Subthreshold membrane potential dynamics of posterior parietal cortical neurons coupled with hippocampal ripples.

During behavioral states of immobility, sleep, and anesthesia, the hippocampus generates high-frequency oscillations called ripples. Ripples occur simultaneously with synchronous neuronal activity in the neocortex, known as slow waves, and contribute to memory consolidation. During these ripples, various neocortical regions exhibit modulations in spike rates and local field activity irrespective of whether they receive direct synaptic inputs from the hippocampus. However, little is known about the subthreshold dynamics of the membrane potentials of neocortical neurons during ripples. We patch-clamped layer 2/3 pyramidal cells in the posterior parietal cortex (PPC), a neocortical region that is involved in allocentric spatial representation of behavioral exploration and sequential series of relevant action potentials during ripples. We simultaneously monitored the membrane potentials of post hoc-identified PPC neurons and the local field potentials of the hippocampus in anesthetized mice. More than 50% of the recorded PPC neurons exhibited significant depolarizations and/or hyperpolarizations during ripples. Histological inspections of the recorded neurons revealed that the ripple-modulated PPC neurons were distributed in the PPC in a spatially non-biased fashion. These results suggest that hippocampal ripples are widely but selectively associated with the subthreshold dynamics of the membrane potentials of PPC neurons even though there is no monosynaptic connectivity between the hippocampus and the PPC.

Experimental febrile seizures induce age-dependent structural plasticity and improve memory in mice.

Population-based studies have demonstrated that children with a history of febrile seizure (FS) perform better than age-matched controls at hippocampus-dependent memory tasks. Here, we report that FSs induce two distinct structural reorganizations in the hippocampus and bidirectionally modify future learning abilities in an age-dependent manner. Compared with age-matched controls, adult mice that had experienced experimental FSs induced by hyperthermia (HT) on postnatal day 14 (P14-HT) performed better in a cognitive task that requires dentate granule cells (DGCs). The enhanced memory performance correlated with an FS-induced persistent increase in the density of large mossy fiber terminals (LMTs) of the DGCs. The memory enhancement was not observed in mice that had experienced HT-induced seizures at P11 which exhibited abnormally located DGCs in addition to the increased LMT density. The ectopic DGCs of the P11-HT mice were abolished by the diuretic bumetanide, and this pharmacological treatment unveiled the masked memory enhancement. Thus, this work provides a novel basis for age-dependent structural plasticity in which FSs influence future brain function.

Pharmacologic action of oseltamivir on the nervous system.

Oseltamivir, an antiviral drug used for the treatment of influenza, contains the L-glutamic acid motif in its chemical structure. We focused on this structural characteristic of oseltamivir and examined the pharmacologic effects of the drug on the nervous system in invertebrate and vertebrate animal models. Injection of oseltamivir or L-glutamic acid into silkworm (Bombyx mori) larvae induced muscle relaxation. Oseltamivir and L-glutamic acid inhibited kainate-induced rapid muscle contraction, but neither drug affected insect cytokine paralytic peptide-induced slow muscle contraction. In the mammalian system, mice (Mus musculus) treated intracerebrally with oseltamivir developed convulsive seizures. Hydrolyzed oseltamivir, the active form containing a carboxylic acid, evoked epileptiform firing of hippocampal neurons in rat (Rattus norvegicus) organotypic hippocampal slice cultures. These results are the first to demonstrate that oseltamivir exerts pharmacologic effects on the nervous system in insects and mammals.

Neonatally born granule cells numerically dominate adult mice dentate gyrus.

Hippocampal granule cells (GCs) are continuously generated in the subgranular zone of the dentate gyrus (DG) and functionally incorporated to dentate neural circuits even in adulthood. This raises a question about the fate of neonatally born GCs in adult DG. Do they exist until adulthood or are they largely superseded by adult-born GCs? To investigate this question, we examined the contributions of postnatally born GCs to the adult mouse DG. C57BL/6 mice were grouped in three different postnatal (P) ages (group 1: P0, group 2: P7, and group 3: P35) and received a daily bromodeoxyuridine (BrdU) injection for three consecutive days (P0/1/2, P7/8/9, and P35/36/37, respectively) to label dividing cells. At 6 months old, hippocampal sections were prepared from the animals and immunostained with anti-BrdU antibody and an antibody against the homeobox prospero-like protein Prox1, a marker of GCs. We defined BrdU- and Prox1-double positive cells as newborn GCs and analyzed their density and distribution in the granule cell layer (gcl), revealing that newborn GCs of each group still existed 6 months after BrdU injections and that the density of GCs born during P0-2 (group 1) was significantly higher compared with the other groups. Although the density of newborn GCs in the each group did not differ between male and female, the radial distribution of them in gcl showed some differences, that is, male newborn GCs localized toward the molecular layer compared with female ones in group 1, while to the hilus in group 2. These results suggest that GCs born in early postnatal days numerically dominate adult DG and that there exist sex differences in GC localizations which depend on the time when they were born.

