Parvalbumin Immunoreactivity and Protein Level are Altered in the Gerbil Hippocampus During Normal Aging

Hippocampal interneurons are local circuit neurons which are responsible for inhibitory activity in the hippocampus. Parvalbumin (PV) is one of useful markers for GABAergic interneurons, not for principle cells, in the hippocampus. In the present study, we investigated age-related changes in PV immunoreactive neurons and protein levels in the gerbil hippocampus during normal aging. PV immunoreactive neurons were detected in all hippocampal subregions of all groups. PV immunoreactive neurons, which innervated principal neurons, were non-pyramidal neurons in the hippocampal CA1-3 regions, and were polymorphic neurons in the dentate gyrus. In the hippocampal CA1 region, the number of PV immunoreactive neurons was significantly reduced in the postnatal month 3 (PM 3) group, which was sustained by PM 18, and, at PM 24, the number of PV immunoreactive neurons was significantly decreased. In the CA2/3 region and dentate gyrus, the number of PV immunoreactive neurons was significantly decreased at PM 6: Thereafter, the number of PV immunoreactive neurons was sustained until PM 24. In addition, changes in PV protein levels in the gerbil hippocampus were similar to immunohistochemical changes during normal aging: PV protein levels were significantly decreased with age by PM 6: Thereafter, PV protein levels were sustained by PM 24. These results suggest that PV immunoreactive interneurons were decreased in the hippocampus with age in gerbils.     

Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family

Cells expressing mRNA for hippocampus-derived neurotrophic factor (HDNF/NT-3) or brain-derived neurotrophic factor (BDNF) were identified by in situ hybridization. In the rat brain, HDNF mRNA was predominantly found in pyramidal neurons in CA1 and CA2 of the hippocampus. Lower levels of HDNF mRNA were found in granular neurons of the dentate gyrus and in neurons of the taenia tecta and induseum griseum. BDNF mRNA-expressing cells were more widely distributed in the rat brain, with high levels in neurons of CA2, CA3, and the hilar region of the dentate gyrus, in the external and internal pyramidal layers of the cerebral cortex, in the claustrum, and in one brainstem structure. Lower levels were seen in CA1 and in the granular layer of the hippocampus, in the taenia tecta, and in the mammillary complex. In peripheral tissues, HDNF mRNA was found in glomerular cells in the kidney, secretory cells in the male rat submandibular gland, and epithelial cells in secondary and tertiary follicles in the ovary. Cells expressing BDNF mRNA were found in the dorsal root ganglia, where neurons of various sizes were labeled.     

Immunohistochemical localization of insulin-like growth factor binding protein 2 in the central nervous system of SOD1<Superscript>G93A</Superscript> transgenic mice

In the present study, we performed immunohistochemical studies to investigate the changes of insulin-like growth factor binding protein 2 (IGFBP2) in the central nervous system of SOD1^G93A mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). Decreased immunoreactivity for IGFBP2 was observed in the cerebral cortex, hippocampus and brainstem of SOD1^G93A transgenic mice. In the cerebral cortex, the number of IGFBP2-positive cells was decreased in the somatomotor area, somatosensory area, auditory area, visual area, entorhinal area, piriform area and prefrontal area. In the hippocampal formation, IGFBP2 immunoreactivity was significantly decreased in the CA1-3 areas and the dentate gyrus. In the brainstem, few IGFBP2-immunoreactive cells were observed in the medullary and pontine reticular formation, vestibular nucleus, trigeminal motor nucleus, facial nucleus, hypoglossal nucleus and raphe nucleus. In the spinal cord, IGFBP2 immunoreactivity was not significantly decreased in SOD1^G93A transgenic mice. This study showing decreased IGFBP2 in different brain regions of SOD1^G93A transgenic mice may provide clues for understanding differential susceptibility of neural structures in ALS. S. E. Sim and Y. H. Chung have contributed equally to this work.     

The role of trace elements in the pathogenesis and progress of pilocarpine-induced epileptic seizures

X-ray fluorescence microscopy was applied for topographic and quantitative elemental analysis within the areas of the rat brain that undergo neurodegenerative changes in consequence of pilocarpine-induced seizures. Significant changes in levels of selected elements were observed in epileptic animals. They included an increased tissue content of Ca in the CA1 and CA3 regions of the hippocampus and in the cerebral cortex. The opposite relation was observed for the Cu level in the dentate gyrus and for Zn in the CA3 region of the hippocampus and in the dentate gyrus.     

