Changes in expression of BDNF and its receptors TrkB and p75NTR in the hippocampus of a dog model of chronic alcoholism and abstinence

Chronic ethanol consumption can produce learning and memory deficits. Brain-derived neurotrophic factor (BDNF) and its receptors affect the pathogenesis of alcoholism. In this study, we examined the expression of BDNF, tropomyosin receptor kinase B (TrkB) and p75 neurotrophin receptor (p75NTR) in the hippocampus of a dog model of chronic alcoholism and abstinence. Twenty domestic dogs (9-10 months old, 15-20 kg; 10 males and 10 females) were obtained from Harbin Medical University. A stable alcoholism model was established through ad libitum feeding, and anti-alcohol drug treatment (Zhong Yao Jie Jiu Ling, the main ingredient was the stems of watermelon; developed in our laboratory), at low- and high-doses, was carried out. The Zhong Yao Jie Jiu Ling was effective for the alcoholism in dogs. The morphology of hippocampal neurons was evaluated using hematoxylin-eosin staining. The number and morphological features of BDNF, TrkB and p75NTR-positive neurons in the dentate gyrus (DG), and the CA1, CA3 and CA4 regions of the hippocampus were observed using immunohistochemistry. One-way ANOVA was used to determine differences in BDNF, TrkB and p75NTR expression. BDNF, TrkB and p75NTR-positive cells were mainly localized in the granular cell layer of the DG and in the pyramidal cell layer of the CA1, CA3 and CA4 regions (DG>CA1>CA3>CA4). Expression levels of both BDNF and TrkB were decreased in chronic alcoholism, and increased after abstinence. The CA4 region appeared to show the greatest differences. Changes in p75NTR expression were the opposite of those of BDNF and TrkB, with the greatest differences observed in the DG and CA4 regions.     

Heme oxgenase-1 is expressed in viable astrocytes and microglia but in degenerating pyramidal neurons in the kainate-lesioned rat hippocampus

The present study aimed to elucidate the distribution of heme oxygenase-1 (HO-1) in the hippocampus after intracerebroventricular injections of kainate. Very little or no staining of HO-1 was observed in the normal CA1, whilst moderate staining of dentate hilar neurons was observed in the dentate gyrus, in the normal hippocampus. At postinjection day 1, a slight increase in immunoreactivity in the neuropil of the lesioned CA fields and a marked increase in HO-1 immunoreactivity in glial cells of the stratum lacunosum moleculare of CA fields and the stratum moleculare of the dentate gyrus was observed. Electron microscopy showed that the glial cells had features of viable astrocytes. At postinjection day 3, glial cells in the dentate gyrus continued to express HO-1, whilst pyramidal neurons in the degenerating CA fields started to express intense HO-1 immunoreactivity in their cell bodies. At postinjection weeks 1–3, HO-1 was observed in glial cells in the center of the lesion, but also in neurons at the perifocal region of the glial scar. The glial cells were found to have features of viable astrocytes and microglia, whilst the neurons contained discontinuous cell membranes and nuclear outlines, and had features of degenerating neurons. Intense immunoreactivity was observed in the cytoplasm of the degenerating neurons. The density of staining was greater than that observed in astrocytes or microglia. Recent in vitro results on fibroblasts transfected with HO-1 cDNA showed that, despite cytoprotection with low (less than fivefold compared with untransfected cells) HO-1 activity, high levels of HO-1 expression (more than 15-fold) were associated with significant oxygen toxicity. These and the present observations suggest a destructive effect of increased expression of HO-1 in neurons, and possible novel therapeutic approaches involving overexpression of HO-1 must therefore be approached with caution. Electronic Publication     