Hippocampal CA1 synaptic plasticity as a gamma transfer function.

The capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output conversion efficiency at the synapses. In the adult mouse hippocampus in vivo, we carefully compared the input-output relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e. conforming to the so-called gamma curve. In extreme cases, long-term potentiation and depression coexist in the same synaptic pathway with long-term potentiation dominating over long-term depression at higher levels of presynaptic activity. These findings predict a novel function of synaptic plasticity, i.e. a contrast-enhancing filtering of neural information through a gamma correction-like process.

Hepatocyte growth factor as an enhancer of nmda currents and synaptic plasticity in the hippocampus.

Hepatocyte growth factor (HGF) promotes the survival and migration of immature neurons, but its role in the mature brain has remained elusive. In the hippocampus of juvenile rats, we found that the HGF receptor c-Met was expressed in neurons. Furthermore, it was highly Tyr-phosphorylated, more so than in the liver under normal conditions, suggesting that the receptor is activated and that HGF may act continuously in the intact brain. Exogenously applied HGF enhanced synaptic long-term potentiation (LTP) in the CA1 region of hippocampus, but did not affect long-term depression. We further found that HGF augmented N-methyl-D-aspartate receptor-mediated currents in both slices and dissociated neurons. This augmentation is likely to underlie the enhancement of LTP. Considering that the expression of both HGF and c-Met are known to be induced by ischemic stimuli, this modulation would provide a novel understanding of a neuronal regulatory systems shared with pathogenic ischemic states.

Regionally selective neurotoxicity of NMDA and colchicine is independent of hippocampal neural circuitry.

The mechanisms by which cerebral ischemia and several neurotoxins cause regionally selective damages to the hippocampal formation are largely unknown. The CA1-selective toxicity of N-methyl--aspartate (NMDA), the CA3-selective toxicity of kainate, and the dentate gyrus (DG)-selective toxicity of colchicine were observed in organotypic entorhino-hippocampal cultures. The selective neurotoxicity of NMDA and colchicine but not kainate was present in isolated tissue cultures of each hippocampal subregion, suggesting that the regional vulnerability is irrespective of the hippocampal trisynaptic pathway. Dispersed cultures of neurons prepared from Ammon's horn and the DG still exhibited a preference for susceptibility to NMDA and colchicine, respectively. Thus, the neurons per se appear to be inherently susceptible to specific toxins independently of their original loci, intrinsic neural circuits, vascular system, or other systemic factors.

Rapid and reversible changes in dendrite morphology and synaptic efficacy following NMDA receptor activation: implication for a cellular defense against excitotoxicity.

Postsynaptic neuronal dendrites undergo functional and morphological changes in response to pathologically excessive synaptic activation. Although rapid formation of segmental focal swelling (varicosity) is the most prominent hallmark in such excitotoxic injury, little is known about the pathophysiological function of these structural alterations. We used cultured rat hippocampal slices to evaluate the relationship between the formation of varicosities and subsequent neuronal death. Substantial numbers of segmental dendritic varicosities were observed all over the hippocampus within 5 minutes of exposure to 30 microM NMDA, although neuronal death was detected only in the CA1 region 24 hours after NMDA exposure. Sublethal NMDA concentrations (1-10 microM) induced reversible focal swelling in all hippocampal subregions. NMDA-induced neuronal death was prevented either by NMDA receptor antagonists or by the use of Ca(2+)-free medium, whereas varicosity formation was virtually independent of Ca(2+) influx. Rather, the Ca(2+)-free conditions per se produced dendritic focal swelling. Also, NMDA-induced varicosity formation was dependent on extracellular Na+ concentration. Thus, we believe that varicosity formation is not causally related to neuronal injury and that the two phenomena are separable and involve distinct mechanisms. Interestingly, dendrite swelling was accompanied by AMPA receptor internalization and a rapid, long-lasting depression in synaptic transmission. Moreover, low Na+ conditions or treatment with ethacrynic acid or proteinase inhibitors, which effectively prevent varicosity formation, aggravated NMDA-induced excitotoxicity, and eliminated the regional specificity of the toxicity. Therefore, the pathological changes in dendrite morphology and function may be associated with an early, self-protective response against excitotoxicity.

