The enhanced expression of c-Jun immunoreactivity in the adrenalectomized gerbil hippocampus

Recent in vitro and in vivo studies have shown that glucocorticoids have a profound influence on the survival of hippocampal neurones, and that the depletion of glucocorticoids as a result of adrenalectomy (ADX) reduces nerve growth factor levels in the hippocampus. It is also believed that ADX is associated with the seizure susceptibility of the Mongolian gerbil. In the present study, the choronological changes of c-jun immunoreactivity were investigated after ADX in the hippocampal formations in the seizure-prone gerbil model. In the sham hippocampus, c-jun immunoreactivity was not observed in the neurones of the hippocampus proper and dentate gyrus. C-jun immunoreactive neurones appeared 3 h after ADX in the neurones of the CA1 area and dentate gyrus, and these immunoreactivities peaked 24 h after ADX and then gradually decreased. These results suggest that, in the adrenalectomized gerbil, c-jun may be expressed in the neurones of the hippocampus in compensation for glucocorticoid deficit. The result of enhanced c-jun expression of the hippocampal formation provides anatomical support for the hypothesis that c-jun may play a role in the reduction of seizure activity.     

Time-course changes of hippocalcin expression in the mouse hippocampus following pilocarpine-induced status epilepticus

Hippocalcin participates in the maintenance of neuronal calcium homeostasis. In the present study, we examined the time-course changes of neuronal degeneration and hippocalcin protein level in the mouse hippocampus following pilocarpine-induced status epilepticus (SE). Marked neuronal degeneration was observed in the hippocampus after SE in a time-dependent manner, although neuronal degeneration differed according to the hippocampal subregions. Almost no hippocalcin immunoreactivity was detected in the pyramidal neurons of the cornu ammonis 1 (CA1) region from 6 h after SE. However, many pyramidal neurons in the CA2 region showed hippocalcin immunoreactivity until 24 h after SE. In the CA3 region, only a few hippocalcin immunoreactive cells were observed at 12 h after SE, and almost no hippocalcin immunoreactivity was observed in the pyramidal neurons from 24 h after SE. Hippocalcin immunoreactivity in the polymorphic cells of the dentate gyrus was markedly decreased from 6 h after SE. In addition, hippocalcin protein level in the hippocampus began to decrease from 6 h after SE, and was significantly decreased at 24 h and 48 h after pilocarpine-induced SE. These results indicate that marked reduction of hippocalcin level may be closely related to neuronal degeneration in the hippocampus following pilocarpine-induced SE.     

Changes in glial fibrillary acidic protein immunoreactivity in the dentate gyrus and hippocampus proper of adult and aged dogs

Astrocytes perform neuron-supportive tasks, repair and scarring process in the central nervous system. In this study, we observed glial fibrillary acidic protein (GFAP), a marker for astrocytes, immunoreactivity in the dentate gyrus and hippocampus proper (CA1-3 region) of adult (2-3 years of age) and aged (10-12 years of age) dogs. In the adult group, GFAP immunoreactive astrocytes were distributed in all layers of the dentate gyrus and CA1-3 region, except in the stratum pyramidale of the CA1-3 region. In the aged group, GFAP immunoreactivity decreased markedly in the molecular layer of the dentate gyrus. However, GFAP immunoreactivity in the CA1-3 region increased in all layers, and the cytoplasm of GFAP immunoreactive astrocytes was hypertrophied. GFAP protein levels in the aged dentate gyrus decreased; however, GFAP levels in the CA1-3 region increased. These results suggest that the morphology of astrocytes and GFAP protein levels in the hippocampal dentate gyrus and CA1 region are changed, respectively, with age.     

Time-dependent changes of calbindin D-28K and parvalbumin immunoreactivity in the hippocampus of rats with streptozotocin-induced type 1 diabetes

The hippocampus is affected by various stimuli that include hyperglycemia, depression, and ischemia. Calcium-binding proteins (CaBPs) have protective roles in the response to such stimuli. However, little is known about the expression of CaBPs under diabetic conditions. This study was conducted to examine alterations in the physiological parameters with type 1 diabetes induced with streptozotocin (STZ) as well as time-dependent changes in the expression of two CaBPs changes of were being evaluated. Rats treated with STZ (70 mg/kg) had high blood glucose levels (>21.4 mmol/L) along with increased food intake and water consumption volumes compared to the sham controls. In contrast, body weight of the animals treated with STZ was significantly reduced compared to the sham group. CB-specific immunoreactivity was generally increased in the hippocampal CA1 region and granule cell layer of the dentate gyrus (DG) 2 weeks after STZ treatment, but decreased thereafter in these regions. In contrast, the number of PV-immunoreactive neurons and fibers was unchanged in the hippocampus and DG 2 weeks after STZ treatment. However, this number subsequently decreased over time. These results suggest that CB and PV expression is lowest 3 weeks after STZ administration, and these deficits lead to disturbances in calcium homeostasis.     

