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Center for Information and Neural Networks, Suita City,Osaka 565-0871, JapanSummaryThe frontal association cortex (FrA) is implicated in higherbrain function [1]. Aberrant FrA activity is likely to beinvolved in dementia pathology [2–4]. However, the func-tional circuits both within the FrA and with other regionsare unclear. A recent study showed that inactivation of theFrAimpairsmemoryconsolidationofanauditoryfearcondi-tioninginyoungmice[5].Inaddition,dendriticspineremod-eling of FrA neurons is sensitive to paired sensory stimulithat produce associative memory [5]. These findings sug-gest that the FrA is engaged in neural processes criticalto associative learning. Here we characterize stimulus inte-gration in the mouse FrA during associative learning. Weexperimentally separated contextual fear conditioning intocontext exposure and shock, and found that memory forma-tion requiresprotein synthesisassociatedwith bothcontextexposure and shock in the FrA. Both context exposure andshock trigger Arc, an activity-dependent immediate-earlygene, expression in the FrA, and a subset of FrA neuronswas dually activated by both stimuli. In addition, we foundthat the FrA receives projections from the perirhinal (PRh)and insular (IC) cortices and basolateral amygdala (BLA),which are implicated in context and shock encoding [6–8].PRh and IC neurons projecting to the FrA were activatedby context exposure and shock, respectively. Arc expres-sion in the FrA associated with context exposure and shockdepended on PRh activity and both IC and BLA activities,respectively.ThesefindingsindicatethattheFrAisengagedinstimulusintegrationandcontributestomemoryformationin associative learning.ResultsThe Frontal Association Cortex Is Required for MemoryFormation in Contextual Fear ConditioningAsamodelforassociativelearning,weusedacontextualfear-conditioning task, which establishes an association betweencontext and shock. To test whether the frontal associationcortex (FrA) is involved in memory formation in contextualfear conditioning, we infused (2R)-amino-5-phosphonovalericacid(APV),anN-methyl-D-aspartate(NMDA)receptorantago-nist, anisomycin, a protein synthesis inhibitor, or vehicle intothe FrA. APV and anisomycin were infused 30 min prior to, orimmediately after, contextual fear conditioning, respectively(experiment 1; Figures 1A and 1B). Contextual fear memorywas assessed by measuring the percentage of freezing timein the conditioning context 1 day after conditioning. BothAPV and anisomycin infusions disrupted freezing behavior.When anisomycin infusions were administered into the dorso-medial prefrontal cortex, which is close to the FrA (experiment2; Figure S1A available online), freezing behavior was compa-rable to that of mice administered vehicle infusions (Fig-ureS1B).TheseresultsindicatethatNMDAreceptoractivationandproteinsynthesisintheFrAarerequiredforcontextualfearconditioning.ProteinSynthesisintheFrAIsRequiredforEncodingBothContext and ShockWe aimed to determine whether the FrA encodes context,shock, or both. To this end, we separated 10 min of contextexposureandimmediateshockbya1-dayintervalandinfusedanisomycinintotheFrAaftereithercontextexposureorshock(experiment 3; Figures 1C and 1D). Anisomycin infusions intothe FrA immediately after both context exposure and immedi-ate shock disrupted freezing behavior during the test. How-ever, when anisomycin infusions were administered into theFrA 6 hr after context exposure (Figure 1E), freezing behaviorwas comparable to that of mice administered vehicle infu-sions. These results indicate that protein synthesis in the FrAis required for encoding both context and shock.FrA Neurons Receive Convergent Information RegardingContext and Shock during Fear ConditioningBecause protein synthesis in the FrA is required for encodingboth context and shock, we hypothesized that paired stimuliconverge in a subset of FrA neurons to potentially contributeto the memory trace. To visualize stimulus convergence, weanalyzed the temporal dynamics of nuclear versus cyto-plasmic Arc localization by fluorescent in situ hybridization[9]. Arc is an activity-dependent immediate-early gene thatis essential for synaptic plasticity and long-term memory[10–13]. Transcribed Arc mRNA first appears in neuronalnuclei, and processed Arc mRNA then accumulates in thecytoplasm. Thus, an analysis of the subcellular localization ofArc enabled us to identify active neuronal ensembles duringtwo behavioral tasks [9, 14, 15]. We first examined the timecourse of the nuclear and cytoplasmic Arc signal after neuralactivity in the FrA. Mice were exposed to a context for 5 minand sacrificed either immediately or 30 min later (experiment4; Figures S2A and S2B). We observed more nuclear Arc

