Dependence of rat hippocampal c-Fos expression on intensity and duration of exercise

The expression of c-Fos, an immediately early gene, is a marker of neural activity. In the present study, the effect of treadmill exercise on c-Fos expression was investigated in various regions of the rat hippocampus via immunohistochemistry. The first part of the experiment was aimed at determining the dependence of c-Fos expression on the intensity of treadmill exercise. In most of the hippocampal regions studied, increasing c-Fos expression was observed with increasing exercise intensity. In the second part of the experiment, the dependence of c-Fos expression on the duration of treadmill exercise was investigated. The c-Fos expression induced by mild-intensity exercise increased until the 7th day of exercise and subsequently decreased. Results of the present study suggest that the effect of treadmill exercise on neuronal activity in the hippocampus is intensity-and duration-dependent.     

Hippocampal functional hyperemia mediated by NMDA receptor/NO signaling in rats during mild exercise

Current studies have demonstrated that exercise increases regional cerebral blood flow (rCBF), an index of neuronal activity. However, neuronal regulation of the increased rCBF in the brain parenchyma is poorly understood. We developed a running model with rats for monitoring hippocampal cerebral blood flow (Hip-CBF) and found that mild treadmill running increases Hip-CBF in a tetrodotoxin-dependent manner, suggesting that functional hyperemia, an increase in rCBF in response to neuronal activation, occurs in the running rat's hippocampus (Nishijima T and Soya H. Neurosci Res 54: 186-191, 2006). To further support our hypothesis, it was important to discover the neurogenic pathways behind the increase in Hip-CBF that occurred during running. Here, we examine the possible role of N-methyl-d-aspartate (NMDA) receptor/nitric oxide (NO) signaling and group I metabotropic glutamate receptors in mediating the Hip-CBF increase. Hip-CBF during running was measured by laser-Doppler flowmetry. Intrahippocampal drug administration was performed by microdialysis. Mild treadmill running (10 m/min) increased Hip-CBF, which was remarkably attenuated by either NMDA receptor antagonists (1 mM MK-801) or NO synthase inhibitors (2 mM N(G)-nitro-l-arginine methyl ester). However, group I metabotropic glutamate receptor antagonists {1 mM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester + 1 mM 2-methyl-6-(phenylethynyl)pyridine hydrochloride} augmented the running-induced Hip-CBF increase. We also found that rCBF in the olfactory bulb was unchanged with running. These results strongly suggest that Hip-CBF during mild exercise is regulated locally under hippocampal neuronal activity, mediated mainly through NMDA receptor/NO signaling. Collectively, these results, together with our previous findings, support our hypothesis that mild exercise elicits neuronal activation, which then triggers functional hyperemia in the rat hippocampus.     

Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus.

The expression of the neurotrophins and trk receptors in the hippocampus has directed attention toward their roles in the development and maintenance of this region. We have examined the effects of the neurotrophins NT-3, BDNF, and NGF in cultures of developing rat hippocampal cells by two criteria: rapid induction of c-fos and neurotrophic responses. The selective induction of c-fos mRNA suggests the presence of functional receptors for NT-3 and BDNF, but not NGF, in embryonic hippocampal cultures. The NT-3-responsive cells were localized in pyramidal neurons of areas CA1 through CA3 and dentate granular and hilar cells of postnatal organotypic slices, as detected by c-Fos immunocytochemistry. In addition to immediate early responses, NT-3 caused a 10-fold increase in the number of cells expressing the neuronal antigen calbindin-D28k. This increase was dose dependent, with maximal stimulation at 10 ng/ml. In contrast, BDNF elicited small but significant calbindin responses. These results indicate biological responses to NT-3 in the CNS and suggest roles for for this neurotrophin during hippocampal neurogenesis.     