C57black/6小鼠全脑缺血模型的海马区Bcl-2和Bax的表达

使用 C5 7black/6小鼠制造的全脑缺血模型 ,观察全脑缺血后海马 Bcl-2和 Bax的表达 ,为这种新的模型在脑缺血研究中的应用提供实验依据。双侧颈总动脉夹闭 15 min诱发全脑缺血模型 ,2 4h后取脑组织进行 Bcl-2和 Bax免疫组织化学染色。缺血组海马 Bax阳性反应神经元数目增多 ,主要分布在 CA1 区 ,胞浆深染 ,假手术组 Bax阳性细胞数目少 ,染色浅。缺血组 CA3区和齿状回 Bcl-2阳性反应神经元数目增多 ,胞浆染色深 ,假手术组 Bcl-2阳性细胞数目少 ,染色浅。C5 7black/6小鼠全脑缺血模型的海马内 Bcl-2和 Bax表达发生改变 ,Bax表达增加在 CA1 区 ,Bcl-2表达增加在 CA3区和齿状回 ,提示二者在该模型的脑缺血后神经细胞死亡过程中发挥作用     

Cellular hybridization for BDNF. trkB, and NGF mRNAs and BDNF-immunoreactivity in rat forebrain after pilocarpine-induced status epilepticus

The messenger RNAs (mRNAs) for the neurotrophins, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), are upregulated during epileptic seizure activity, as visualized by in situ hybridization techniques. Neurotrophins might be protective against excitotoxic cell stress, and the upregulation during seizures might provide such cell protection. In this study, a high dose of pilocarpine (300 mg/kg) was used to induce long-lasting, limbic motor status epilepticus and a selective pattern of brain damage. The regulation of BDNF, trkB, and NGF mRNA was studied by in situ hybridization at 1, 3, 6, and 24 h after induction of limbic motor status epilepticus. BDNF immunoreactivity was examined with an anti-peptide antibody and the neuropathological process studied in parallel. BDNF mRNA increased in hippocampus, neocortex, piriform cortex, striatum, and thalamus with a maximum at 3–6 h. Hybridization levels increased earlier in the resistant granule and CA1 cells as compared to the vulnerable CA3 neurons. BDNF immunoreactivity was elevated in dentate gyrus at 3–6 h. trKB mRNA increased in the entire hippocampus. NGF mRNA in hippocampus appeared in dentate gyrus at 3–6 h and declined in hilar neurons at 6–24 h. Cell damage was found in the CA3 area, entire basal cortex, and layers II/III of neocortex. Endogenous neurotrophins are upregulated during status epilepticus caused by pilocarpine, which is related to the coupling between neuronal excitation and trophic factor expression. This upregulation of neurotrophic factors may serve endogenous protective effects; however, the excessive levels of neuronal hyperexcitation resulting from pilocarpine seizures lead to cell damage which cannot be prevented by endogenous neurotrophins.     

Modulation of BDNF and TrkB expression in rat hippocampus in response to acute neurotoxicity by diethyldithiocarbamate

In this study, we examined the expression profile of brain-derived neurotrophic factor (BDNF) and its receptor TrkB in adult rat hippocampus following acute administration of diethyldithiocarbamate (DDTC), a neurotoxic compound which was previously shown to induce microglia activation and cell death. Semiquantitative RT-PCR analysis detected significant variations of BDNF mRNA levels in whole hippocampus homogenates, with a peak at 24h after DDTC injection. Increased BDNF protein expression was demonstrated by immunohistochemistry in various hippocampal subfields. The most relevant increase was observed in the hilus of the dentate gyrus where BDNF levels at 120h were found to be almost four times those of basal levels. Full-length TrkB (TrkB.FL) encoding mRNA was also shown to undergo an earlier increase in the hippocampus of DDTC-treated rats. TrkB immunostaining with an antibody binding both full-length and truncated (TrkB.T) isoforms was found to increase at 120h in the hippocampal CA2 and CA3 regions. These results demonstrate that DDTC modulates the expression of BDNF and its receptor in the adult rat hippocampus and suggest a possible involvement of this neurotrophin in the protective response to DDTC-induced neuronal damage.     