Antihistamines are widely used to treat allergy symptoms. First-generation antihistamines have adverse effects on the central nervous system (CNS), such as hypnotic and amnesic effects, whereas second-generation antihistamines have poor brain penetration, and therefore, have fewer CNS-related adverse effects. Memory consists of several phases, including acquisition, consolidation, expression, and extinction. It remains unclear whether these phases are affected by antihistamines. We investigated the effects of diphenhydramine, a first-generation antihistamine, and levocetirizine and olopatadine, second-generation antihistamines, on memory phases. Mice were subjected to fear conditioning on day 1 and tested on day 2. Antihistamines were administered before conditioning, immediately after conditioning, or before the test session. Diphenhydramine (30 mg/kg) decreased freezing time when administered immediately after conditioning or before the test session. These effects were not attributable to a change in locomotor activity. Levocetirizine (0.1, 1, 10 mg/kg) and olopatadine (1, 10, 20 mg/kg) had no effects on conditioned fear. We also examined the effect of diphenhydramine and levocetirizine on the expression of an activity-dependent gene associated with the test session. Diphenhydramine, but not levocetirizine, increased Arc transcription in the central nucleus of the amygdala. These data indicate that diphenhydramine, but not levocetirizine or olopatadine, impairs the consolidation and expression of conditioned fear.

The hippocampus plays a critical role in contextual fear conditioning. Population activity in the hippocampal CA1 encoding the surrounding environment is thought to be responsible for retrieval of contextual fear memory. However, the characteristics of CA1 neuronal ensemble activity during retrieval of contextual fear memory remain unclear. Here, we examined CA1 ensemble activity during contextual fear memory expression in male C57Bl/6J mice, using Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization. The “Shock” group was conditioned with a footshock in two separate chambers, whereas the “No shock” group was not exposed to shocks in the chamber. Animals were then re-exposed to either the same chamber twice or two different conditioning chambers. In the No shock group, exposure to the same chamber twice activated a more significantly overlapping neuronal population than exposure to two different chambers. In the Shock group, exposure to the same conditioning chamber twice activated a similarly overlapping neuronal population as exposure to two different chambers, with overlap smaller than in nonshocked mice exposed to the same chamber twice. Thus, population activity in the hippocampal CA1 encoding the surrounding environment is detected during spatial exploration, but absent during contextual fear memory expression. Even the variable ensemble activity of CA1 may contribute to retrieval of contextual fear memory.

A large number of aberrant hilar granule cells (GCs) are found in the patients and animal models of adult temporal lobe epilepsy (TLE), and these “ectopic” GCs have synchronous epileptiform bursting with other hippocampal neurons. In this study, we investigated whether early-life status epilepticus (SE) induces hilar ectopic GCs that remain in the adulthood because TLE patients frequently experience seizures in the early childhood when a large number of postnatally born GCs migrate in the hilus. To label newborn GCs, bromodeoxyuridine (BrdU) was injected daily for three consecutive days to C57BL/6J mice at different postnatal days starting at postnatal-0-day-old (P0) (Group1), P7 (Group2), or P35 (Group3). Mice in each group underwent pilocarpine-induced SE at P14. Six months later, to determine whether SE induces ectopic GCs, we plotted the distribution of postnatally born GCs which were immunohistochemically defined as BrdU- and the GC marker Prox1-colabeled cells. We also examined whether SE causes the granule cell layer (GCL) dispersion and/or the mossy fiber (MF) sprouting, other representative pathologies of TLE hippocampus. Only SE-experiencing mice in Group1 had significantly more neonatally born ectopic GCs compared with control mice. Neither control nor SE mice had dispersed GCL. All mice that underwent SE had sprouted MFs in CA3. We conclude that early-life SE disrupts a normal incorporation of GCs born pre-SE but not post-SE, inducing ectopic GCs in the adult hilus. Interestingly, the results also indicate that developmentally earlier born GCs are more responsive to early-life SE in terms of the emergence of ectopic GCs.

Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.

We examined behaviorally induced expression of brain-derived neurotrophic factor (BDNF) in area CA1 of the hippocampus. Sprague-Dawley rats were trained in a contextual fear conditioning (CFC) task, sacrificed 4h later, and their brains were processed for immunohistochemistry. We found distinctively high levels of BDNF immunoreactivity in a small number ( approximately 1%) of CA1 neurons in untrained animals. The number of these exceptional neurons, which are identified as BDNF(++) in this study, was increased by up to approximately 3% after CFC. This increase was blocked in the presence of a memory-impairing dose of a NMDA receptor antagonist (MK801 0.3 mg/kg, i.p.) given 30 min prior to training. The BDNF signal intensity in BDNF(++) neurons correlated with that of surrounding glutamic acid decarboxylase (GAD) 65. This correlation between GAD65 and BDNF signal intensities suggests that BDNF upregulation was associated with increased signaling via inhibitory GABAergic synapses that would lessen further intervening neuronal activity. Our observation that neurons which upregulate BDNF expression following a learning experience are rich in GAD65-enriched afferent synapses suggests that these neurons may have distinct roles in memory consolidation.

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.

We investigated the effect of beta-estradiol (E2) on synaptogenesis in the hippocampus using organotypic hippocampal slice cultures and subregional hippocampal neuron cultures. E2 increased the expression of PSD95, a postsynaptic marker, specifically in stratum lucidum of Cornu Ammonis 3 (CA3SL) in cultured hippocampal slices. E2 also increased the spine density at the proximal site of CA3 apical dendrites in CA3SL and PSD95 was clustered on these spine heads. The effects of E2 on the expression of PSD95 and the spine density disappeared when the dentate gyrus (DG) had been excised at 1 day in vitro (DIV). FM1-43 analysis of subregional hippocampal neuron cultures which were comprised of Ammon's horn neurons, DG neurons, or a mixture of these neurons, revealed that E2 increased the number of presynaptic sites in the cultures that contained DG neurons. K252a, a potent inhibitor of the high affinity receptor of brain-derived neurotrophic factor (BDNF), and function-blocking antibody to BDNF (BDNFAB) completely inhibited the effects of E2 in hippocampal slice cultures and subregional neuron cultures, whereas ICI182,780 (ICI), a strong antagonist of nuclear estrogen receptors (nERs), did not. Expression of BDNF in DG neurons was markedly higher than that in Ammon's horn neurons and E2 did not affect these expression levels. E2 significantly increased the BDNF release from DG neurons. KT5720, a specific inhibitor of 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), and Rp-adenosine 3', 5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMP), a non-hydrolyzable diastereoisomer and a potent inhibitor of PKA, completely suppressed the E2-induced increase in BDNF release, whereas ICI and U0126, a potent inhibitor of MAP kinase kinase (MEK), did not. These results suggest that E2 induces synaptogenesis between mossy fibers and CA3 neurons by enhancing BDNF release from DG granule cells in a nER-independent and PKA-dependent manner.

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.

Although amyloid beta-protein (A beta) has long been implicated in the pathogenesis of Alzheimer's disease, little is known about the mechanism by which A beta causes dementia. A beta leads to neuronal cell death in vivo and in vitro, but recent evidence suggests that the property of the amnesic characteristic of Alzheimer's disease can be explained by a malfunction of synapses rather than a loss of neurons. Here we show that prolonged treatment with A beta augments the glutamate clearance ability of cultured astrocytes and induces a dramatic decrease in glutamatergic synaptic activity of neurons cocultured with the astrocytes. Biotinylation assay revealed that the enhancement of glutamate uptake activity was associated with an increase in cell-surface expression of GLAST, a subtype of glial glutamate transporters, without apparent changes in the total amount of GLAST. This phenomenon was blocked efficiently by actin-disrupting agents. Thus, A beta-induced actin-dependent GLAST redistribution and relevant synaptic malfunction may be a cellular basis for the amnesia of Alzheimer's disease.