Immunohistochemical changes in vulnerable rat brain regions after reversible global brain ischaemia

Human global ischaemia was simulated in adult rats by inducing 20 min brain ischaemia and 60 min post-ischaemic recirculation. Immunohistochemical expression of MMP-9, TIMP-3, Bax and Bcl-2, and DNA fragmentation (with the TUNEL reaction) were investigated. The morphological data showed different neuronal responses in the hippocampus compared with the cerebral and cerebellar cortices. MMP-9 immunoreactivity was different in the hippocampus, particularly in dentate gyrus and the CA1 region, compared with these cortices. Negative TIMP-3 staining in ischaemic hippocampal neurons may indicate a loss of its inhibitory activity on MMP-9 that could enhance cell death. Bcl-2 down regulation, Bax positivity and TUNEL+ type II cells in the dentate gyrus granular layer could be responsible for induction of apoptotic death in CA1 hippocampal pyramidal cells via loss of fibre input. Results suggest differential behaviours of neural cells after 60 min reperfusion.     

Amygdala kindling differentially regulates the expression of the elements involved in TRH transmission

Subthreshold electrical stimulation of the amygdala (kindling) activates neuronal pathways increasing the expression of several neuropeptides including thyrotropin releasing-hormone (TRH). Partial kindling enhances TRH expression and the activity or its inactivating ectoenzyme; once kindling is established (stage V), TRH and its mRNA levels are further increased but TRH-binding and pyroglutamyl aminopeptidase II (PPII) activity decreased in epileptogenic areas. To determine whether variations in TRH receptor binding or PPII activity are due to regulation of their synthesis, mRNA levels of TRH receptors (R1, R2) and PPII were semi-quantified by RT-PCR in amygdala, frontal cortex and hippocampus of kindled rats sacrificed at stage II or V. Increased mRNA levels of PPII were found at stage II in amygdala and frontal cortex, and of pro-TRH and TRH-R2, in amygdala and hippocampus. At stage V, pro-TRH mRNA levels increased and those of PPII, decreased in the three regions; TRH-R2 mRNA levels diminished in amygdala and frontal cortex and of TRH-R1 only in amygdala. In situ hybridization analyses revealed, at stage II, enhanced TRH-R1 mRNA levels in dentate gyrus and amygdala while decreased in piriform cortex; those of TRH-R2 increased in amygdala, CA2, dentate gyrus, piriform cortex, thalamus and subiculum and of PPII, in CAs and piriform cortex. In contrast, at stage V decreased expression of TRH-R1 occurred in amygdala, CA2/3, dentate gyrus and piriform cortex; of TRH-R2 in CA2, thalamus and piriform cortex, and of PPII in CA2, and amygdala. The magnitude of changes differed between ipsi and contralateral side. These results support a trans-synaptic modulation of all elements involved in TRH transmission in conditions that stimulate the activity of TRHergic neurons. They show that reported changes in PPII activity or TRH-binding caused by kindling relate to regulation of the expression of TRH receptors and degrading enzyme.     

Chronological Distribution of Rip Immunoreactivity in the Gerbil Hippocampus During Normal Aging

Age-dependent studies on oligodendrocytes, which are the myelinating cells in the central nervous system, have been relatively less investigated. We examined age-dependent changes in Rip immunoreactivity and its protein level in the gerbil hippocampus during normal aging using immunohistochemistry and Western blot analysis with Rip antibody, an oligodendrocyte marker. Rip immunoreactivity and its protein level in the hippocampal CA1 region significantly increased at postnatal month 3 (PM 3). Thereafter, they decreased in the hippocampal CA1 region with age. At PM 24, Rip immunoreactive processes in the hippocampal CA1 region markedly decreased in the stratum radiatum. In the hippocampal CA2/3 region and dentate gyrus, the pattern of changes in Rip immunoreactivity and its protein level was similar to those in the hippocampal CA1 region; however, no significant changes were found in the CA2/3 region and dentate gyrus at various age stages. These results indicate that Rip immunoreactivity and protein level in the hippocampal CA1 region decreases significantly at PM 24 compared to the CA2/3 region and dentate gyrus.     