Development of full-length Trk B-immunoreactive structures in the hippocampal formation of the macaque monkey

Distribution and morphological changes of cells containing the signal transducing neurotrophin receptor, full-length Trk B (fl-Trk B), were investigated in the hippocampal formation of the macaque monkey between embryonic day 140 and the adult stage. Western blot analysis showed that one main protein band, which migrated at 141 kDa, was detected in both the embryonic and adult hippocampal formation. In the pyramidal cells in CA1 and CA3 subfields, the subiculum, and the entorhinal cortex, fl-Trk B-immunoreactive dendrites were observable in the embryonic stage. In contrast, in the granule cells of the dentate gyrus, few dendrites were immunoreactive during embryonic and early developmental stages. This difference may be due to the later growth of the granule cells of the dentate gyrus. The existence of fl-Trk B immunoreactivity in the cell body and dendrites in the embryonic hippocampal neurons, suggests that BDNF and/or NT4/5 act on the hippocampal cells by autocrine/paracrine mechanisms. In the entorhinal cortex, fl-Trk B immunoreactivity became localized in the stellate cells in layer II and the pyramidal cells in layers III, V and VI in adulthood. This indicates that BDNF and/or NT4/5 are important for the maintenance of the projection neurons in the entorhinal cortex at the adult stage. The strongest fl-Trk B immunoreactivity in the hippocampal neurons occurred at postnatal month 4, corresponding to the period of greatest synapse production in the monkey hippocampus, suggesting that BDNF and/or NT4/5 with fl-Trk B may play a role in synapse formation in the monkey hippocampus.     

Long-term changes in calbindin D(28K) immunoreactivity in the rat hippocampus after cardiac arrest

Calbindin D(28K) (CB) expression was analyzed in the rat hippocampus following 10-min-cardiac arrest-induced ischemia within a year after reperfusion. In rats examined 3 days after ischemia, CB immunoreactivity disappeared completely from CA1 pyramidal neurons and from most CA2 pyramids. In the stratum granulosum of the dentate gyrus, mossy fibers, and hippocampal interneurons, CB immunoreactivity was preserved, although staining was somewhat paler than that in control rats. A similar pattern of CB immunoreactivity was found in rats sacrificed 14 days and 1 month after cardiac arrest. From the 14th postischemic day, neuronal loss in the stratum pyramidale of CA1 but not in that of CA2 became apparent. The reappearance of CB immunoreactivity in CA1 and CA2 pyramidal neurons was noticed 6 months after ischemia, and the pattern was identical to that observed in animals sacrificed 12 months after the ictus. The prolonged loss and delayed reappearance of CB immunoreactivity in the hippocampus demonstrate that ischemia may induce long-term disturbances of protein expression, which may in turn result in impairment of hippocampal functioning.     

脑缺血后大鼠海马及纹状体一氧化氮合酶阳性神经元的变化

夹闭大鼠双侧颈总动脉２ｈ后，用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶反应观察了大鼠海马及纹状体一氧化氮合酶阳性神经元的变化及神经损伤。结果显示：在缺血损伤严重的ＣＡ１区及齿状回一氧化氮合酶阳性神经元较正常动物明显增多并深染，而在同样严重受损的纹状体一氧化氮合酶阳性神经元较正常者明显减少。结合文献及本结果提示：一氧化氮在脑缺血所致的神经损伤中起着重要作用，而海马、纹状体的一氧化氮合酶阳性神经元本身可能具有不同的抗伤害机制．     

Altered regulation of brain-derived neurotrophic factor protein in hippocampus following slice preparation

