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Cerebellar Purkinje cells are key elements in motor learning and motor coordination, and therefore, it is important to clarify the mechanisms by which Purkinje cells integrate information and control cerebellar function. Gene transfer into neurons, followed by the assessment of the effects on neural function, is an effective approach for examining gene function. However, this method has not been used fully in the study of the cerebellum because adenovirus vectors, the vectors most commonly used for in vivo gene transfer, have very low affinity for Purkinje cells. In this study, we used a human immunodeficiency virus (HIV)-derived lentiviral vector and examined the transduction profile of the vector in the cerebellum. A lentiviral vector carrying the GFP gene was injected into the cerebellar cortex. Seven days after the injection, Purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. GFP was also expressed, though less efficiently, in other cortical interneurons and Bergmann glias. In contrast to reported findings with other viral vectors, no transduced cells were observed outside of the cerebellar cortex. Thus, when HIV-derived lentiviral vectors were injected into the cerebellar cortex, transduction was limited to the cells in the cerebellar cortex, with the highest tropism for Purkinje cells. These results suggest that HIV-derived lentiviral vectors are useful for the study of gene function in Purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect Purkinje cells.

The present study examined the effects of the proinflammatory cytokine interleukin-18 (IL-18) on mouse hippocampal synaptic transmission. IL-18 (100 ng/ml) significantly increased amplitude and frequency of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated miniature excitatory postsynaptic currents (AMPA-mEPSCs), that are monitored from CA1 pyramidal neurons of mouse hippocampal slices. IL-18 (100 ng/ml) enhanced slope of basal field excitatory postsynaptic potentials (fEPSPs) that are recorded from the CA1 region of mouse hippocampal slices. There was no significant difference in the expression of Schaffer collateral/CA1 long-term potentiation (LTP) between in the presence and absence of IL-18, although IL-18 tended to inhibit saturation levels of the potentiation induced by tetanic stimulation in a dose-dependent manner at concentrations ranged from 10 ng/ml to 1 microg/ml. Paired-pulse facilitation in the presence of IL-18 (100 ng/ml) was not influenced after tetanic stimulation, while that in the absence of IL-18 was depressed. The results of the present study, thus, suggest that IL-18 stimulates synaptically released glutamate and enhances postsynaptic AMPA receptor responses in CA1 pyramidal neurons of mouse hippocampal slices, thereby facilitating basal hippocampal synaptic transmission without affecting the LTP., (Copyright 2004 Elsevier B.V.)

The present study aimed at understanding the effects of arachidonic acid peroxides on neuronal cell death using the mouse neuroblastoma cell line, Neuro-2A cells. Arachidonic acid peroxides were produced by ultraviolet (UV) radiation. UV-radiated arachidonic acid significantly reduced Neuro-2A cell viability at concentrations of more than 0.1 muM, with being more potential than non-radiated arachidonic acid. Nuclei of Neuro-2A cells killed with UV-radiated arachidonic acid were reactive to Hoechst 33342, a marker of apoptosis, and the effect was much greater than that achieved with non-radiated arachidonic acid. UV-radiated arachidonic acid persistently increased intracellular Ca(2+) concentrations and dissipated mitochondrial membrane potential in Neuro-2A cells. UV-radiated arachidonic acid-induced Neuro-2A cell death, whereas it was not affected by a pancaspase inhibitor or a caspase-3 inhibitor, was significantly inhibited by an inhibitor of caspase-1, -8, or -9. The results of the present study suggest that arachidonic acid peroxides induce apoptotic neuronal cell death in association with intracellular Ca(2+) rise and mitochondrial damage, in part via a caspase-dependent pathway regardless of caspase-3.

Pipecolic acid, a lysine metabolite, is thought to be a factor responsible for hepatic encephalopathy; however, the underlying mechanism is far from understood. Twenty minutes treatment with D-, L-, and DL-pipecolic acid at concentrations ranging from 1 to 100 microM, except for 1 microM L-pipecolic acid, had no inhibitory effect on excitatory postsynaptic responses in the dentate gyrus of rat hippocampal slices. In a whole-cell voltage-clamp configuration, DL-pipecolic acid (10 and 100 microM) did not affect voltage-sensitive Na(+) channel currents and K(+) channel currents, but it potentiated voltage-sensitive Ca(2+) channel currents, but to a lesser extent, in cultured rat cortical neurons and Neuro-2A cells, a mouse neuroblastoma cell line. Notably, 72-h treatment with D-, L-, and DL-pipecolic acid reduced Neuro-2A cell viability in a dose-dependent manner at concentrations ranging from 1 to 100 microM in a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, in parallel with reactions to propidium iodide, a marker of cell death, and Hoechst 33,342, a marker of apoptosis in a fluorescent microscopic study, with DL-pipecolic acid being the most potent. The results of the present study suggest that pipecolic acid could cause hepatic encephalopathy by inducing neuronal cell death, perhaps apoptosis, rather than by depressing neurotransmissions.