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-D-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.

Colchicine, a potent microtubule-depolymerizing agent, is well known to selectively kill dentate granule cells in the hippocampal formation in vivo. Using organotypic cultures of rat entorhino-hippocampal slices, we confirmed that in vitro exposure to 1 μM and 10 μM of colchicine reproduced a specific degeneration of the granule cells after 24 h. Similar results were obtained with other types of microtubule-disrupting agents, i.e., nocodazole, vinblastine, and Taxol. Interestingly, the actin-depolymerizing agents cytochalasin D and latrunculin A also elicited selective neurotoxicity in the dentate gyrus without affecting survival of hippocampal pyramidal cells. The selective pattern of degeneration was observable 24 h after a brief treatment with the toxins as short as 5 min, but this delayed neuronal death was unlikely to be a result of excitotoxicity because it was virtually unaffected by glutamate receptor antagonists, tetrodotoxin, or extracellular Ca 2+ -free conditions. The damaged tissues contained a large number of TUNEL-positive neurons and exhibited an increased level in caspase-3-like activity, suggesting that cytoskeleton disruption triggers an apoptosis-like process in dentate granule cells. Thus, this study may provide a basis for understanding the distinctive mechanism that supports granule cell survival.

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.

Behavioral and cognitive deficits are one of the most frequent sequelae of childhood epilepsy. Accumulating evidence indicates that epilepsy induces aberrant development of the mossy fibers in the hippocampus, the region that is commonly accepted to play a key role in learning and memory. We have therefore proposed that such abnormal maturation of the central nervous system may cause the adverse prognoses following epilepsy. Based on this hypothesis, using primary cultures of the dentate granule cells, we showed that the L-type Ca(2+) channel blocker nicardipine was neuroprotective against excessive mossy fiber synaptogenesis induced by prolonged depolarization that was assumed to mimic epileptiform conditions. Therefore, we evaluated the in vivo effect of nicardipine on aversive sequelae following epileptiform seizures. We found aberrant sprouting of the mossy fibers and poor performance of spatial and contextual tasks in the mice that had received treatment with pilocarpine at their early postnatal age. Repetitive administration of nicardipine prevented the mossy fiber sprouting and ameliorated the cognitive deterioration, although it did not show anticonvulsant actions against pilocarpine seizures. In the present study, we proposed two in vitro and in vivo models for evaluating epilepsy sequelae and noticed that L-type Ca(2+) channel blocker nicardipine was effective in both models. L-type Ca(2+) channel blocker may be a good candidate for a preventive for childhood epilepsy sequelae. Likewise, these useful systems will disclose additional candidates in future.

Docosahexaenoic acid (DHA), a 22-carbon fatty acid with six double bonds, is one of the major polyunsaturated fatty acids in fish oils or in the mammalian central nervous system and is believed to be essential for neuronal plasticity and development. In the present study, we evaluated the effect of DHA on hippocampal neurotransmissions using anesthetized rats. Field excitatory postsynaptic potential (fEPSP) evoked by stimulation of the Schaffer collaterals was recorded from the CA1 stratum radiatum. Following intracerebroventricular injection of DHA 25 nmol, the fEPSP slope decreased gradually in 30 min and was eventually suppressed by about 30%. On the other hand, when fEPSP was evoked by stimulation of the perforant path was recorded in the molecular layer of the dentate gyrus, an increase in fEPSP slope occurred over a similar time course after DHA injection. These phenomena were independent of N -methyl- d -aspartate receptor activity. Linoleic acid, one of polyunsaturated fatty acids, was virtually ineffective. Furthermore, we investigated the effect of DHA on hippocampal synaptic plasticity. Although DHA did not alter the profile of paired-pulse facilitation, it inhibited the induction of long-term potentiation in the CA1 area but not in the dentate gyrus. Thus, DHA exerts regionally different effects on hippocampal neurotransmission and may be a good tool for clarifying physiological functions of the hippocampus.