Transient upregulation of the glial glutamate transporter GLAST in response to fibroblast growth factor, insulin-like growth factor and epidermal growth factor in cultured astrocytes.

Although expression of the glial glutamate transporter GLAST is tightly regulated during development and under pathophysiological conditions, little is known about endogenous modulators of GLAST expression. Because growth factors are generally believed to regulate glial functions, we addressed their possible contribution to GLAST regulation in cultured rat astrocytes. Of the six growth factors tested (basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), insulin, platelet-derived growth factor, and hepatocyte growth factor), bFGF, IGF-1 and EGF enhanced [(3)H]glutamate transport activity in a concentration-dependent manner. These effects were accompanied by an increase in the V(max) value for transport activity and in GLAST protein and mRNA levels, which suggests that GLAST expression is transcriptionally regulated by the growth factors. Interestingly, the effects reached a peak after 36 hours of exposure to growth factors, and rapidly returned to baseline by 48 hours. A combination of IGF-1 with either bFGF or EGF showed an additive effect on the glutamate uptake activity, but a combination of bFGF and EGF did not. Pharmacological blockade of protein kinase C inhibited the effects of IGF-1 and EGF, but not bFGF. By contrast, genistein, an inhibitor of tyrosine kinases, blocked the effects of bFGF and EGF without affecting the effect of IGF-1. These results suggest that the growth factors activate different signaling pathways for GLAST upregulation. The present study may indicate a novel regulatory system of glial glutamate transporters.

Withdrawal from chronic morphine administration causes prolonged enhancement of immobility in rat forced swimming test.

RATIONALE: Opiate-dependent subjects experience severe depression as one of the subjective symptoms during withdrawal. No experimental work, however, has focused on the ability of opiate-withdrawal to produce depression-like behavior in dependent animal. OBJECTIVES. We therefore investigated whether withdrawal from chronic morphine treatment affects immobility in forced swimming test in rats. METHODS: Morphine was administered in a dose escalation fashion using doses ranging from 20 to 140 mg/kg twice daily for 14 days, followed by 1-6 days of withdrawal, and their duration of immobility was assessed. RESULTS: After the last morphine treatment. an increase in immobility occurred late on day 3 and persisted to at least day 6 of withdrawal without any change in ambulatory activity. CONCLUSIONS: The results suggest that the morphine withdrawal resulted in prolonged enhancement of depression-like behavior in drug-dependent laboratory animals.

Cyclic nucleotide-mediated regulation of hippocampal mossy fiber development: a target-specific guidance.

The mossy fibers (MFs) arising from dentate granule cells project primarily onto a narrow segment of the proximal dendrites of hippocampal CA3 pyramidal cells. The mechanisms underlying this specific MF target selection are not fully understood. To investigate the cellular basis for development of the stereotyped MF trajectories, we have arranged the fascia dentata and hippocampal Ammon's horn tissues in diverse topographical patterns in organotypic explant coculture systems. Here we show that cyclic nucleotide signaling pathways regulate the MF pathfinding. When the dentate gyrus explants were ectopically placed facing the CA3 stratum oriens of hippocampal slices, MFs crossed the border between cocultures and reached their appropriate target area in the Ammon's horn, as assessed by membrane tracer labeling, Timm staining, electrophysiological recording of synaptic responses, and optical analyses using a voltage-sensitive dye. This lamina-specific MF innervation was disrupted by pharmacological blockade of cGMP pathway. Similar apposition of the dentate grafts near the CA1 region of host slices rarely resulted in MF ingrowth into the Ammon's horn. Under blockade of cAMP pathway, however, the MFs were capable of making allopatric synapses with CA1 neurons. These data were further supported by the pharmacological data obtained from granule cells dispersed over hippocampal slice cultures. Thus, our findings suggest that the stereotyped MF extension is mediated by at least two distinct factors, i.e., an attractant derived from the CA3 region and a repellent from the CA1 region. These factors may be regulated differently by cAMP and cGMP signaling pathways.

Neuroprotective effects of lipoxygenase inhibitors against ischemic injury in rat hippocampal slice cultures.

Using organotypic cultures of rat hippocampal slices, we investigated the possible involvement of arachidonate cascades in neuronal death following ischemic insult. Oxygen/glucose deprivation-induced neuronal damage was efficiently attenuated by various inhibitors of lipoxygenase, whereas cyclooxygenase inhibitors were less effective. Interestingly, 5- and 12-lipoxygenases are likely to separately mediate ischemic injury in the hippocampus. The present study will provide novel therapeutic targets for the development of neuroprotective agents.