In a whole-cell patch-clamp configuration, currents through N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels were monitored in cultured rat hippocampal neurons, and those currents were depressed to 25 and 28% of basal levels, respectively, by 3-min treatment with tunicamycin (10 microM), an inhibitor of protein N-glycosylation. Tunicamycin (10 microM) reduced amplitude of population spikes elicited in the dentate gyrus of rat hippocampal slices, reaching 78% of basal levels 60 min after the beginning of treatment, and long-term potentiation (LTP) of the perforant path was never induced in the presence of tunicamycin. Tunicamycin, thus, appears to serve as a modulator for NMDA and AMPA receptors, regardless of N-glycosylation, thereby inhibiting neurotransmission and LTP in the dentate gyrus.

The present study investigated the effects of (-)-epigallocatechin gallate (EGCG), which is the major component of polyphenol in green tea, on nitric oxide (NO) stress-induced neuronal damage, by monitoring NO mobilizations in the intact rat hippocampus and assaying the viability of cultured rat hippocampal neurons. A 10-min ischemia increased NO (NO(3)(-)/NO(2)(-)) concentrations in the intact rat hippocampus, while EGCG (50 mg/kg i.p.) inhibited the increase by 77% without affecting hippocampal blood flow. The NO donor, sodium nitroprusside (SNP; 50 microM), produced NO (NO(3)(-)/NO(2)(-)), while EGCG inhibited it in a dose-dependent manner at concentrations ranging from 50 to 200 microM. Treatment with SNP (100 microM) reduced the viability of cultured rat hippocampal neurons to 22% of control levels, while EGCG caused it to recover to 51% for 10 microM, 73% for 20 microM, and 70% for 50 microM. Taken together, it appears that EGCG could protect against ischemic neuronal damage by deoxidizing peroxynitrate/peroxynitrite, which is converted to NO radical or hydroxy radical., (Copyright 2002 Elsevier Science B.V.)

The present study was designed to assess whether the facilitatory action of nefiracetam, a pyrrolidone derivative, on hippocampal postsynaptic responses is dependent upon N-methyl-D-aspartate (NMDA) receptors or not, by monitoring population spikes (PSs) in the dentate gyrus of hippocampal slices from mice lacking the NMDA receptor epsilon1 subunit. Nefiracetam (1 microM) induced a sustained facilitation of postsynaptic responses in the dentate gyrus of hippocampal slices from wild-type mice. The facilitation occluded the potentiation induced by high-frequency stimulation at the perforant path, and vice versa, suggesting a common mechanism between them. The perforant path long-term potentiation (LTP) was not induced in epsilon1 subunit knock-out mice, but nefiracetam (1 microM) persistently potentiated PS amplitude, reaching 280% of basal levels 50 min after 10-min treatment, similar to the potentiation achieved with wild-type mice. The results of the present study, thus, suggest that nefiracetam exerts its facilitatory action on hippocampal postsynaptic responses in an NMDA receptor-independent manner.

The present study was designed to assess the effects of adenosine triphosphate (ATP) on hippocampal neurotransmissions under the normal and hypoxic/hypoglycemic conditions. ATP reversely depressed population spikes (PSs), which were monitored in the dentate gyrus of guinea pig hippocampal slices, in a dose-dependent manner at concentrations ranged from 0.1 micro M to 1 mM. A similar depression was obtained with the P(2) receptor agonist, alpha,beta-methylene ATP (alpha,beta-MeATP), and the effect was inhibited by the P(2) receptor antagonists, suramin and PPADS. The inhibitory action of ATP or alpha,beta-MeATP was inhibited by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonist, bicuculline, but it was not affected by theophylline, a broad inhibitor of adenosine (P(1)) receptors, tetraethylammonium, a broad inhibitor of K(+) channels, or ecto-protein kinase inhibitors. ATP or alpha,beta-MeATP enhanced GABA release from guinea pig hippocampal slices, that was inhibited by deleting extracellular Ca(2+) or in the presence of tetrodotoxin, while ATP had no effect on GABA release from cultured rat hippocampal astrocytes or postsynaptic GABA-gated channel currents in cultured rat hippocampal neurons. Twenty-minutes deprivation of glucose and oxygen from extracellular solution abolished PSs, the amplitude recovering to about 30% of basal levels 50 min after returning to normal conditions. ATP or alpha,beta-MeATP accelerated the recovery after hypoxic/hypoglycemic insult (approximately 80% of basal levels). Adenosine diphosphate and adenosine monophosphate accelerated the recovery, but to a much lesser extent, and adenosine had no effect. The results of the present study thus suggest that ATP inhibits neuronal activity by enhancing neuronal GABA release via a P(2) receptor, perhaps a P2X receptor, thereby protecting against hypoxic/hypoglycemic perturbation of hippocampal neurotransmission.