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.

Murine neuroblastoma cell line Neuro-2A cells and rat brain astrocytes showed a dose-dependent increase in intracellular Ca2+ in response to bradykinin, when assessed by a single cell image analyzing system. The Ca2+ increase in Neuro-2A cells by bradykinin was also examined by a suspension fluorescent assay using fura-2 loading. The Ca2+ increase in both cases was suppressed by a bradykinin B2 receptor antagonist, Hoe 140, but not by a B1 receptor antagonist, des-Arg-Hoe 140, suggesting that the effect occurred via specific B2 receptor activation. RT-PCR for bradykinin B2 receptor mRNA showed that both Neuro-2A cells and the astrocytes expressed B2 receptor mRNA. Binding of [3H]bradykinin to Neuro-2A cells was assessed, and a specific binding constant of 0.75 nM was determined. Furthermore, the increase in [Ca2+]i by bradykinin could be caused by a release of Ca2+ from storage sites in the endoplasmic reticulum, since thapsigargin and U-73122 attenuated the effect of bradykinin in Neuro-2A as well as in astrocytes. These results indicate that both astrocytes and neuroblastoma Neuro-2A cells stimulated by bradykinin could express a bradykinin B2 receptor-mediated intracellular Ca2+ increase leading to signal transduction.

It has previously been shown that fatty liver was easily induced in suncus by starvation and that it was easily reversed by refeeding. It was also previously reported that a defect in the assembling process of apolipoprotein (apo) B-containing lipoprotein (very low density lipoprotein, VLDL) may be one of the reasons for induction of fatty liver by starvation in suncus. Microsomal triglyceride transfer protein (MTP) is recently proposed to be an important protein during the assembly of plasma VLDL. In this study, the hepatic and intestinal MTP activities in suncus and the effect of nutritional state on them were investigated. The results indicate that the MTP activity in suncus liver was significantly lower than that in rat liver. On the other hand, the basal MTP activity in suncus intestinal mucosa was comparable to that of rat. The hepatic activity was not affected by starvation but was enhanced by refeeding significantly in suncus. This evidence suggests that the low activity of MTP is specific to the liver in suncus and may play a role in induction of fatty liver observed in starved suncus because refeeding led to enhancement of the activity as well as reversal of fatty liver.

Effects of proteases and protease inhibitors on generation of long-term potentiation (LTP) were investigated in the CA1 and dentate regions of rat hippocampus. Plasmin, a serine protease, and its precursor plasminogen significantly enhanced short-term potentiation (STP) induced by a weak tetanic stimulation, without affecting basal responses. The STP-enhancing effect of plasmin disappeared by concomitant perfusion of α2-antiplasmin, an endogenous plasmin inhibitor. Other proteases, such as thrombin, trypsin and cathepsin B, did not affect STP. On the other hand, α2-antiplasmin and leupeptin significantly attenuated LTP induced by a strong tetanus though plasminogen or plasmin itself did not influence LTP. Furthermore, plasminogen and plasmin did not affect NMDA receptor-mediated synaptic responses in the absence of extracellular Mg2+. These results suggest that endogenous plasmin is involved in the mechanism of LTP in CA1 and dentate regions of rat hippocampus and that the STP-enhancing effect of plasmin is independent of NMDA receptors.