Brain-derived neurotrophic factor (BDNF) and its cognate receptor tyrosine kinase B (TrkB) play important roles in regulating survival, structure, and function of CNS neurons. One method of studying the functions of these molecules has utilized in vitro hippocampal slice preparations. An important caveat to using slices, however, is that slice preparation itself might alter the expression of BDNF, thereby confounding experimental results. To address this concern, BDNF immunoreactivity was examined in rodent slices using two different methods of slice preparation. Rapid and anatomically selective regulation of BDNF content followed slice preparation using both methodologies; however, different patterns of altered BDNF immunoreactivity were observed. First, in cultured slices, BDNF content decreased in the dentate molecular layer and increased in the CA3 pyramidal cell layer and the mossy fiber pathway of the hippocampus after 30 min. Furthermore, an initially "punctate" pattern of BDNF labeling observed in the mossy fiber pathway of control sections changed to homogenous labeling of the pathway in vitro. In contrast to these findings, slices prepared as for acute slice physiology exhibited no change in BDNF content in the molecular layer and mossy fiber pathway 30 min after slicing, but exhibited significant increases in the dentate granule and CA3 pyramidal cell layers. These findings demonstrate that BDNF protein content is altered following slice preparation, that different methods of slice preparation produce different patterns of BDNF regulation, and raise the possibility that BDNF release and TrkB activation may also be regulated. These consequences of hippocampal slice preparation may confound analyses of exogenous or endogenous BDNF on hippocampal neuronal structure or function.     

Immunohistochemical study of glial reaction and serum-protein extravasation in relation to neuronal damage in rat hippocampus after ischemia.

Transient forebrain ischemia of 30 min duration was produced in anaesthetized rats by four-vessel occlusion. After survival periods of 3 h to three days brains were perfusion-fixed and sections through the mid-dorsal hippocampus were processed for conventional staining and immunohistochemical analysis. Neuronal damage in the hilus was manifested 3-8 h after ischemia; neurons in the CA1 and CA2 sector suffered delayed neuronal death after 48-72 h whereas the dentate gyrus and the CA3 sector were normal. Vasogenic edema formation was visualized using antibodies against rat serum-proteins, serum albumin and immunoglobulins. By 3 h after ischemia, only faint and diffuse serum-staining was detected. At 8 h survival, weak astrocytic-staining was present. After 24-72 h CA1-CA2 exhibited massive serum extravasation. The molecular layer of the dentate gyrus showed edema formation in the absence of granule cell damage. The glial reaction was studied using antibodies against glial fibrillary acidic protein, vimentin and S-100 protein. Glial fibrillary acidic protein and S-100 protein-staining increased in areas with either edema or neuronal damage. In contrast, changes in vimentin were only detected in areas with neuronal necrosis. The observations demonstrate that following 30 min of ischemia neuronal damage is accompanied by changes in blood-brain barrier function and reactive glial alterations. The dissociation between neuronal necrosis and astroglial hypertrophy and hyperplasia reflects differences in cellular responsiveness which constitute inherent features of postischemic hippocampal injury.     

Effects of chronic forced swim stress on hippocampal brain-derived neutrophic factor (BDNF) and its receptor (TrkB) immunoreactive cells in juvenile and aged rats

A type of stress stimulation and age are claimed to affect the expression of brain-derived neurotrophic factor (BDNF) and its receptor - tyrosine kinase B (TrkB) in the hippocampal regions differentially. This study aimed to explore the influence of chronic (15 min daily for 21 days) forced swim stress (FS) exposure on the BDNF and TrkB containing neurons in the hippocampal CA1, CA3 pyramidal cell layers and dentate gyrus (DG) granule cell layer in juvenile (P28) and aged (P360) rats. An immunofluorescence (-ir) method was used to detect BDNF-ir and TrkB-ir cells. Under chronic FS exposure, in the group of juvenile rats a significant decrease in the density of BDNF immunoreactive neurons was observed in CA1 and DG (P less than <0.001), unlike CA3, where it remained unaltered just as the density of TrkB-ir cells in CA1 and DG, but in CA3 the number of TrkB-ir cells was found to grow (P less than 0.05) in comparison with control groups. After chronic FS exposure of aged (P360) rats, the density of BDNF-ir and TrkB-ir cells did not decline in any of the subregions of the hippocampus. In all subfields of the hippocampus, the denseness of BDNF-positive neurons was significantly higher in P360 stressed group, compared with P28 stressed group, but the density of TrkB-ir fell more markedly in P360 than in P28. In conclusion, chronic FS stress influenced the number of BDNF and TrkB immunoreactive neurons only in juvenile animals. The age of rats tested in the chronic forced swim test was a decisive factor determining changes in the density of BDNF-ir and TrkB-ir in the hippocampal structures.     