Adult neurogenesis in the mammalian dentate gyrus

Earlier observations in neuroscience suggested that no new neurons form in the mature central nervous system. Evidence now indicates that new neurons do form in the adult mammalian brain. Two regions of the mature mammalian brain generate new neurons: (a) the border of the lateral ventricles of the brain (subventricular zone) and (b) the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. This review focuses only on new neuron formation in the dentate gyrus of the hippocampus. During normal prenatal and early postnatal development, neural stem cells (NSCs) give rise to differentiated neurons. NSCs persist in the dentate gyrus SGZ, undergoing cell division, with some daughter cells differentiating into functional neurons that participate in learning and memory and general cognition through integration into pre-existing neural networks. Axons, which emanate from neurons in the entorhinal cortex, synapse with dendrites of the granule cells (small neurons) of the dentate gyrus. Axons from granule cells synapse with pyramidal cells in the hippocampal CA3 region, which send axons to synapse with CA1 hippocampal pyramidal cells that send their axons out of the hippocampus proper. Adult neurogenesis includes proliferation, differentiation, migration, the death of some newly formed cells and final integration of surviving cells into neural networks. We summarise these processes in adult mammalian hippocampal neurogenesis and discuss the roles of major signalling molecules that influence neurogenesis, including neurotransmitters and some hormones. The recent controversy raised concerning whether or not adult neurogenesis occurs in humans also is discussed.     

Effects of dizocilpine pretreatment on parvalbumin immunoreactivity and Fos expression after cerebral ischemia in the hippocampus of the Mongolian gerbil

The mechanisms of ischemic neuronal death have been focused on glutamate receptor activation and subsequent elevation of intracellular Ca2+ concentration. The purpose of this study was to evaluate the effects of dizocilpine, an NMDA receptor antagonist, pretreatment on Fos expression and parvalbumin (PV, calcium binding protein) immunoreactivity in the hippocampus of the mongolian gerbil after global ischemic insults. The number of PV-immunoreactive (PV-ir) neurons in CA1 were significantly decreased from 1 day after cerebral ischemia, while dizocilpine pretreatment completely suppressed the loss of PV-ir neurons in CA1. Dizocilpine pretreatment also protected the structural loss of microtubule-associated protein 2 immunoreactivity in CA1 after ischemic insults. In addition, dizocilpine pretreatment increased Fos expression in both hippocampal CA3 and CA4 after 3 hr ischemic reperfusion as compared to that of the saline pretreated group. Subsequently, the Fos-defined cellular activity of PV-ir neurons was slightly increased by dizocilpine pretreatment in the hippocampal area. This study demonstrated that NMDA receptor mediated calcium influx was associated with the loss of PV-ir neurons in CA1 hippocampal region, and that dizocilpine pretreatment increased Fos expression and the neuronal activity of PV-ir neurons in the non-vulnerable region of hippocampus after cerebral ischemia. Based on this data, we conclude that the protective effect of dizocilpine may be induced by the regulation of calcium overload, or by the upregulation of a neuroregenerative initiator such as Fos protein.     

Age-related changes of calbindin D-28k-immunoreactive neurons in the myenteric plexus of gerbil duodenum

We examined the age-related changes of calbindin D-28k (CB)-immunoreactive neurons and overall populations of neurons in the myenteric plexus of gerbil duodenum using whole mount preparations and immunohistochemistry. The circumference of duodenum increased age-dependently. CB-immunoreactive neurons were observed in all groups, and most of them had the Dogiel type II morphology. The fully developed cobweb-like structures were observed in the myenteric plexus of duodenum at postnatal month (PM) 3 to 24. Although the highest numbers of CB-immunoreactive neurons and overall population were observed in PM 1.5, it is related with significant increase of the size of circumference between PM 1.5 to PM 3. CB-immunoreactive neurons were slightly decreased with age between PM 3 to PM 24. We have also found that whole numbers of myenteric neurons were also significantly decreased in PM 24 group. These results suggest that loss of overall numbers of myenteric neurons and CB-immunoreactive neurons may be related with age-related neurodegeneration and functional loss of duodenum in the gerbil.     

