hippocampus is observed only in the dentate gyrus granule cell layer, while alpha1D-AR mRNA expression is observed only in the pyramidal cell layers of CA1, CA2 and CA3, regions where 3H-prazosin did not bind. alpha1B-AR mRNA is not expressed at detectable levels in the human hippocampus. These results confirm a difference in hippocampal alpha1-AR localization between rat and humans and further describe a difference in the localization of the alpha1A- and alpha1D-AR mRNA subtype between rats and humans.     

Increased Expression of GAP-43, Somatostatin and Neuropeptide Y mRNA in the Hippocampus During Development of Hippocampal Kindling in Rats

The expression and distribution of the mRNA coding for the growth-associated protein-43 (GAP-43), a putative marker for neuritic growth, for preprosomatostatin and the preproneuropeptide Y (ppNPY) were analysed in the rat hippocampus during the development of hippocampal kindling by an in situ hybridization technique and computer-assisted grain counting in the cell. The levels of GAP-43 mRNA increased significantly in the CA3 pyramidal neurons and hilar polymorphic neurons of the dentate gyrus 2 days after stage 2 of kindling (preconvulsive stage) but not stage 5 (full seizure expression) in the stimulated hippocampus. The distribution of GAP-43 mRNA was the same in the hippocampus of kindled rats as in sham-stimulated animals. Preprosomatostatin mRNA and ppNPY mRNA contents rose significantly in the hilar polymorphic neurons of the dentate gyrus of the stimulated and contralateral hippocampus at both stages of kindling, with the greatest effect at stage 5. In addition, the number of ppNPY mRNA neurons in the fascia dentata was significantly higher in kindled rats than in controls, but there were no differences in the number of preprosomatostatin mRNA-positive cells. Preprosomatostatin and ppNPY mRNAs were also increased in the neurons of the stratum oriens of the CA1 - CA3 subfield of fully kindled animals, whereas at stage 2 only neurons of the CA1 stratum oriens showed a significant increase of preprosomatostatin mRNA. No changes in preprosomatostatin and ppNPY mRNA expression were observed in the various regions of the hippocampus after a single afterdischarge or 1 month after stage 5. These data show that synthesis of somatostatin and neuropeptide Y increases in certain neurons of the hippocampus during the development of hippocampal kindling, and support the suggestion that these peptides are involved in epileptogenesis. Moreover, the increased synthesis of GAP-43 may contribute to the synaptic remodelling of certain hippocampal neurons during kindling.     

Synapsin I Gene expression in the adult rat brain with comparative analysis of mRNA and protein in the hippocampus

Synapsin I is the best characterized member of a family of neuron-specific phosphoproteins thought to be involved in the regulation of neurotransmitter release. In this report, we present the first extensive in situ hybridization study detailing the regional and cellular distribution of synapsin I mRNA in the adult rat brain. Both the regional distribution and relative levels of synapsin I mRNA established by in situ hybridization were confirmed by RNA blot analysis. Our data demonstrate the widespread yet regionally variable expression of synapsin I mRNA throughout the adult rat brain. The greatest abundance of synapsin I mRNA was found in the pyramidal neurons of the CA3 and CA4 fields of the hippocampus, and in the mitral and internal granular cell layers of the olfactory bulb. Other areas abundant in synapsin I mRNA were the layer II neurons of the piriform cortex and layer II and V neurons of the entorhinal cortex, the granule cell neurons of the dentate gyrus, the pyramidal neurons of hippocampal fields CA1 and CA2, and the cells of the parasubiculum. In general, the pattern of expression of synapsin I mRNA paralleled those encoding other synaptic terminal-specific proteins, such as synaptophysin, VAMP-2, and SNAP-25, with noteworthy exceptions. To determine specificall how synapsin I mRNA levels are related to levels of synapsin I protein, we examined in detail the local distribution patterns of both synapsin I mRNA and protein in the rat hippocampus. These data revealed differential levels of expression of synapsin I mRNA and protein within defined synaptic circuits of the rat hippocampus. © 1993 Wiley-Liss, Inc.     

Restoration of calbindin after fetal hippocampal CA3 cell grafting into the injured hippocampus in a rat model of temporal lobe epilepsy

Degeneration of the CA3 pyramidal and dentate hilar neurons in the adult rat hippocampus after an intracerebroventricular kainic acid (KA) administration, a model of temporal lobe epilepsy, leads to permanent loss of the calcium binding protein calbindin in major fractions of dentate granule cells and CA1 pyramidal neurons. We hypothesize that the enduring loss of calbindin in the dentate gyrus and the CA1 subfield after CA3-lesion is due to disruption of the hippocampal circuitry leading to hyperexcitability in these regions; therefore, specific cell grafts that are capable of both reconstructing the disrupted circuitry and suppressing hyperexcitability in the injured hippocampus can restore calbindin. We compared the effects of fetal CA3 or CA1 cell grafting into the injured CA3 region of adult rats at 45 days after KA-induced injury on the hippocampal calbindin. The calbindin immunoreactivity in the dentate granule cells and the CA1 pyramidal neurons of grafted animals was evaluated at 6 months after injury (i.e. at 4.5 months post-grafting). Compared with the intact hippocampus, the calbindin in "lesion-only" hippocampus was dramatically reduced at 6 months post-lesion. However, calbindin expression was restored in the lesioned hippocampus receiving CA3 cell grafts. In contrast, in the lesioned hippocampus receiving CA1 cell grafts, calbindin expression remained less than the intact hippocampus. Thus, specific cell grafting restores the injury-induced loss of calbindin in the adult hippocampus, likely via restitution of the disrupted circuitry. Since loss of calbindin after hippocampal injury is linked to hyperexcitability, re-expression of calbindin in both dentate gyrus and CA1 subfield following CA3 cell grafting may suggest that specific cell grafting is efficacious for ameliorating injury-induced hyperexcitability in the adult hippocampus. However, electrophysiological studies of KA-lesioned hippocampus receiving CA3 cell grafts are required in future to validate this possibility.     