Inhomogeneous hippocampal activation along the rostrocaudal axis in mice after exploration of novel environment

Levels of the c-Fos protein expression in neurons were used as an index of neural activation in the hippocampus of C57BL/6 mice after their exploration of novel environments. C-Fos expression was measured at 8 levels along the rostrocaudal axis of the hippocampus. In Experiment 1, C57BL/6 mice were trained in a modified 8-arm radial maze to find the entry to a home cage through a target arm (1 day, 6 trials). Animals of control group were trained to enter the home cage through an isolated arm. In mice trained in 8-arm maze, functional rostrocaudal inhomogeneity of hippocampus was found. C-Fos expression was increased, mainly, in the caudal parts of CA1, CA3 and dentate gyrus as compared to the control group. In Experiment 2, C57BL/6 mice were tested (1 day, 6 trials) in a novel open-field arena. In this case, c-Fos activity was increased in CA1 (to a greater extent in the caudal than in rostral parts) and CA3 and dentate gyrus (equally in rostrocaudal direction). Significant positive correlations between the exploration activity and density of c-Fos positive cells were found in both experiments. The findings suggest that exploration in novel environment differentially affects the hippocampal subfields along the hippocampal rostrocaudal axis.     

神经途径在肢体缺血预处理抗脑缺血/再灌注损伤中的作用

目的：探讨神经途径在肢体缺血预处理（limbi schemic preconditioning,LIP）抗脑缺血／再灌注损伤中的作用。方法：脑缺血采用四血管闭塞模型,重复短暂夹闭放松大鼠双侧股动脉3次作为LIP。将凝闭椎动脉的大鼠随机分为sham组、脑缺血组、股神经切断＋脑缺血组、LIP＋脑缺血组、股神经切断＋LIP＋脑缺血组。于Sham手术和脑缺血后7d处死大鼠,硫堇染色观察海马CA1区锥体神经元迟发性死亡的变化。于Sham手术和脑缺血后6h心脏灌注固定大鼠,免疫组化法测定海马CAI区c-Fos表达的变化。结果：硫堇染色结果显示,与sham组比较。脑缺血组和股神经切断＋脑缺血组大鼠海马CAI区均有明显组织损伤。LIP＋脑缺血组CAI区无明显细胞缺失,神经元密度明显高于脑缺血组（P〈0．01）。而股神经切断＋LIP＋脑缺血组大鼠海马CA1区明显损伤,锥体细胞缺失较多,与LIP＋脑缺血组组比较,神经元密度显著降低（P〈O．01）,提示LIP前切断双侧股神经取消了LIP抗脑缺血／再灌注损伤作用。c—Fos免疫组化染色结果显示,Sham组海马CAI区未见明显的c-Fos蛋白表达。脑缺血组海马CAI区偶见c—Fm的阳性表达。LIP＋脑缺血组c—Fos表达增强,数量增加,与Sham组和脑缺血组比较。c-Fos阳性细胞数和光密度均明显升高（P〈0．01）。而股神经切断＋LIP＋脑缺血组c-Fos表达明显减少,仅见少量弱阳性e-Fos表达。结论：LIP可通过神经途径发挥抗脑缺血／再灌注损伤作用,而LIP诱导c—Fos表达增加可能是LIP诱导脑缺血耐受神经途径的一个环节。     

Deletion of Running-Induced Hippocampal Neurogenesis by Irradiation Prevents Development of an Anxious Phenotype in Mice

Recent evidence postulates a role of hippocampal neurogenesis in anxiety behavior. Here we report that elevated levels of neurogenesis elicit increased anxiety in rodents. Mice performing voluntary wheel running displayed both highly elevated levels of neurogenesis and increased anxiety in three different anxiety-like paradigms: the open field, elevated O-maze, and dark-light box. Reducing neurogenesis by focalized irradiation of the hippocampus abolished this exercise-induced increase of anxiety, suggesting a direct implication of hippocampal neurogenesis in this phenotype. On the other hand, irradiated mice explored less frequently the lit compartment of the dark-light box test irrespective of wheel running, suggesting that irradiation per se induced anxiety as well. Thus, our data suggest that intermediate levels of neurogenesis are related to the lowest levels of anxiety. Moreover, using c-Fos immunocytochemistry as cellular activity marker, we observed significantly different induction patterns between runners and sedentary controls when exposed to a strong anxiogenic stimulus. Again, this effect was altered by irradiation. In contrast, the well-known induction of brain-derived neurotrophic factor (BDNF) by voluntary exercise was not disrupted by focal irradiation, indicating that hippocampal BDNF levels were not correlated with anxiety under our experimental conditions. In summary, our data demonstrate to our knowledge for the first time that increased neurogenesis has a causative implication in the induction of anxiety.     