Changes in cyclooxygenase-2 immunoreactivity in the hippocampus in a model of streptozotocin-induced type 1 diabetic rats

In this study, we investigated diabetic stage dependent cyclooxygenase-2 (COX-2) immunoreactivity in the dentate gyrus in streptozotocin (STZ)-induced type 1 diabetic rats. The animals were sacrificed at 2, 3 and 4 weeks after STZ treatment. Blood glucose levels were increased after STZ treatment. COX-2 immunoreactivity in dentate gyrus was significantly increased in these regions 3 weeks after STZ treatment and restored to its basal level to 4 weeks after STZ treatment. In contrast, COX-2 immunoreactivity was not changed in CA3 region in all groups. These results suggest that STZ-induced type 1 diabetes transiently, but not permanently, decreased synaptic transmission and plasticity 3 weeks after STZ treatment in the dentate gyrus.     

Age-related changes in ionized calcium-binding adapter molecule 1 immunoreactivity and protein level in the gerbil hippocampal CA1 region

Microglia are evenly distributed throughout the brain parenchyma. They respond rapidly to a variety of alterations in the microenvironment of the brain and act as sensors for pathological events in the brain. In the present study, we investigated the age-dependent changes in the immunoreactivity and protein level of ionized calcium-binding adapter molecule 1 (Iba-1), a microglial marker, in the CA1 region of the gerbil hippocampus. Iba-1 immunoreactive microglia were detected in the hippocampal CA1 region of the postnatal month 1 (PM 1) group. Iba-1 positive microglia were morphologically inactive between the PM 1 and PM 12 stages. Some Iba-1 immunoreactive microglia were present in the active form in the hippocampal CA1 region of the PM 18 and PM 24 groups. The Iba-1 protein levels in hippocampal CA1 homogenates were decreased in the PM 1 through PM 6 groups and increased in an age-dependent manner thereafter. These results suggest that Iba-1 immunoreactive microglia in the active form were detected in the hippocampal CA1 region in the PM 18 and PM 24 groups. This result may be associated with an age-dependent susceptibility to neurodegenerative diseases associated with the hippocampus.     

去卵巢及雌激素替代治疗后家兔海马nNOS阳性神经元的形态结构及分布变化

用SABC免疫组织化学技术,观察家兔海马各区nNOS阳性神经元在去卵巢及雌激素替代治疗后的形态结构及分布变化,为雌激素类药物防治绝经后老年性痴呆症提供理论依据。结果表明,家兔海马各区都有nNOS阳性神经元分布;去卵巢后海马nNOS阳性神经元的形态结构及分布变化有区域差异性:与假手术对照组相比,在海马CA1区、CA3区、齿状回(DG)阳性神经元数量明显减少(P0.05),而在CA2区数量明显增多(P0.05)。CA1、CA3区和DG的阳性神经元胞体截面积明显变小,最长突起长度明显变短,第一级突起数变少,与假手术组有显著差异(P0.05)。CA2区阳性神经元胞体截面积明显变小(P0.05),最长突起长度、第一级突起数增多,但差异不显著(P0.05);nNOS阳性神经元的4种指标在雌激素替代治疗组与假手术组之间无显著差异(P0.05)。结果提示:雌激素可能通过影响海马nNOS的表达来影响脑的学习和记忆功能。     

Effect of 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, on parvalbumin immunoreactivity after cerebral ischaemia in the hippocampus of the Mongolian gerbil