Nefiracetam, a nootropic agent, enhanced the slope of field excitatory postsynaptic potentials in the CA1 region of rat hippocampal slices to about 170% of basal levels, being evident still at 4-h washing-out of the drug. A similar sustained enhancement (>/=16 h after i.m. injection with nefiracetam) was observed in the population spikes recorded from the granular cell layer of the intact mouse hippocampus. Saturation of the enhancement in the synaptic strength occluded potentiation obtained with long-term potentiation (LTP) induced by high-frequency (tetanic) stimulation, and vice versa. Interestingly, the facilitatory action of nefiracetam was blocked by either the nicotinic acetylcholine (ACh) receptor antagonists, alpha-bungarotoxin and mecamylamine, or the selective protein kinase C (PKC) inhibitor, GF109203X, but in contrast, it was not affected by D-2-amino-5-phosphonovaleric acid (APV), a selective N-methyl-D-aspartate (NMDA) receptor antagonist. The results of the present study suggest that nefiracetam, whereas the action is independent of NMDA receptors, induces an 'LTP-like' facilitation of hippocampal synaptic transmission as a consequence of modulation of nicotinic ACh receptors and PKC. This may represent a likely mechanism underlying the cognition-enhancing actions of nefiracetam., (Copyright 1999 Elsevier Science B.V.)

The effects of Ca2+ /phospholipid dependent (PKC) phosphorylation on the current amplitudes of alpha 1 and alpha 2 glycine receptors expressed in Xenopus oocytes were examined by whole cell voltage clamp recording. In studies using phorbol esters, PKC phosphorylation has been shown to reduce glycine-induced currents. Endogenous PKC activation by pretreatment with serum, however, enhanced the currents to around 140% in both alpha 1 and alpha 2 glycine receptors. This effect was completely blocked by a specific PKC inhibitor, GF109203X. Instead, treatment with a potent PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) revealed a decrease in glycine-gated channel currents. Thus, the present results demonstrate that glycine receptor phosphorylation mediated by endogenous pathway of PKC activation potentiates glycine-induced currents and phorbol esters may have a direct action on glycine receptor channels independent of PKC activation.

The frequency of nucleolar organizer regions (NORs) in each glioma tissue and the relation between the expression of glial fibrillary acidic protein (GFAP) and the frequency of NORs was investigated. The number of Ag-NORs per cell for glioblastoma multiforme was significantly higher than that for anaplastic astrocytoma (P less than 0.05) and that for astrocytoma (P less than 0.01). The number of Ag-NORs per cell for GFAP-positive cells was significantly lower than that for GFAP-negative cells in each histopathological grade (P less than 0.01). Moreover, the linear relationship was demonstrated between the Ag-NORs numbers of GFAP-negative cells and bromodeoxyuridine (BUdR) labeling indices. From these results, it is concluded that many GFAP-positive glioma cells may have low growth potential in glioma tissue and GFAP-negative cells may have a close relation to cell proliferation. The combination of immunohistochemical and silver colloid staining is a useful method for investigating the biological characteristics of brain tumors.

The effects of excitatory amino acids such as glutamate (Glu) and aspartate (Asp), and their receptor agonists, kainate (Ka), N-methyl-D-aspartate (NMDA), and quisqualate (Quis) on the neuronal activity and the oxygen consumption were investigated using hippocampal slices of the guinea pig. Bath application of these excitants elevated the amplitude of the postsynaptic field potential (PSP) to approximately 120% of the original level at low concentrations, although effective doses varied for the different excitants (Ka greater than NMDA greater than Quis greater than Glu greater than Asp). At concentrations over each effective dose the PSP was diminished and subsequently abolished. The application of Ka (1 x 10(-8) to 1 x 10(-6) M), NMDA (1 x 10(-8) to 1 x 10(-4) M), Quis (1 x 10(-7) to 1 x 10(-4) M), Glu (1 x 10(-5) to 5 x 10(-4) M), and Asp (1 x 10(-5) to 5 x 10(-3) M) enhanced the oxygen consumption dose-dependently, to a maximum of 120-146% of the resting level (8.43 mumol/g protein/min). It was notable that the initial increase in the oxygen consumption was associated with neuronal excitation and the doses of the excitants producing maximal oxygen consumption was in good agreement with those demonstrating disappearance of the PSP, probably because of massive depolarization of the neurones. The increase of the oxygen consumption and the neuronal activity induced by the excitants were specifically inhibited by their antagonists, such as glutamic acid diethylester (GDEE), DL-2-amino-5-phosphonovaleric acid (APV), and Joro spider toxin (JSTX). The present results strongly suggest that the enhancement of oxygen consumption due to the excitatory amino acids and agonists must reflect the neuronal activation induced by them.