This paper is the first to describe aspects of the mechanics of retching in the insectivore Suncus murinus (house musk shrew) and in an animal of such a small size (approximately 50 g). In anaesthetised animals using the novel stimulus of mechanical stimulation of the upper gastrointestinal tract as the provocative stimulus the frequency of retching was found to be about 4 retches/s, a much higher frequency than in other species (dog, cat, ferret). These studies show that quantification of retching in Suncus cannot be undertaken using direct observation. The temporal pattern of the emetic response was characterised in conscious Suncus using motion (1 Hz, 5 min) and nicotine (20 mg/kg s.c.). The ultrapotent capsaicin analogue resiniferatoxin (100 micrograms/kg s.c.) was discovered to be highly emetic and comparative studies showed that nicotine and resiniferatoxin induced the most intense responses with episodes (retches and a vomit) occurring every 10-15 s. The retching response to mechanical stimulation in the anaesthetised Suncus was not blocked by a 5-HT3 receptor antagonist (granisetron, 1-5 mg/kg s.c.), a tachykinin NK1 receptor antagonist (CP-99,994 20 mg/kg s.c. dihydrochloride salt (9+) -(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine) or morphine (2 mg/kg s.c.) but was blocked by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT 100 micrograms/kg s.c.). Suncus appears to be a suitable animal in which to study the pharmacology of the emetic response to mechanical stimulation of the gut. The results are discussed in the light of studies of the pharmacology of emesis in other species.

Basic fibroblast growth factor (bFGF) is one of the effective growth factors that protect neurons against excitotoxic/ischemic injury and promote neuronal survival. In the present study, we examined the acute modulative effect of bFGF on synaptic transmission by monitoring spontaneous intracellular Ca 2+ ([Ca 2+ ]i]) oscillation, the amplitudes of which reflect excitatory and inhibitory inputs. The hippocampal cells from embryonic day 18 rats were cultured for 11–14 days, and changes in [Ca 2+ ]i of single neurons were measured by a microfluometrical technique with fura-2. The amplitude of spontaneous oscillation was decreased by 10 ng/ml bFGF, but not by nerve growth factor (10–1000 ng/ml). Acidic FGF (1000 ng/ml) had a weaker depressant effect. The effect of bFGF was counteracted by suramin. bFGF did not affect the increase in [Ca 2+ ]i evoked by glutamate agonists, NMDA or kainate, indicating that glutamate receptors are not involved in the mechanism. This is supported by similar results that kainate-evoked current was not affected by bFGF. On the other hand, bicuculline masked the effect of bFGF on the Ca 2+ oscillation. But GABA-evoked current was slightly decreased by bFGF. These results suggest the possible role of bFGF in modulating GABAergic rather than glutamatergic neurotransmission.

Neurons can form a synaptic network in culture and show spontaneous oscillation of intracellular Ca2+ concentration ([Ca2+]i). In the present study, spontaneous oscillation of [Ca2+]i was characterized in cultured hippocampal neurons. The oscillation was blocked completely by tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and nicardipine, while DL-2-amino-5-phosphonovaleric acid (APV) showed only a partial depression of the increase in [Ca2+]i. These results suggest that the oscillation in [Ca2+]i is mainly mediated by non-N-methyl-D-aspartate (NMDA) type glutamatergic transmission. The oscillation of [Ca2+]i may be a good model for analyzing glutamatergic transmission and synapse formation.

The effects of serotonin (5-hydroxytryptamine: 5-HT) and 5-HT1A agonist on voltage-gated calcium channels (VGCCs) in acutely isolated ventromedial hypothalamic (VMH) neuronal cells were studied using whole-cell configuration of the patch-clamp technique. 5-HT at 10 microM inhibited inward calcium current reversibly in 80% of cells. This inhibition was specific to N-type current. Because pindolol blocked the effect of 5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) mimicked the effect of 5-HT, the inhibitory effect of 5-HT appeared to be mediated via the 5-HT1A receptor. In the fura-2 fluorometry method, 8-OH-DPAT attenuated the [Ca2+]i increase induced by the depolarization stimulus of 50 mM K+. These results indicate that 5-HT suppresses Ca2+ entry through N-type channels in the VMH neurons via the 5-HT1A receptor and that the stimulating effect of 8-OH-DPAT on feeding behavior may be mediated by the blocking of Ca2+ entry through N-type channels.