Subregion-specific vulnerability to endoplasmic reticulum stress-induced neurotoxicity in rat hippocampal neurons

It is well known that in certain disease states, including ischemia and Alzheimer's disease, neurodegeneration occurs in the hippocampus and that vulnerability to neuronal death is area dependent. The present study investigated the mechanism of area-dependent vulnerability to neuronal death under endoplasmic reticulum stress conditions induced by tunicamycin (TM), using rat organotypic hippocampal cultures (OHC) and hippocampal slices. Analysis of propidium iodide uptake showed that TM-induced neuronal death in a concentration-dependent manner (20-80 microg/mL) and that the rank order of vulnerability among hippocampal subregions was dentate gyrus (DG)>CA1>CA3. Results of immunohistochemistry using hippocampal slices also showed that procaspase-12-positive cells in area CA3 were significantly fewer than those in area CA1 and the DG. Moreover, procurement of neurons in areas CA1, CA3 and the DG by laser microdissection, followed by Western blot analysis, also revealed that the level of procaspase-12 in area CA3 was significantly lower than those in area CA1 and the DG. Pretreatment with z-ATAD-fmk, a cell-permeable caspase-12-selective inhibitor significantly attenuated the TM-induced increase of PI fluorescence in the CA1 and DG subregion but not in area CA3. These results suggest that TM elicits subregion-specific neuronal toxicity in OHC and that the vulnerability to TM-induced toxicity is at least partly dependent on the expression level of endogenous procaspase-12 in each area of the hippocampus.     

Neuroprotective effects of lactation against kainic acid treatment in the dorsal hippocampus of the rat

Marked hippocampal changes in response to excitatory amino acid agonists occur during pregnancy (e.g. decreased frequency in spontaneous recurrent seizures in rats with KA lesions of the hippocampus) and lactation (e.g. reduced c-Fos expression in response to N-methyl-d,l-aspartic acid but not to kainic acid). In this study, the possibility that lactation protects against the excitotoxic damage induced by KA in hippocampal areas was explored. We compared cell damage induced 24 h after a single systemic administration of KA (5 or 7.5 mg/kg bw) in regions CA1, CA3, and CA4 of the dorsal hippocampus of rats in the final week of lactation to that in diestrus phase. To determine cellular damage in a rostro-caudal segment of the dorsal hippocampus, we used NISSL and Fluorojade staining, immunohistochemistry for active caspase-3 and TUNEL, and we observed that the KA treatment provoked a significant loss of neurons in diestrus rats, principally in the pyramidal cells of CA1 region. In contrast, in lactating rats, pyramidal neurons from CA1, CA3, and CA4 in the dorsal hippocampus were significantly protected against KA-induced neuronal damage, indicating that lactation may be a natural model of neuroprotection.     

Activation of calcium/calmodulin-dependent protein kinase IV in long term potentiation in the rat hippocampal CA1 region

The importance of well characterized calcium/calmodulin-dependent protein kinase (CaMK) II in hippocampal long term potentiation (LTP) is widely well established; however, several CaMKs other than CaMKII are not yet clearly characterized and understood. Here we report the activation of CaMKIV, which is phosphorylated by CaMK kinase and localized predominantly in neuronal nuclei, and its functional role as a cyclic AMP-responsive element-binding protein (CREB) kinase in high frequency stimulation (HFS)-induced LTP in the rat hippocampal CA1 region. CaMKIV was transiently activated in neuronal nuclei after HFS, and the activation returned to the basal level within 30 min. Phosphorylation of CREB, which is a CaMKIV substrate, and expression of c-Fos protein, which is regulated by CREB, increased during LTP. This increase was inhibited mainly by CaMK inhibitors and also by an inhibitor for mitogen-activated protein kinase cascade, although to a lesser extent. Our results suggest that CaMKIV functions as a CREB kinase and controls CREB-regulated gene expression during HFS-induced LTP in the rat hippocampal CA1 region.     