Caspase-3 immunoreactivity in different cortical areas of young and aging macaque (Macaca mulatta) monkeys

It has been shown that cytochrome-c-dependent caspase-3 activation is significantly elevated in the aging macaque brain. To assess the underlying age-related changes in the cellular distribution of caspase-3, we have examined the motor cortex, cerebellum and hippocampus of young (4-year-old, n = 4) and old (20-year-old, n = 4)rhesus monkeys by immunohistochemistry. Western blot analyses of brain homogenate showed that the antibody reacted only with inactive 32-kDa procaspase and its active 20- and 17-kDa subunits, formed after granzyme B exposure. In the motor cortex, pyramidal cells of layers III and V were moderately labeled; the underlying white matter contained weakly stained astrocytes. In the hippocampus, hilar neurons and pyramidal cells in CA3 showed the strongest immunoreaction, pyramidal cells in CA1 and granule cells of the dentate gyrus were also strongly labeled. In contrast, CA2 pyramidal cells were only weakly stained, and neurons of the molecular layer were unlabeled. Weak caspase-3 immunoreaction of CA2 neurons parallels known decreased susceptibility to apoptosis. In the cerebellar cortex, clusters of strongly labeled Purkinje cells were observed next to groups of weakly and unstained cells; granule cells were generally unstained. The brains of aging monkeys displayed a similar pattern of caspase-3 immunoreactivity. In neocortical layer V, however, scattered very strongly labeled pyramidal cells were regularly detected, which were not observed in younger animals. This clustering of caspase-3 indicates increased vulnerability of a subset of pyramidal cells in the aging brain.     

Decreased Glucagon-Like Peptide-1 Receptor Immunoreactivity in the Dentate Granule Cell Layer from Adult in the Gerbil Hippocampus

In this study, we investigated age-related changes in glucagon-like peptide-1 receptor (GLP-1R) immunoreactivity and its protein levels in the gerbil hippocampus during normal aging. In the postnatal month 3 (PM 3) group, GLP-1R immunoreaction was well observed in neurons, especially pyramidal and non-pyramidal cells in the hippocampus proper, and granule and polymorphic cells in the dentate gyrus. In the hippocampus proper, GLP-1R immunoreactivity in neurons was maintained until PM 24. In the dentate gyrus, however, GLP-1R immunoreactivity in granule cells, not polymorphic cells, was hardly detected from PM 6. Western blot analysis also showed that age-dependent change patterns in GLP-1R protein levels in the gerbil hippocampus were similar to the immunohistochemical changes. These results indicate that GLP-1R immunoreactivity was markedly decreased in dentate granule cells from PM 6, showing that GLP-1R immunoreactivity and its protein levels were decreased in the adult and aged gerbil hippocampus.     

Novel control by the CA3 region of the hippocampus on neurogenesis in the dentate gyrus of the adult rat

The dentate gyrus is a site of continued neurogenesis in the adult brain. The CA3 region of the hippocampus is the major projection area from the dentate gyrus. CA3 sends reciprocal projections back to the dentate gyrus. Does this imply that CA3 exerts some control over neurogenesis? We studied the effects of lesions of CA3 on neurogenesis in the dentate gyrus, and on the ability of fluoxetine to stimulate mitotic activity in the progenitor cells. Unilateral ibotenic-acid generated lesions were made in CA3. Four days later there was no change on the number of either BrdU or Ki67-positive progenitor cells in the dentate gyrus. However, after 15 or 28 days, there was a marked reduction in surviving BrdU-labelled cells on the lesioned side (but no change in Ki-67+ cells). pCREB or Wnt3a did not co-localise with Ki-67 but with NeuN, a marker of mature neurons. Lesions had no effect on the basal expression of either pCREB or Wnt3a. Subcutaneous fluoxetine (10 mg/kg/day) for 14 days increased the number of Ki67+ cells as expected on the control (non-lesioned) side but not on that with a CA3 lesion. Nevertheless, the expected increase in BDNF, pCREB and Wnt3a still occurred on the lesioned side following fluoxetine treatment. Fluoxetine has been reported to decrease the number of “mature” calbindin-positive cells in the dentate gyrus; we found this still occurred on the side of a CA3 lesion. We then showed that the expression GAP-43 was reduced in the dentate gyrus on the lesioned side, confirming the existence of a synaptic connection between CA3 and the dentate gyrus. These results show that CA3 has a hitherto unsuspected role in regulating neurogenesis in the dentate gyrus of the adult rat.     

