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Parkinson disease (PD) is a neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the substantia nigra, and oxidative stress is thought to contribute to this pathogenesis. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, which induces the production of antioxidant enzymes, is thereby a potential target for therapeutics to reduce neurodegeneration in PD. Previously, we identified TPNA10168 from a chemical library as an activator of the Nrf2-ARE pathway, and the present study examined the effects of TPNA10168 on an in vivo PD model. Subcutaneous administration of TPNA10168 was associated with inhibited dopaminergic neuronal loss and behavioral impairment in 6-hydroxydopamine-induced PD model mice. Heme oxygenase-1 (HO-1) is an antioxidant enzyme expressed downstream of the Nrf2-ARE signaling pathway, and we observed that HO-1 protein levels were upregulated by TPNA10168 in the mouse brain. These results suggest that TPNA10168 inhibits dopaminergic neuronal death in PD model mice, and that upregulation of HO-1 might participate in this effect.

Excitatory neuronal activity produces beneficial influences on neuronal survival under several circumstances. We show that cultivation of rat midbrain slices in the presence of elevated extracellular Mg(2+) resulted in a marked decrease in the number of dopaminergic neurons. The effect was prominent when Mg(2+) was added to the medium during the first week of cultivation. Chronic treatment with antagonists of N-methyl-D-aspartate receptors such as 2-amino-5-phosphonovaleric acid, MK-801 and ifenprodil also resulted in a marked loss of dopaminergic neurons, whereas nicotinic receptor antagonists showed no effect. The effect of MK-801 was abolished by chronic depolarization by elevated extracellular K(+), or by application of forskolin or dibutyryl cyclic AMP. Thus, tonic activation of N-methyl-D-aspartate receptors driven by neuronal activity may play an important role in the maintenance of dopaminergic neurons.

Inhibition of histone deacetylases (HDACs) is a promising approach for the treatment of mood disorders. However, the effects of HDAC inhibition on the serotonin (5-HT) system, a common target for psychiatric disorders, are poorly understood. Here, we show that a broad-spectrum HDAC inhibitor, trichostatin A (TSA), enhances the function of 5-HT neurons in organotypic raphe slice cultures. Sustained treatment with TSA (1μM) for 2 or 4 days significantly increased the 5-HT tissue content and tryptophan hydroxylase 2 (TPH2) expression, which were accompanied by hyper-acetylation of histone H3 in the promoter region of the TPH2 gene. TSA treatment for 4 days increased the extracellular 5-HT level, which was significantly suppressed in the presence of the selective AMPA receptor (AMPAR) antagonist NBQX. Moreover, the expression of both the AMPAR subunit GluA2 and Ca(2+)/calmodulin-dependent kinase II α (CaMKIIα) mRNAs were significantly increased by TSA treatment. Co-treatment with the CaMKII inhibitors KN-62 and KN-93 prevented the TSA-induced increase in 5-HT release, but had no effect on the increases in 5-HT tissue content. These results suggest that inhibition of HDACs increases 5-HT synthesis and release by epigenetic mechanisms, and that 5-HT release is mediated by the enhancement of AMPAR-mediated excitatory inputs and CaMKII signaling.

Transverse slices of the spinal cord with dorsal root attached were prepared from adult rats and used to record the response of the spinal substantia gelatinosa neurons evoked by repetitive stimulation of the dorsal root with a whole cell patch clamp method. The repetitive stimulation of 10 trains (20 pulses at 100 Hz/train) with an intensity necessary to activate C-fibers, but not A-fibers alone, evoked slow depolarization (SD) during it and the SD disappeared within 1 min after the termination of it. The SD was completely inhibited by 500 nM of tetrodotoxin (TTX), but not significantly changed by 50 nM of TTX, nor 20 microM CNQX + 50 microM AP5, nor 1 microM CP-99994. DAMGO inhibited the SD in a concentration dependent manner (10 nM-1 microM), but 1 microM DPDPE and 1 microM U-50488H did not. The inhibitory effect of DAMGO (1 microM) was reversed by naloxone (1 microM). These results suggest that the SD of substantia gelatinosa neurons evoked by repetitive stimulation of C-fibers of the dorsal root is an event relevant to nociception in the spinal dorsal horn.

Xenopus oocytes were used for investigating the cAMP-dependent modulation of N- and Q-type Ca2+ channels. Treatments to increase intracellular cAMP concentration with forskolin (FK) and 3-isobutyl-1-methylxanthine (IBMX) markedly potentiated Q-type Ca2+ channel current in oocytes coexpressing alpha 1A and beta subunits, and the enhancement was reversed by protein kinase A inhibitors. Moderate enhancement was observed by FK+IBMX in N-type channel current, of which potentiation was equivalent to that of endogenous Ca2+ channel current being activated by exogenously-expressed beta subunits. No potentiation was observed in the oocyte-native Ca2+ channel current. These results suggest that Q-type Ca2+ channels are more susceptible to the protein kinase A-mediated facilitation than N-type channels. A significant role of Ca2+ channel beta subunits for the cAMP-dependent positive modulation was also suggested.