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the “spatial” novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.     

Relationships among seizures, extracellular amino acid changes, and neurodegeneration induced by 4-aminopyridine in rat hippocampus: a microdialysis and electroencephalographic study

4-Aminopyridine is a powerful convulsant that induces the release of neurotransmitters, including glutamate. We report the effect of intrahippocampal administration of 4-aminopyridine at six different concentrations through microdialysis probes on EEG activity and on concentrations of extracellular amino acids and correlate this effect with histological changes in the hippocampus. 4-Aminopyridine induced in a concentration-dependent manner intense and frequent epileptic discharges in both the hippocampus and the cerebral cortex. The three highest concentrations used induced also a dose-dependent enhancement of extracellular glutamate, aspartate, and GABA levels and profound hippocampal damage. Neurodegenerative changes occurred in CA1, CA3, and CA4 subfields, whereas CA2 was spared. In contrast, microdialysis administration of a depolarizing K+ concentration and of tetraethylammonium resulted in increased amino acid levels but no epileptic activity and no or moderate neuronal damage. These results suggest that seizure activity induced by 4-aminopyridine is due to a combined action of excitatory amino acid release and direct stimulation of neuronal firing, whereas neuronal death is related to the increased glutamate release but is independent of seizure activity. In addition, it is concluded that the glutamate release-inducing effect of 4-aminopyridine results in excitotoxicity because it occurs at the level of nerve endings, thus permitting the interaction of glutamate with its postsynaptic receptors, which is probably not the case after K+ depolarization.     

Hippocampal apoptosis involved in learning deficits in the offspring exposed to maternal high sucrose diets

The hippocampus plays a crucial role in learning and memory, and neuronal apoptosis in the hippocampus contributes to learning deficits. Metabolism problems in pregnancy related to excessive fuel consumption (e.g., high fat, high sugar) may influence cognitive and behavioral functions in the offspring by affecting developing brain cells. This study determined the influence of maternal high sucrose (HS) diets on behavior and hippocampal neurons in the young offspring. The ratio of brain weight to body weight in the offspring exposed to prenatal HS diets was significantly decreased; the Morris water maze showed that the offspring exposed to prenatal HS diets exhibited increased escape latencies and path length during navigation testing, while there were no changes in time spent in the target quadrant and number of target approaches. In the offspring exposed to prenatal HS, TUNEL-positive cells were significantly increased in CA1, CA2 and CA3 of the hippocampus; protein expression of insulin-like growth factor-I, PI3K and phosphorylated Akt was significantly decreased, while caspase-3 and N-methyl-d-aspartate receptors were significantly increased in the hippocampus, and there was no change in expression of Bcl-2 and Akt. The results demonstrated that prenatal HS diets could induce the spatial acquisition deficits in the young offspring associated with hippocampal apoptosis, and altered signaling factors for antiapoptosis in the hippocampus might play a critical role in cognition disorders in young children.     