Phospholipase A2 mediates ischemic injury in the hippocampus: a regional difference of neuronal vulnerability.

Although it is well known that the hippocampal CA1 subfield is highly vulnerable to ischemic injury, cellular mechanisms leading to this neuronal degeneration are not fully understood. Using organotypic cultures of rat hippocampal slices, we determined whether phospholipase A2 (PLA2) is activated in response to ischemic conditions (OGD; oxygen and glucose deprivation). The PLA2 activity in the pyramidal cell layer increased immediately following a 35-min exposure to OGD, which was likely to be mediated by selective activation of cytosolic Ca2+-dependent PLA2 subtype (cPLA2). This enhancement lasted for at least 24 h. Interestingly, no apparent increase was detected in the dentate gyrus. Twenty-four hours after the OGD exposure, neuronal death was detected mainly in the CA1 region of hippocampal slices. To examine whether the PLA2 activation is causally or protectively involved in the ischemic injury, we investigated the effect of pharmacological blockade of PLA2 on the OGD-induced neuronal death. The PLA2 inhibitor bromophenacyl bromide efficiently prevented the cell death in a concentration-dependent manner. Similar results were obtained for the selective cPLA2 inhibitor AACOCF3. However, the Ca2+-independent PLA2 inhibitor bromoenol lactone and the secretory PLA2 inhibitor LY311727 were virtually ineffective. These results suggest that cPLA2 plays a causative role in the neuronal death following OGD exposure. Thus, the present study may provide novel therapeutic targets for the development of neuroprotective agents.

Spatial performance correlates with long-term potentiation of the dentate gyrus but not of the CA1 region in rats with fimbria-fornix lesions.

Although hippocampal long-term potentiation (LTP) is generally assumed to be a cellular mechanism of learning and memory, there has not been definitive evidence for this hypothesis. In the present study, therefore, we addressed the possible relationship between spatial learning ability and LTP by using rats with bilateral fimbria-fornix lesions. The animals were tested for spatial performance in spontaneous alternation behaviors with further in vivo investigation of LTP. The behavioral parameters of spatial memory showed a significant correlation with LTP in the dentate gyrus, but we found no evidence for a linkage with LTP in the CA1 region. Thus, LTP in the dentate gyrus may be important for spatial cognitive ability.

Docosahexaenoic acid improves long-term potentiation attenuated by phospholipase A(2) inhibitor in rat hippocampal slices.

1. We investigated the possible involvement of phospholipase A(2) (PLA(2)) and its products in long-term potentiation (LTP) in the CA1 neurotransmission of rat hippocampal slices. 2. Inhibitors of Ca(2+)-independent PLA(2) (iPLA(2)) prevented the induction of LTP without affecting the maintenance phase of LTP whereas Ca(2+)-dependent PLA(2) inhibitors were virtually ineffective, which suggests a pivotal role of iPLA(2) in the initiation of LTP. 3. We then investigated the effect of docosahexaenoic acid (DHA) and arachidonic acid (AA) on BEL (bromoenol lactone, an iPLA(2)-inhibitor) -impaired LTP, and found that either DHA or AA abolished the effect of BEL. However, DHA did not restore BEL-attenuated LTP when applied after the tetanus. DHA per se affected neither the induction nor maintenance of LTP. Linoleic acid had no effects, either. 4. These results suggest that DHA is crucial for the induction of LTP and that endogenously released DHA during tetanus is sufficient to trigger the formation of LTP.

Aberrant synaptic transmission in the hippocampal CA3 region and cognitive deterioration in protein-repair enzyme-deficient mice.

L-aspartate is the amino-acid residue most susceptible to spontaneous isomerization. This denaturation causes an alteration in the biological activity of the protein and is regarded as an aging process of the protein. Protein L-isoaspartyl methyltransferase (PIMT) repairs this post-translational modification and thus is implicated in retarding the aging process of proteins. PIMT is highly expressed in the brain, and its deficiency results in progressive epilepsy after 4 weeks of age, with a fatal seizure in mice. Here we report the pathophysiological role of this repair system in the hippocampal slice of PIMT-deficient mice. The hippocampal mossy fiber-CA3 synapses of PIMT-deficient mice showed hyperexcitation that was repressed by a gamma-aminobutyric acid (GABA)A receptor agonist muscimol. In addition, the mossy fiber-CA3 synapses failed to show long-term potentiation or paired-pulse facilitation. No abnormality, however, was observed in Schaffer collateral-CA1 synapses or in perforant path-dentate gyrus synapses. Electron microscopic study revealed aberrant distribution of synaptic vesicles in the mossy fiber terminals and vacuolar degeneration at the axon hillock of dentate granule cells in PIMT-deficient mice. Furthermore, the PIMT-deficient mice showed impaired spatial memory in Morris water maze test and exhibited fewer anxiety-related behaviors in the elevated-plus test. These results suggest that the mossy fiber-CA3 system is vulnerable to aspartate isomerization and that the PIMT-mediated repair system is essential for maintenance of normal functions of the hippocampus.