This paper reports that vasopressin is emetogenic in the house musk shrew Suncus murinus. Either intravenous or intracerebroventricular administration of vasopressin caused vomiting within a few minutes. The ED50 of intravenous vasopressin was as high as 4.67 microg/kg, whereas intracerebroventricularly injected vasopressin was effective at a low dose of 20 ng/brain. The emetogenic target of vasopressin may therefore be present in the central nervous system. We propose the Suncus as a useful animal for investigation of vasopressin-mediated emesis, including motion sickness.

The participation of free radicals in cisplatin-induced emesis was investigated in the house musk shrew, Suncus murinus. Thiobarbituric acid (TBA) values, which indicate the degree of lipid peroxidation, in brain, liver and small intestine were increased significantly 60 min after the treatment with cisplatin (20 mg/kg, i.p.). Moreover, cisplatin (20 mg/kg, i.p.)-induced emesis was prevented by intraperitoneal injection of N-(2-mercaptopropionyl)glycine (MPG), a radical scavenging agent, with ID50 value of 130 mg/kg. However, MPG did not block the emesis induced by copper sulfate (40 mg/kg, p.o.), veratrine (0.5 mg/kg, s.c.) or serotonin (10 mg/kg, i.p.). We also investigated the effects of superoxide dismutase conjugated to polyethylene glycol and catalase, but the number of vomiting episodes and latency did not change significantly when these agents were intraperitoneally injected 30 min prior to or 20 min after the administration of cisplatin. MPG did not affect the antitumor effect of cisplatin tested in vitro. These results suggest that free radicals mediate emesis caused by cisplatin and that radical scavengers may become a new class of prophylactic drug against cancer-chemotherapeutic drug-induced emesis.

Effects of aniracetam on whole-cell calcium currents were studied in acutely isolated neuronal cells from postnatal rat ventromedial hypothalamus. There were three types of inward calcium currents, one low-threshold transient current and two high-threshold sustained currents. The nicardipine sensitive L-type current was activated at -20 mV or more depolarized potentials, and the omega-conotoxin sensitive N-type current was recorded at more positive potentials than the L-type. Aniracetam inhibited the N-type current in a dose-dependent manner without affecting the other two types of calcium currents. The effect appeared soon after the addition and lasted for several minutes during washing. Since the N-type current is thought to regulate the release of transmitters, the inhibitory effect may contribute to the nootropic property of aniracetam by modifying the neurotransmission.

It is difficult to comprehend the entorhino-hippocampal information processing using acute transverse hippocampal slice, because the dorsally inclined connections of the entorhino-hippocampal projections can be damaged easily. Therefore, we investigated the spatial-temporal propagation in organotypic cultures of the hippocampus attaching to the entorhinal cortex using a real-time optical recording system with a voltage-sensitive dye and suitability as an in vitro model. Real-time imaging demonstrated that the stimulation of the perforant pathway induced excitatory propagation in trisynaptic pathway of the hippocampus and sequentially in the layer V from the medial to the lateral entorhinal cortex. The horizontal propagation from the lateral to the medial site was also seen after the stimulation of the lateral entorhinal cortex. The analysis of the entorhino-hippocampal organotypic culture would contribute to understanding of the mechanism of learning and memory.

The prophylactic effect of serotonin uptake inhibitors, imipramine and fluoxetine, against motion sickness was investigated in Suncus murinus. Imipramine (s.c.) and fluoxetine (i.p.) inhibited motion-induced emesis dose dependently with ID50 values of 1.7 and 26 mg/kg, respectively. The results suggest that increasing the concentration of serotonin in the synaptic cleft can prevent motion-induced emesis and that serotonin uptake inhibitors are effective as anti-motion sickness drugs.

The effects of ferric chloride and deferoxamine, an iron chelator, on cisplatin-induced emesis were studied in Suncus murinus . Pretreatment with ferric chloride (FeCl 3 , 16–64 mg/kg) increased the number of 20 mg/kg cisplatin-induced vomiting episodes. Deferoxamine (256 mg/kg) significantly reduced the number of vomiting episodes and prolonged the latency. These results further support the involvement of free radicals in the generation of cisplatin-induced emesis.