Expression of brain-derived neurotrophic factor mRNA in rat hippocampus after treatment with antipsychotic drugs

Typical and atypical antipsychotic drugs, though both effective, act on different neurotransmitter receptors and are dissimilar in some clinical effects and side effects. The typical antipsychotic drug haloperidol has been shown to cause a decrease in the expression of brain-derived neurotrophic factor (BDNF), which plays an important role in neuronal cell survival, differentiation, and neuronal connectivity. However, it is still unknown whether atypical antipsychotic drugs similarly regulate BDNF expression. We examined the effects of chronic (28 days) administration of typical and atypical antipsychotic drugs on BDNF mRNA expression in the rat hippocampus using in situ hybridization. Quantitative analysis revealed that the typical antipsychotic drug haloperidol (1 mg/kg) down-regulated BDNF mRNA expression in both CA1 (P < 0.05) and dentate gyrus (P < 0.01) regions compared with vehicle control. In contrast, the atypical antipsychotic agents clozapine (10 mg/kg) and olanzapine (2.7 mg/kg) up-regulated BDNF mRNA expression in CA1, CA3, and dentate gyrus regions of the rat hippocampus compared with their respective controls (P < 0.01). These findings demonstrate that the typical and atypical antipsychotic drugs differentially regulate BDNF mRNA expression in rat hippocampus.     

匹罗卡品致疒间大鼠海马GAP-43与TrkB基因表达及其意义

目的探讨生长相关蛋白(GAP-43)和脑源性神经营养因子(BDNF)受体TrkB基因在匹罗卡品致疒间大鼠海马的表达及其意义.方法应用原位杂交组织化学方法研究匹罗卡品(PILO)致疒间大鼠海马GAP-43及TrkB mRNA表达的变化.结果匹罗卡品致癫疒间持续状态后3～6小时,海马齿状回颗粒细胞、CA3区及CA1区锥体细胞层TrkB mRNA表达显著高于对照组(P<0.01或0.05);在慢性期第7～30天,呈现第2次表达增强.致疒间后6～12小时,正常状态下并不表达GAP-43的大鼠海马颗粒细胞其GAP-43 mRNA表达较对照组显著增高(P<0.01),24小时～7天表达减少,在癫疒间慢性期表达再次高于对照组.结论 GAP-43及TrkB是颞叶癫疒间病理基础--海马苔藓纤维出芽的重要分子机制;BDNF对苔藓纤维的作用部分是通过GAP-43实现的.     

Differential expression of HSC73 and HSP72 mRNA and proteins between young and adult gerbils after transient cerebral ischemia: relation to neuronal vulnerability.

This study presents a quantitative comparison of the time courses and regional distribution of both constitutive HSC73 and inducible HSP72 mRNA expression and their respective encoded proteins between young (3-week-old) and adult (3-month-old) gerbil hippocampus after transient global ischemia. The constitutive expression of HSC73 mRNA and protein in the hippocampus of the young sham-operated gerbils was significantly higher than in the adults. The HSC73 mRNA expression after ischemia in the CA1 layer of young gerbils was greater than in adult gerbils. HSC73 immunoreactivity was not significantly changed after ischemia-reperfusion in adult hippocampus, whereas it decreased in young gerbils. Ischemia-reperfusion led to induction of HSP72 mRNA expression throughout the hippocampus of both young and adult gerbils. HSP72 mRNA induction was more intense and sustained in the CA1 subfield of young gerbils; this was associated with a marked induction of HSP72 proteins and neuronal survival. The transient expression of HSP72 mRNA in the CA1 layer of adult gerbils was not associated with a subsequent synthesis of HSP72 protein but was linked to neuronal loss. Expression of HSP72 mRNA was shifted to an earlier period of reflow in CA3 and dentate gyrus (DG) subfields of young animals. These findings suggest that the induction of both HSP72 mRNA and proteins in the CA1 pyramidal neurons of young gerbils, as well as the higher constitutive expression of HSC73, may partially contribute to higher neuronal resistance of young animals to transient cerebral ischemia.     

Administration of quinolinic acid in the rat hippocampus induces expression of c-fos and NGFI-A.

We have studied the effect of intrahippocampal administration of quinolinic acid (QUIN) on the temporal expression of mRNAs encoding the immediate early genes (IEGs) c-fos and NGFI-A, by in situ hybridization histochemistry. After administration of QUIN to the left hippocampus, expression of mRNA of both IEGs was transiently stimulated. Maximal expression was found between 1 and 3 h. mRNA of both IEGs was simultaneously expressed in the ipsilateral and contralateral sides in the granule cell layer of the dentate gyrus, the pyramidal cell layer of the CA1 and CA3 fields as well as in the cortex. After pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg i.p. -30 min) the increased expression of both IEGs was partially prevented in the hippocampus and completely in the cortex. No inhibition was observed after treatment with the AMPA antagonist NBQX (30 mg/kg i.p. -15, -5 and +10 min). Additional delayed expression of both IEGs was observed in the ipsilateral hippocampus. This expression was related to cell damage. Twelve h after QUIN administration, c-fos and NGFI-A mRNAs were present in the dentate gyrus. After 4 days, only c-fos mRNA was observed in the dentate gyrus and CA1 field while no NGFI-A mRNA was detected. The present results show that the effect of QUIN is mediated by NMDA and not by AMPA receptors.