Previous studies have demonstrated that a loss of parvalbumin-immunoreactive (PV-ir) neurones is observed in the hippocampus after transient cerebral ischaemia. However, whether the loss of parvalbumin (PV) immunoreactivity is related to the over-production of nitric oxide (NO) during cerebral ischaemia has not been evaluated. This study was designed to test the effect of 7-nitroindazole pre-treatment (7-NI, 50 mg/kg), a selective neuronal NO synthase inhibitor, on PV immunoreactivity and its cellular activity following forebrain ischaemia. PV-ir neurones in the hippocampus of the control group were widely distributed in the pyramidal cell layer and stratum oriens of CA1 and CA3, and the granular cell layer of dentate gyrus. 7-NI pre-treatment completely suppressed the reduction of PV immunoreactivity in CA1 that was observed in the ischaemia-induced group. Subsequently, 7-NI pre-treatment also protected against the structural loss of microtubule-associated protein 2 (MAP2) immunoreactivity in CA1 after ischaemic insult. In addition, the Fos-defined neuronal activity of PV-ir neurones was slightly increased by the 7-NI pre-treatment 3 h after ischaemia. Based on these data, we conclude that the neuronal toxicity of NO may be involved in the loss of PV-ir neurones after cerebral ischaemia.     

Ribosomal protein s3 immunoreactivity in the young, adult and aged gerbil hippocampus

Ribosomal protein S3 (rpS3) is well known to participate in DNA repair mechanisms. In the present study, we compared rpS3 immunoreactivity and its protein levels in the hippocampus among young, adult and aged gerbils. In the postnatal month (PM) 3 group as the young, rpS3 immunoreaction was observed in pyramidal and non-pyramidal cells of the hippocampus proper and in granule and polymorphic cells of the dentate gyrus. In the PM 12 group as adult and 24 group as the aged, rpS immunoreactivity in the hippocampus was decreased compared to the PM 3 group. Western blot analysis showed that rpS3 levels were decreased with time; lowest at PM 24. These results indicate that rpS3 immunoreactivity and protein levels were markedly decreased in the aged gerbil hippocampus.     

白肉灵芝水提物对脑缺血大鼠海马神经元的保护作用

试验旨在探讨白肉灵芝水提物（Ganoderma leucocontextum aqueous extracts,GLAE）对脑缺血后海马神经元的保护作用及机制。将50只健康大鼠分为对照组、模型组、GLAE低（0.05 mg/（g·BW））、中（0.1 mg/（g·BW））、高（0.2 mg/（g·BW））剂量组。利用双侧颈总动脉夹闭法建立大鼠脑缺血模型,GLAE组灌胃不同剂量的GLAE干预,对照组和模型组灌胃同体积的生理盐水,连续2周。用跳台试验方法检测记忆获得、记忆巩固和记忆再现障碍大鼠的学习记忆能力,HE染色观察大鼠海马组织的病理形态的变化,比色法检测海马组织一氧化氮合酶（nitric oxide synthase,NOS）活性和一氧化氮（nitric oxide,NO）含量,Western blotting和实时荧光定量PCR法分别检测海马组织生长相关蛋白-43（growth associated protein-43,GAP-43）和脑源性神经生长因子（brain derived neurotrophic factor,BDNF）的水平。结果显示,与对照组相比,模型组大鼠跳台试验的逃避潜伏期显著缩短、电击次数显著增加（P<0.05）;海马神经元细胞出现明显核固缩、排列松散紊乱等退行性改变,细胞数量显著减少（P<0.05）;海马组织NOS活性和NO含量均显著降低（P<0.05）;大鼠海马组织GAP-43蛋白表达量显著升高（P<0.05）;海马组织BDNF mRNA表达量显著下调（P<0.05）。与模型组相比,GLAE干预后,大鼠逃避潜伏期均显著延长、电击次数均显著减少（P<0.05）;GLAE高剂量组大鼠CA1区和齿状回锥体神经元细胞形态明显改善,神经元数量显著增加（P<0.05）;GLAE低剂量组对NOS活性影响不明显（P>0.05）,显著增加NO含量（P<0.05）,GLAE中、高剂量组NOS活性和NO含量均显著升高（P<0.05）;GLAE低、中、高剂量组海马组织GAP-43蛋白表达量均显著增加（P<0.05）;GLAE低、中、高剂量组海马组织BDNF mRNA表达量均显著增加（P<0.05）。以上结果表明,GLAE可通过提高NOS活性和NO水平、促进海马神经发生和功能恢复对脑缺血后海马神经元损伤有一定的保护作用,从而改善大鼠认知功能,0.2 mg/g GLAE效果最好。     