Effects of MK-801 and CNQX on various neurotoxic responses induced by kainic acid in mice

Effects of MK-801 (a NMDA receptor blocker) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; a non-NMDA receptor blocker) on several neurotoxic responses induced by kainic acid (KA) were examined in ICR mice. In a lethality test, intracerebroventricular (i.c.v.) pretreatment of MK-801 (1 microg), but not CNQX (0.5 microg), attenuated the time to lethality induced by KA (0.5 microg) administered i.c.v. In the memory test (a passive avoidance test), MK-801, but not CNQX, prevented the memory loss induced by KA (0.1 microg). The damage induced by KA (0.1 microg) administered i.c.v. in the hippocampus was markedly concentrated in the CA3 pyramidal neurons. Both MK-801 and CNQX blocked the pyramidal cell death in CA3 hippocampal region induced by KA. In the immunocytochemical study, KA dramatically increased the phosphorylated ERK (p-ERK) and decreased the phosphorylated CREB (p-CREB) in the hippocmapus. Both MK-801 and CNQX attenuated, in part, the increased p-ERK and the decreased p-CREB induced by KA. In addition, both MK-801 and CNQX partially reduced the increased c-Fos and c-Jun protein expression in hippocampus induced by KA. Our results suggest that both NMDA and non-NMDA receptors are involved in supraspinally administered KA-induced pyramidal cell death in CA3 region of hippocampus in the mouse and the p-ERK and the dephosphorylation of CREB protein may play an important role in CA3 region cell death of the hippocampus induced by KA administered supraspinally. Furthermore, c-Fos and c-Jun proteins may serve as third messengers responsible for CA3 pyramidal cell death induced by supraspinally administered KA.     

c-Fos Expression by Dopaminergic Receptor Activation in Rat Hippocampal Neurons

While dopamine is likely to modulate hippocampal synaptic plasticity, there has been little information about how dopamine affects synaptic transmission in the hippocampus. The expression of IEGs including c-fos has been associated with late phase LTP in the CA1 region of the hippocampus. The induction of c-fos by dopaminergic receptor activation in the rat hippocampus was investigated by using semiquantitative RT-PCR and immuno-cytochemistry. The hippocampal slices which were not treated with dopamine showed little expression of c-fos mRNA. However, the induction of c-fos mRNA was detected as early as 5 min after dopamine treatment, peaked at 60 min, and remained elevated 5 h after treatment. Temporal profiles of increases in c-fos mRNA by R(+)-SKF-38393 (50 M) and forskolin (50 M) were similar to that of dopamine. An increase in [cAMP] was observed in dopamine-, SKF-, or forskolin-treated hippocampal slices. By immunocytochemical studies, control hippocampal cells showed little expression of c-Fos immunoreactivity. However, when cells were treated with dopamine, an increase in the expression of c-Fos immunoreactivity was observed after treatment for 2 h. The treatment of hippocampal neurons with R(+)-SKF38393 (50 M) or forskolin (50 M) also induced a significant increase in c-Fos expression. These results indicate that the dopamine D1 receptor-mediated cAMP dependant pathway is associated with the expression of c-Fos in the hippocampal neurons. These data are consistent with the possible role of endogenous dopamine on synaptic plasticity via the regulation of gene expression. Furthermore, these results imply that dopamine might control the process of memory storage in the hippocampus through gene expression.     

Lateral entorhinal cortex lesions rearrange afferents, glutamate receptors, increase seizure latency and suppress seizure-induced c-fos expression in the hippocampus of adult rat

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurones in chronic epilepsy. Here we analysed the effects of one-sided lateral EC (LEC) and temporoammonic (alvear) path lesion on the development and properties of 4-aminopyridine-induced seizures. Electroencephalography (EEG) analysis of freely moving rats identified that the lesion increased the latency of the hippocampal seizure significantly and decreased the number of brief convulsions. Seizure-induced neuronal c-fos expression was reduced in every hippocampal area following LEC lesion. Immunocytochemical analysis 40 days after the ablation of the LEC identified sprouting of cholinergic and calretinin-containing axons into the dentate molecular layer. Region and subunit specific changes in the expression of ionotropic glutamate receptors (iGluRs) were identified. Although the total amount of AMPA receptor subunits remained unchanged, GluR1(flop) displayed a significant decrease in the CA1 region. An increase in NR1 and NR2B N-methyl-d-aspartate (NMDA) receptor subunits and KA-2 kainate receptor subunit was identified in the deafferented layers of the hippocampus. These results further emphasize the importance of the lateral entorhinal area in the spread and regulation of hippocampal seizures and highlight the potential role of the rewiring of afferents and rearrangement of iGluRs in the dentate gyrus in hippocampal convulsive activity.     