Deterioration of spatial learning performances in lipopolysaccharide-treated mice.

It is well demonstrated that acute or chronic stress leads to reduction of learning ability. Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, induces profound physiological and behavioral changes, including fever, decrease in food motivation, and decrease in social behavior. These changes might be interpreted as an acute stress reaction to the LPS. In the present study, therefore, we investigated the effects of LPS (400-800 microg/kg, i.p.) on spatial learning performances using C57BL/6J male mice. In the Morris water-maze task, spatial learning performances were examined in six trials of training for two consecutive days. LPS-treated mice took a longer time to reach the hidden platform than control mice (F(1,60)=4.80801, P<0.05 at 600 microg/kg). In addition, injection of LPS decreased the percent of correct choices in the Y-maze test (P<0.05 at 800 microg/kg). LPS, however, did not alter the body weight, grip tone, motor activity or swimming speed. Taken together, these results indicate that LPS treatment specifically impaired spatial learning performances.

Cytotoxicity of tributyltin in rat hippocampal slice cultures.

The neurotoxic effects of tributyltin (TBT), an endocrine-disrupting chemical, were evaluated in organotypic slice cultures of immature rat hippocampus. Confocal microscopy study with propidium iodide showed that TBT induced severe neuronal death in a concentration- and time-dependent manner with CA3 > CA1 > dentate gyrus ranking of vulnerability of the hippocampal subfields. Dead or damaged neurons exhibited chromatin condensation, which is one of the morphological characteristics of apoptosis, as revealed by acridine orange staining. TBT neurotoxicity was alleviated by application of free radical scavengers or antioxidants, such as catalase, superoxide dismutase, Trolox and alpha-tocopherol but not by ascorbic acid or N-acetyl-L-cysteine, which suggests an involvement of free radicals, particularly reactive oxygen species. Neurons displayed a long-lasting increase in intracellular Ca2+ concentrations after TBT treatment. Although neither N-methyl-D-aspartate (NMDA) receptor inhibitors nor voltage-sensitive Ca2+ channel blockers protected hippocampal neurons against TBT neurotoxicity, non-NMDA receptor antagonist completely prevented TBT-induced neurodegeneration. These data suggest that TBT provokes apoptosis-like neuronal cell death, which might be mediated by intracellular Ca2+ elevation and free radical generation via non-NMDA receptor activation.

Ca2+-independent phospholipase A2 inhibitor impairs spatial memory of mice.

Pharmacological blockade of Ca2+-independent phospholipase A2 (PLA2) is reported to disintegrate hippocampal synaptic plasticity, which is thought to be the cellular mechanism underlying learning and memory. Therefore, we investigated the effect of the Ca2+-independent PLA2 inhibitor bromoenol lactone (BEL) on spontaneous alteration behaviors of mice. When 3 nmol BEL was intracerebroventricularly injected 30 min prior to the test, the mice showed a poor alternation ratio, compared with control animals. The data suggest that Ca2+-independent PLA2 activity is required for spatial memory.

Pharmacological property of tributyltin in vivo and in vitro.

Tributyltin (TBT), an assumed endocrine-disrupting chemical, is widely known to show harmful effects in invertebrates including the dioecious snails. As for mammals, there are several reports concerning TBT toxicology, but few indications about general pharmacology of TBT. In the present study, we comprehensively examined the pharmacological effects of TBT both in vivo and in vitro. TBT (0.3 or 1.0 mg/kg) attenuated the small intestinal propulsive activity measured by the charcoal method in vivo, and caused concentration-dependent relaxation of isolated guinea-pig ileum in vitro (1.0x10(-8)-3.0x10(-6) M). TBT induced concentration-dependent relaxation of guinea-pig trachea, which was not inhibited by pre-treatment with a beta-adrenoceptor antagonist. TBT caused a concentration-dependent contraction of rat aortae, and also evoked endothelium-dependent relaxation in the presence of an alpha-adrenoceptor antagonist. The relaxation was inhibited by a muscarinic receptor antagonist. TBT reduced the electrically evoked, sympathetic contractile responses of rat vas deferens, which were slightly prevented by an alpha(2)-adrenoceptor antagonist. These results suggest that TBT possesses diverse pharmacological properties in mammalian organs.