神经干细胞与BDNF联合治疗对Alzheimer病鼠海马突触素表达的影响

本研究目的在于探讨神经干细胞(NSCs)与脑源性神经营养因子(BDNF)联合应用对老年性痴呆鼠海马突触素(SYH)表达的影响。切断成年老年性痴呆模型鼠左侧穹窿海马伞,基底前脑行神经干细胞移植(移植组),或同时持续侧脑室内注射BDNF(联合组),4周后用免疫组织化学染色结合图像分析技术对各组大鼠海马突触素进行校正光密度(COD)测定。结果显示:损伤组损伤侧海马CA1区辐射层和分子层SYH的COD分别下降到正常组的54.6%和59.0%(P<0.01);移植组COD恢复到正常组的68.9%和67.9%,与损伤组相比差异显著(P<0.05);联合组COD恢复到正常组的82.9%和81.0%,与移植组相比差异显著(P<0.05)。以上结果提示:神经干细胞移植与BDNF注射联合治疗,对老年性痴呆模型鼠海马CA1辐射层和齿状回分子层突触素的表达有明显促进作用,且联合治疗效果比单纯神经干细胞移植效果好。     

High level of adenosine A1 receptor-like immunoreactivity in the CA2/CA3a region of the adult rat hippocampus.

We describe the immunocytochemical distribution of adenosine A1 receptors in the rat hippocampus. Adenosine A1 receptor-like immunoreactivity was seen on the cell soma and dendrites of pyramidal cells and the cell soma and proximal part of dendrites of granule cells, but not on glial cells. Developmentally, adenosine A1 receptor-like immunoreactivity was diffuse on postnatal day 7 and increased in intensity in individual cells by day 21. In the CA2/CA3a region, the adult pattern of A1 receptor distribution was established by day 28. In the adult rat hippocampus, rostrocaudal inspection revealed that immunoreactivity in CA2/CA3a was greatest. Confocal microscopy revealed differences in the staining patterns for the adenosine A receptor and synaptophysin, a marker of presynaptic terminals. This result suggests that the adenosine A1 receptor might have postsynaptic physiological functions. Double-labeling of adenosine A1 receptors and anterogradely-labeled fibers from the supramammillary nucleus showed that the fibers from the supramammillary nucleus terminate directly on the cell soma of the A1 receptor-immunopositive neurons in CA2/CA3a and the dentate gyrus. These results indicate that the adenosine A 1 receptor in CA2/CA3a and the dentate gyrus are in a position to regulate hippocampal theta activity and that resultant strong synaptic depression in CA2/CA3a could play a role in regulating the intrinsic signal flow between CA3 and CA1.     

Differential distribution of 68 Kd and 200 Kd neurofilament proteins in the gerbil hippocampus and their early distributional changes following transient forebrain ischemia

Summary The distribution of neurofilament (NF) proteins was studied immunohistochemically in the gerbil hippocampus with antibodies against NF68 (68 Kd molecular weight) and NF200 proteins, and changes in the distribution of NF proteins after transient ischemia were observed in order to investigate the temporal correlation between NF and delayed neuronal death. In the normal hippocampus, NF68-like immunoreactivity (NF68-LI) was densely distributed in nerve processes in CA2, CA3 and the hilus of the dentate gyrus but was less intense in CA1. In contrast, processes with NF200-LI appeared to be evenly distributed in CA1, CA2, CA3 and the dentate gyrus. Mongolian gerbils were subjected to transient ischemia for 5 min by bilateral carotid occlusion and subjected to immunohistochemistry 1, 2, 3 and 4 days after ischemia. Following transient ischemia, prior to neuronal cell death, the intensity of both NF68-LI and NF200-LI decreased in the whole hippocampal formation. This decrease was more obvious in the case of NF68-LI than NF200-LI. Four days after ischemia, when neuronal death of CA1 pyramidal cells had occurred, processes in CA1, particularly 68 Kd components, showed marked decreases in number and staining intensity, although processes in most layers of CA2, CA3 and the dentate gyrus appeared to be stained similarly to normal brain. Since NF68 protein is considered to be the major component of NF proteins and NF200 is an associated accessory protein, the current observations suggest that the poor distribution of NF68 in CA1 and the early loss of NF proteins may be closely related to selective vulnerability of CA1 pyramidal cells and delayed neuronal death.     