Comparison of Ionized Calcium-binding Adapter Molecule 1 Immunoreactivity of the Hippocampal Dentate Gyrus and CA1 Region in Adult and Aged Dogs

Similarities between age-related changes in the canine and human brain have resulted in the general acceptance of the canine brain as a model of human brain aging. The hippocampus is essentially required for intact cognitive ability and appears to be particularly vulnerable to the aging process. We observed changes in ionized calcium-binding adapter molecule 1 (Iba-1, a microglial marker) immunoreactivity and protein levels in the hippocampal dentate gyrus and CA1 region of adult (2-3 years) and aged (10-12 years) dogs. We also observed the interferon-gamma (IFN-gamma), a pro-inflammatory cytokine, protein levels in these groups. In the dentate gyrus and CA1 region of the adult dog, Iba-1 immunoreactive microglia were well distributed and their processes were highly ramified. However, in the aged dog, the processes of Iba-1 immunoreactive microglia were hypertrophied in the dentate gyrus. Moreover, Iba-1 protein level in the dentate gyrus in the aged dog was higher than in the adult dog. IFN-gamma expression was increased in the dentate gyrus homogenates of aged dogs than adult dogs. In addition, we found that some neurons were positive to Fluoro-Jade B (a marker for neuronal degeneration) in the dentate polymorphic layer, but not in the hippocampal CA1 region in the aged dog. These results suggest that Iba-1 immunoreactive microglia are hypertrophied in the dentate gyrus in the aged dog.     

Expression and Localization of Type II Diacylglycerol Kinase Isozymes δ and η in the Developing Mouse Brain

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II–VI of the cerebral cortex; CA–CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II–VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.     

Gas7在大鼠海马和齿状回发育过程中的动态表达

目的研究生长休止蛋白7（Gas7）在大鼠海马和齿状回不同发育阶段的表达。方法采用免疫组织化学方法观察Gas7在SD大鼠胚胎第18d（E18）、新生（P0）、生后第7d（P7）、P14、P21和成年海马和齿状回中的表达和分布。结果在大鼠脑海马和齿状回部位的冠状切片上,Gas7免疫反应阳性产物主要表达在海马的锥体细胞、齿状回的颗粒细胞和门区的多形层细胞。随着发育的进程,在海马,Gas7较早表达在CA3区,其次是CA2和CA1区;在齿状回,Gas7在外臂的表达早于内臂,在颗粒细胞层的表达是按先外层后内层的顺序。在围生期,Gas7在海马和齿状回各区的表达逐渐增强,至P14达到高峰,后逐渐降低,至P21其表达强度和分布趋于恒定至成年水平。结论 Gas7在大鼠海马和齿状回发育过程中的动态表达具有时间和空间上的特异性,提示Gas7可能参与了海马和齿状回形态形成和功能成熟的调控。     

Alterations of Glial Cells in the Mouse Hippocampus During Postnatal Development

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.     

Expression of death-associated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures

Death-associated protein (DAP) kinase is calcium-regulated and known to function downstream of death receptors, prompting us to examine its role in the mechanism of seizure-induced neuronal death. Brief seizures were focally evoked in rats, eliciting neuronal death within the CA3 subfield of the hippocampus, and to a lesser extent, cortex. Western blotting confirmed expression of DAP kinase within hippocampus and cortex at the predicted weight of approximately 160 kDa. Immunohistochemistry revealed seizures triggered a significant increase in numbers of DAP kinase-expressing cells within CA3 and cortex, without affecting cell counts within seizure-resistant CA2 or the dentate gyrus. Numbers of DAP kinase-expressing cells were increased in relation to specific patterns of injury-causing seizure activity, electrographically defined. Seizures caused an early increase in DAP kinase binding to actin, and association with calmodulin. Co-immunoprecipitation studies also revealed seizures triggered binding of DAP kinase to the tumor necrosis factor receptor 1 and the Fas-associated death domain protein, commensurate with caspase-8 proteolysis. In contrast, within surviving fields of the hippocampus, DAP kinase interacted with the molecular chaperone 14-3-3. These data suggest DAP kinase is involved in the molecular pathways activated during seizure-induced neuronal death.     