Late-Onset Running Biphasically Improves Redox Balance,Energy- and Methylglyoxal-Related Status,as well as SIRT1 Expression in Mouse Hippocampus

Despite the active research in this field, molecular mechanisms underlying exercise-induced beneficial effects on brain physiology and functions are still matter of debate, especially with regard to biological processes activated by regular exercise affecting the onset and progression of hippocampal aging in individuals unfamiliar with habitual physical activity. Since such responses seem to be mediated by changes in antioxidative, antiglycative and metabolic status, a possible exercise-induced coordinated response involving redox, methylglyoxal- and sirtuin-related molecular networks may be hypothesized. In this study, hippocampi of CD1 mice undergoing the transition from mature to middle age were analyzed for redox-related profile, oxidative and methylglyoxal-dependent damage patterns, energy metabolism, sirtuin1 and glyoxalase1 expression after a 2- or 4-mo treadmill running program. Our findings suggested that the 4-mo regular running lowered the chance of dicarbonyl and oxidative stress, activated mitochondrial catabolism and preserved sirtuin1-related neuroprotection. Surprisingly, the same cellular pathways were negatively affected by the first 2 months of exercise, thus showing an interesting biphasic response. In conclusion, the duration of exercise caused a profound shift in the response to regular running within the rodent hippocampus in a time-dependent fashion. This research revealed important details of the interaction between exercise and mammal hippocampus during the transition from mature to middle age, and this might help to develop non-pharmacological approaches aimed at retarding brain senescence, even in individuals unfamiliar with habitual exercise.     

Forskolin and 8-cyclopentyltheophylline synergistically facilitate the neuronal activity in the CA2 area of rat hippocampus via cAMP and non-cAMP cascades

High level of adenosine A1 receptor-like immunoreactivity has been found in the CA2/CA3a region of adult rat hippocampus, but its roles in the neuronal activity or signal propagation in hippocampus and its intracellular cascade remain to be studied. In this study, we examined the relation between adenosine-3',5'-cyclic monophosphate (cAMP) cascade and suppression of synaptic transmission by endogenous adenosine through adenosine A1 receptor in the CA2 area. In transverse hippocampal slice, maximal electrical stimulation of the hilus region (0.6 mA) only evoked small population spikes (PSs) in the CA2 area (0.5 mV). In the presence of forskolin (20 micromol/L), a direct adenylate cyclase activator, PSs in CA2 were increased to 1.1 mV. When 8-cyclopentyltheophylline (8CPT, 2 micromol/L), an adenosine A1 receptor antagonist, was added in the presence of 20 micromol/L forskolin, PSs with an average amplitude of 4.7 mV were recorded in the CA2 area, much higher than the sum of the amplitude of PSs in the presence of forskolin and 8CPT alone. To test whether this synergistic potentiation results from the additive activation of cAMP cascade, the cAMP content in hippocampal slices was measured with enzyme immunoassay (EIA). Results showed that 8CPT did not increase the cAMP content in CA2 with or without forskolin. Co-application of forskolin and Ro 20-1724, a cAMP-specific phosphodiesterase-IV inhibitor, only increased PSs in CA2 to 1.3 mV but increased cAMP content by 4.4 times. On the other hand, co-application of 8CPT and 1, 9-dideoxyforskolin, a forskolin analog which has no effect on adenylate cyclase, did not mimic the synergistic effect of 8CPT and forskolin on PSs in CA2. These results indicate that up-regulation of adenylate cyclase activity and inhibition of adenosine A1 receptor activity synergistically facilitate the neuronal activity in the CA2 area and the effect of adenosine A1 receptor antagonist is via non-cAMP cascade. These data also suggest that acting on adenosine A1 receptors, endogenous and extragenous adenosine/adenosine A1 agonist(s) inhibit neuronal activity through different pathways.