Alteration in the immunoreactivity of the calcineurin subunits after ischemic hippocampal damage.

Dephosphorylation processes of target proteins are critical to the reversible regulation of intracellular signal transduction systems. Further, brain damage such as ischemic insult induces marked changes in protein kinase activity. To study these changes more thoroughly, specific monoclonal antibodies of the A and B subunits of calcineurin (protein phosphatase 2B) were raised, and regional alterations in the immunoreactivity of calcineurin in the rat hippocampus were investigated after a transient forebrain ischemic insult causing selective and delayed hippocampal CA1 pyramidal cell damage. In normal rats it was found that both the calcineurin A and the B subunits showed high immunoreactivity in the dendritic fields of the hippocampal formation. The immunoreactivity of subunit A in the strata oriens, the radiatum of the CA1 subfield and in the stratum lucidum of the CA3 subfield was most intense, whereas the immunoreactivity in the other CA3 subfields and in the dentate gyrus was relatively low. In contrast, the dendritic fields of the hippocampal formation were equally immunoreactive to calcineurin subunit B, although the stratum lucidum of the CA3, where the mossy fibers from the dentate granule cells terminate, showed a very high immunoreactivity of the B subunit. After transient forebrain ischemia in the CA1 subfield, where selective pyramidal cell death occurred two days after this ischemia, a marked loss of immunoreactivity in both subunits was observed, along with morphological pyramidal cell damage. A recovery of the immunoreactivity of A and B subunits in the strata oriens and radiatum was later noted 30 days after ischemia. In the stratum lucidum of the CA3, the immunoreactivity of both the A and B subunits was transiently depressed from 6 to 24 h, followed by a marked immunoreactivity enhancement from four to 30 days after ischemia. Further, in the histologically intact dentate gyrus, both the immunoreactivity of the A and B subunits in the molecular layer were transiently enhanced from four to 14 days after ischemia, particularly in the supragranular layer. The results clearly indicate that the protein dephosphorylation systems were markedly altered in the whole hippocampal formation during the recirculation period following ischemia. Further, the transient depression in the calcineurin immunoreactivity seen in the mossy fiber terminals may reflect modulated synaptic activity of the dentate granule cells, which may play a pivotal role in the delayed and selective death of the CA1 pyramidal cells. Thus, calcineurin appears to be an excellent marker enzyme for the detection of neuronal activity and synaptic plasticity after brain damage, such as an ischemic insult.     

Seizure-induced changes of mineralocorticoid and glucocorticoid receptors in the hippocampus in seizure sensitive gerbils

Abnormal corticosteroid hormone levels during stress and resultant mineralocorticoid receptor (MR)/glucocorticoid receptor (GR) imbalance enhance the vulnerability of specific hippocampal neurons. In the present study, we investigated the distribution of MR and GR in seizure resistant (SR) and seizure sensitive (SS) gerbils, and observed the seizure-induced changes of MR and GR in the hippocampus of SS gerbils using immunohistochemistry and western blot analysis. MR and GR immunoreactivities were higher in the SS pre-seizure gerbils than that in SR gerbils. In the SR gerbils, the immunodensity of GR was high compared to that of MR. The changes of MR and GR immunoreactivities were significant in the stratum pyramidale of the hippocampal CA1 region and the infrablade of the dentate gyrus after seizure on-set. MR immunoreactivity in the CA1 region was significantly increased at 12h after seizure on-set, thereafter MR immunoreactivity was decreased. MR immunoreactivity in the dentate gyrus was decreased time-dependently after seizure on-set. GR immunoreactivity was decreased in the CA1 region and dentate gyrus time-dependently after seizure on-set. At 12h after seizure on-set, differences in MR and GR immunodensity diminished in the CA1 region and dentate gyrus. This imbalance of MR and GR immunoreactivity in these regions may be associated with seizure generation in the Mongolian gerbil, which is a hereditary seizure model.     