Reverberation of excitation in "hippocampus-entorhinal cortex" slices in rats. Optical recording

Using RH155 voltage-sensitive dye and photodiode array for optical recording, responses to electrical stimuli were investigated in rat brain slices, which included hippocampus and entorhinal cortex. It was shown that single electrical stimulation of the entorhinal cortex, subiculum, or dentate gyrus evoked a potential consecutively spreading from the dentate gyrus to the CA3 and then CA1 hippocampal areas. When the GABAergic inhibition was partially blocked by picrotoxin, the first excitation wave was followed by additional several waves. Such secondary waves were observed in all the hippocampal areas with a constant trial-to-trial latency shift increasing in the direction from the dentate gyrus to CA3 and CA1 areas. Reverberation of activity between the hippocampus and entorhinal cortex is regarded as the most probable cause of appearance of the secondary excitation waves.     

Electroconvulsive Shock Reduces Inositol 1,4,5-Trisphosphate 3-Kinase mRNA Expression in Rat Dentate Gyrus

Abstract: We investigated the expression of inositol 1,4,5-trisphosphate (InsP₃) 3-kinase mRNA after a single electroconvulsive shock (ECS) with in situ hybridization histochemistry in rat brain. At 6 h after ECS, the expression was markedly decreased in the dentate gyrus, and the decrease was maintained until 9 h with a slight recovery. The InsP₃ 3-kinase mRNA content returned to basal levels after 12 h. We could not detect any apparent changes in the expression of InsP₃ 3-kinase mRNA in the CA1–CA3 areas of hippocampus, the striatum, and the cerebral cortex at any time point examined. In the temporal pattern, the reduction of the expression in the dentate gyrus was preceded by the induction of c- fos after ECS. These observations suggest that the InsP₃ 3-kinase might be one of the genes whose expression can be altered by ECS.     

Morphological changes in the hippocampus following nicotine and kainic acid administration

Using histochemical analysis (NADPH-diaphorase, Fluoro-Jade B dye and bis-benzimide 33,342 Hoechst) we studied the influence of intraperitoneal administration of nicotine (NIC), kainic acid (KA) and combination of both these substances on hippocampal neurons and their changes. In experiments, 35-day-old male rats of the Wistar strain were used. Animals were pretreated with 1 mg/kg of nicotine 30 min prior to the kainic acid application (10 mg/kg). After two days, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blades of the dentate gyrus and in the hilus of the dentate gyrus. Fluoro-Jade B positive cells were examined in the same areas in order to elucidate a possible neurodegeneration. In animals exposed only to nicotine the number of NADPH-diaphorase positive neurons in the CA3 area of the hippocampus and in the hilus of the dentate gyrus was higher than in controls. In contrast, KA administration lowered the number of NADPH-diaphorase positive cells in all studied hippocampal areas and in both blades of the dentate gyrus. Massive cell degeneration was observed in CA1 and CA3 areas of the hippocampus and in the hilus of the dentate gyrus after kainic acid administration. Animals exposed to kainic acid and pretreated with nicotine exhibited degeneration to a lesser extent and the number of NADPH-diaphorase positive cells was higher compared to rats, which were exposed to kainic acid only.     

Immunoreactivities and Levels of Mineralocorticoid and Glucocorticoid Receptors in the Hippocampal CA1 Region and Dentate Gyrus of Adult and Aged Dogs

Corticosteroids are important factors in the maintenance of homeostasis in the brain. They are regulated via the interaction with two corticosteroid receptor systems—the mineralocorticoid (MR) and glucocorticoid receptor (GR). In the present study, we observed age-related changes in serum cortisol levels, and immunoreactivities and protein levels of MR and GR in the hippocampal CA1 region and dentate gyrus. The serum cortisol levels were significantly high (about twofold) in the aged group compared to that in the adult group. In the adult dog (2–3 years old), MR and GR immunoreactivity was detected in neurons in the pyramidal layer of the CA1 region, and in the granular and multiform layers of the dentate gyrus. In the aged dog (10–12 years old), MR immunoreactivity in the CA1 region was significantly decreased, especially, in the dentate multiform layer. In contrast, GR immunoreactivity in the aged dog was slightly decreased in the CA1 region and dentate gyrus. In the Western blot analysis, MR protein level in the aged dog was significantly lower compared to that of the adult dog; GR protein level in the aged dog was not significantly decreased. This result indicates that the reduction of MR immunoreactivity and protein level in the hippocampus of the aged dog may be associated with neural dysfunction in the aged hippocampus.