制动应激对孕猪海马结构神经元可塑性的影响

旨在研究限位栏饲养造成的制动应激对妊娠母猪神经元可塑性的影响，本研究选取6头8~9月龄配种成功的巴马小型母猪，随机分成两组，应激组（n=3）限位栏饲养，对照组（n=3）自由环境饲养，其余饲养管理一致。于妊娠第18天处死孕猪，取其海马和血液，使用ELISA、放射免疫法检测相关激素水平，制作海马石蜡切片进行尼氏染色和镀银染色后观察神经元丢失率、树突复杂度以及树突棘数量等，并采用Western blot检测海马脑源性神经营养因子（BDNF）表达水平。结果显示，应激组血浆中促肾上腺皮质激素释放激素（CRH）、促肾上腺皮质激素（ACTH）、皮质醇（COR）水平均显著上升（P≤0.05），齿状回（DG）和CA3区出现神经元丢失现象，CA3区锥形细胞树突复杂度下降，神经元成熟树突棘和未成熟树突棘数量均显著降低，DG的成熟树突棘数量显著降低，且海马内的BDNF蛋白表达量较对照组降低70.6%（P≤0.01）。因此，制动应激减弱了孕猪大脑海马中CA3区和DG的神经元可塑性。     

Involvement of granulin in estrogen-induced neurogenesis in the adult rat hippocampus

Recent studies have demonstrated the presence of neurogenesis in the adult mammalian hippocampus, and it has been suggested that estrogen and various growth factors influence the processes of adult neurogenesis. The present study assessed cell proliferation in the dentate gyrus and the mRNA expression levels of granulin, insulin-like growth factor-I (IGF-I), and brain-derived neurotrophic factor (BDNF) in the hippocampus 4 h after treatment with estradiol benzoate (EB) in 3- and 12-month old ovariectomized rats. At 3 months of age, mRNA expression of granulin precursor and cell proliferation were increased by EB treatment, although the mRNA expressions of IGF-I and BDNF remained unchanged. At 12 months of age, however, neither mRNA expression of the three genes nor cell proliferation in the dentate gyrus were affected by EB treatment. In addition, 17beta-estradiol enhanced the proliferation of neural progenitor cells derived from hippocampal tissue of 3-month-old female rats in vitro; this was inhibited by neutralization of granulin with specific antibody. These results suggest that estrogen induces granulin gene expression in the hippocampus and that the product of this gene is involved in the mitogenic effects of estrogen in the dentate gyrus, although the responses to estrogen decline with age.     

Pathology of domoic acid toxicity in California sea lions (Zalophus californianus)

Over 100 free-ranging adult California sea lions (Zalophus californianus) and one Northern fur seal (Callorhinus ursinus), predominantly adult females, were intoxicated by domoic acid (DA) during three harmful algal blooms between 1998 and 2000 in central and northern California coastal waters. The vector prey item was Northern anchovy (Engraulis mordax) and the primary DA-producing algal diatom was Psuedonitzschia australis. Postmortem examination revealed gross and histologic findings that were distinctive and aided in diagnosis. A total of 109 sea lions were examined, dying between 1 day and 10 months after admission to a marine mammal rehabilitation center. Persistent seizures with obtundation were the main clinical findings. Frequent gross findings in animals dying acutely consisted of piriform lobe malacia, myocardial pallor, bronchopneumonia, and complications related to pregnancy. Gross findings in animals dying months after intoxication included bilateral hippocampal atrophy. Histologic observations implicated limbic system seizure injury consistent with excitotoxin exposure. Peracutely, there was microvesicular hydropic degeneration within the neuropil of the hippocampus, amygdala, pyriform lobe, and other limbic structures. Acutely, there was ischemic neuronal necrosis, particularly apparent in the granular cells of the dentate gyrus and the pyramidal cells within the hippocampus cornu ammonis (CA) sectors CA4, CA3, and CA1. Dentate granular cell necrosis has not been reported in human or experimental animal DA toxicity and may be unique to sea lions. Chronically, there was gliosis, mild nonsuppurative inflammation, and loss of laminar organization in affected areas.     