Physiological variability in brain-derived neurotrophic factor expression predicts dendritic spine density in the mouse dentate gyrus

Dendritic spines are the predominant sites of excitatory neurotransmission in the adult brain, and brain-derived neurotrophic factor (BDNF) is a well-characterized determinant of dendritic spine number and morphology. The relationship between BDNF expression and dendritic spine number is particularly evident in the hippocampus, where environmental conditions that enhance hippocampal BDNF levels also promote local increases in dendritic spine density. However, the relationship between physiological variability in hippocampal BDNF expression and spine number has yet to be assessed. To determine whether natural variability in BDNF expression is associated with hippocampal dendritic spine number, correlations between BDNF protein levels and dendritic spine density among Golgi-impregnated neurons in the hippocampal dentate gyrus and CA1 subfields were assessed in adult male C57Bl/6J mice. In the dentate gyrus, but not in the apical oblique dendrites of CA1 pyramidal cells, BDNF protein expression was significantly correlated with dendritic spine density. This observation suggests that there may be a subregionally specific relationship between hippocampal BDNF expression and the density of spines.     

Effects of age and insulin-like growth factor-1 on neuron and synapse numbers in area CA3 of hippocampus.

Age-related effects associated with the hippocampus include declines in numbers of neurons and synapses in the dentate gyrus and area CA1, and decreased cognitive ability as assessed with the Morris water maze. The present study quantified both neuron and synapse number in the same tissue block of area CA3 of the hippocampus. No investigations of both density of neurons and synapses together in area CA3 of hippocampus have been performed previously, despite its importance as the terminal field of dentate gyrus mossy fibers, the second synapse in the trisynaptic circuit in the hippocampus. Numerical density of neurons and synapses were assessed in 4-, 18-, and 29-month-old rats receiving infusions of saline into the lateral ventricle and in 29-month-old rats receiving infusions of insulin-like growth factor-1 (IGF-1). Numerical density of neurons of the stratum pyramidale of CA3 of hippocampus remained constant across the life span as did the numerical density of synapses in stratum lucidum of area CA3. Despite the reported role of IGF-1 in synaptogenesis and improvements in behavior with age, ventricular infusion of this growth factor did not affect the numerical density of neurons or synapses in 29-month-old rats when compared to saline-infused old rats. Further, reported effects of IGF-1 on adult neurogenesis in the dentate gyrus are not reflected in an IGF-1-related increase in synapse density in this region.     

Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.     

Effects of global cerebral ischemia and preconditioning on heat shock protein 27 immunocontent and phosphorylation in rat hippocampus.

Global cerebral ischemia, with or without preconditioning, leads to an increase in heat shock protein 27 (HSP27) immunocontent and alterations in HSP27 phosphorylation in CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (1, 4, 7, 14, 21 and 30 days) using 2 min, 10 min or 2+10 min of ischemia. The results showed an increase in HSP27 immunocontent of about 300% after 10 min of ischemia in CA1 and dentate gyrus. CA1, a hippocampal vulnerable area, showed an increase in HSP27 phosphorylation, parallel with immunocontent. In dentate gyrus, a resistant area, the increase in HSP phosphorylation was lower than immunocontent. After preconditioned ischemia (2+10 min), when CA1 neurons are protected to a lethal, 10 min insult, we observed an increase in HSP immunocontent and a decrease in phosphorylation in both regions of the hippocampus, suggesting that, when there is no neuronal death, HSP27 in a vulnerable area responds similarly to the resistant area.When dephosphorylated, HSP27 acts as a chaperone, protecting other proteins from denaturation. As it is markedly expressed in astrocytes, we suggest that HSP27 could be protecting hippocampal astrocytes, which could then be helping neurons to resist to the insult, maintaining tissue normal homeostasis.