Absence of temporal lobe epilepsy pathology in dogs with medically intractable epilepsy

Epilepsy is a common neurological problem in dogs. In some dogs, seizures cannot be controlled adequately with anticonvulsant medication. Temporal lobe epilepsy is the most common type of epilepsy in adult humans, it is frequently resistant to anticonvulsant therapy, and it is commonly associated with characteristic neuropathological abnormalities in the hippocampal dentate gyrus. We sought to test the hypothesis that dogs with medically intractable epilepsy have temporal lobe epilepsy. The hippocampi of 6 dogs that were euthanized because of chronic, recurrent seizures were compared with those of 8 nonepileptic controls. In control and epileptic dogs, stereological cell counting showed similar numbers of neurons in the hilus of the dentate gyrus, somatostatin immunoreactivity identified numerous immunopositive neurons in the hilus, and Timm staining showed the normal pattern of granule cell axon projections. These findings demonstrate a lack of hilar neuron loss and granule cell axon reorganization, suggesting that temporal lobe epilepsy is not a common cause of medically intractable epilepsy in dogs.     

Age-dependent changes in progranulin expression in the mouse brain

The progranulin (PGRN) gene is involved in sexual differentiation of the brain during the perinatal period and estrogen-induced adult neurogenesis in the hippocampus. Mutations in the PGRN gene are also implicated in human frontotemporal lobar degeneration. Thus, while PGRN appears to play important roles as a growth factor in the brain, the localization of PGRN-expressing cells throughout the brain has not been fully established. In the present study, we examined the localization of PGRN proteins in the brain using adult male wild-type mice and PGRN-deficient mice we had generated previously. We also evaluated age-dependent changes in PGRN expression at the mRNA and protein levels. As expected, no immunoreactivity was observed in the brains of the PGRN-deficient mice. In the wild-type mice, intense immunoreactivity was observed in several brain regions including the cingulate and piriform cortices, the pyramidal cell layer and dentate gyrus of the hippocampus, the amygdala, the ventromedial and arcuate nuclei of the hypothalamus and the Purkinje cell layer in the cerebellum. Moreover, PGRN mRNA and protein expression decreased in the cortex, hippocampus and hypothalamus in an age-dependent manner. Since many of these brain regions are involved in emotion, memory and recognition, PGRN may play roles as a growth factor in these brain functions that decline with age.     

Transient ischaemia affects plasma membrane glutamate transporter, not vesicular glutamate transporter, expressions in the gerbil hippocampus

In the present study, we investigated expressions of vesicular glutamate transporter (VGLUT) and of the plasma membrane glutamate transporters [glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST) and excitatory amino acid carrier 1 (EAAC-1)] in the gerbil hippocampus following transient ischaemia. The expressional levels and distribution patterns of VGLUT immunoreactivities were unaltered until 3 days after ischaemic-insults. However, VGLUT-2 immunoreactivity in the CA1 region was reduced at 4 days after ischaemia due to delayed neuronal death. In addition, both GLT-1 and GLAST immunoreactivities in the CA1 region were enhanced at 30 min - 12 h after ischaemia-reperfusion and their expression began to reduce at 24 h after ischaemia-reperfusion. In contrast, EAAC-1 immunoreactivity was transiently reduced in the CA1 region at 30 min after ischaemia, re-enhanced at 3-12 h after ischaemia, and re-reduced at 24 h after ischaemia. These findings suggest that malfunctions of plasma membrane glutamate transporters, not of VGLUT, may play an important role in the elevation of extracellular glutamate concentration following ischaemic insults.     

Pituitary adenylate cyclase-activating polypeptide-immunoreactive cells in the ageing gerbil hippocampus

In the present study, we investigated age-related changes in pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactivity and its protein levels in the gerbil hippocampus at various ages using immunohistochemistry and western blot analysis. In the post-natal month 1 (PM 1) group, PACAP-immunoreactive cells were found in all hippocampal subregions. The number of PACAP-immunoreactive cells was decreased in the PM 3 group and was still more decreased in the PM 6 and 12 groups. Thereafter, in the PM 18 and 24 groups, PACAP-immunoreactive cells were significantly increased again. However, in the mossy fibre zone, PACAP immunostaining was very strong in the adult group, especially in the PM 6 group. In addition, PACAP protein level was highest at PM 6, showing a slight decrease at PM 24. These results indicate that PACAP-immunoreactive cells are lowest in the adult stage and highest in the aged stage. However, PACAP immunoreactivity in the mossy fibre zone and PACAP protein level in the hippocampus are highest in the adult stage.