To estimate the functional role of endogenous somatostatin in the production of long-term potentiation (LTP) in mossy fiber-CA3 system, an influence of depletion of somatostatin on the magnitude of it was examined in guinea-pig hippocampal slices. Administration of cysteamine (200 mg/kg, s.c.), a depletor of somatostatin, to guinea-pigs 13 h prior to preparing slices resulted in a significant decrease in the magnitude of LTP of population spikes in mossy fiber-CA3 system, being associated with a significant depletion of the content of somatostatin in the hippocampus. Furthermore, bath-applied somatostatin (1–14) at a concentration, at which the substance did not influence LTP in slices prepared from saline-treated animals, significantly augmented LTP in slices from cysteamine-treated animals. Cyclo-somatostatin (0.32 and 3.2 μM), a putative antagonist of somatostatin receptors, failed to affect the magnitude of LTP of mossy fiber-CA3 system when applied alone; however, the combined application of cyclo-somatostatin with somatostatin (0.32 μM) significantly inhibited the augmenting action of somatostatin on LTP. From these observations, it is suggested that endogenous somatostatin plays a facilitatory role in the production of LTP in mossy fiber-CA3 system in guinea-pig hippocampus.

Dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA) have been demonstrated to be the inducers of morphological differentiation in SH-SY5Y cells, a human catecholaminergic neuroblastoma cell line. However, it remains unclear whether morphologically differentiated SH-SY5Y cells by these compounds acquire catecholaminergic properties. We focused on the alteration of tyrosine hydroxylase (TH) expression and intracellular content of noradrenaline (NA) as the indicators of functional differentiation. Three days treatment with dbcAMP (1mM) and RA (10microM) induced morphological changes and an increase of TH-positive cells using immunocytochemical analysis in SH-SY5Y cells. The percentage of TH-expressing cells in dbcAMP (1mM) treatment was larger than that in RA (10microM) treatment. In addition, dbcAMP increased intracellular NA content, whereas RA did not. The dbcAMP-induced increase in TH-expressing cells is partially inhibited by KT5720, a protein kinase A (PKA) inhibitor. We also investigated the effect of butyrate on SH-SY5Y cells, because dbcAMP is enzymatically degraded by intracellular esterase, thereby resulting in the formation of butyrate. Butyrate induced the increase of NA content at lower concentrations than dbcAMP, although the increase in TH-expressing cells by butyrate was smaller than that by dbcAMP. The dbcAMP (1mM)- and butyrate (0.3mM)-induced increase in NA content was completely suppressed by alpha-methyl-p-tyrosine (1mM), an inhibitor of TH. These results suggest that dbcAMP induces differentiation into the noradrenergic phenotype through both PKA activation and butyrate.

3,4-Methylenedioxymethamphetamine (MDMA) selectively releases serotonin (5-HT) from neurons after uptake by the serotonin transporter (SERT) and causes psychostimulant effects accompanied by hyperthermia. Since rapid increases in CNS lactate levels are suggested in response to MDMA, we investigated the effects of the physiologically-relevant increase in H(+) concentration on the two conducting states of SERT expressed in Xenopus oocytes. Perfusion with SERT substrates at pH 7.4 dose-dependently evoked a transport-associated inward current response with the following rank order of potency: 5-HT>MDMA>dopamine>>norepinephrine. In the absence of transport substrates, a step hyperpolarization pulse activated a transient Na(+) leak current, which was inhibited by SERT substrates with the same rank, as well as by a SERT inhibitor, citalopram. At pH 6.4, the K(m) values of 5-HT and dopamine for the transport-associated current were not altered, while that of MDMA was three-fold lower. In contrast, the K(i) values of all these substrates for transient leak current were unchanged at pH 6.4, suggesting that the affinities to SERT binding sites are not influenced. These results suggest that the effect of MDMA on SERT is enhanced by acidic conditions.

The ataxic groggy rat (strain name; GRY) is an autosomal recessive neurological mutant found in a closed colony of Slc:Wistar rats. Recent genetic analysis has identified the missense (M251K) mutation in the alpha(1) subunit of the Ca(V)2.1 (P/Q-type) voltage-dependent Ca(2+) channel gene (Cacna1a) of GRY rat. In this study, we found that high-voltage-activated (HVA) Ca(2+) channel currents in acutely dissociated Purkinje cells of GRY rats showed increased (not decreased) current density and depolarizing shift of the activation and inactivation curves compared with those of normal Wistar rats. In contrast low-voltage-activated (LVA) Ca(2+) channel currents of GRY rats showed no significant changes. These results suggest that functional alteration of Ca(2+) channel currents in cerebellar Purkinje cells of GRY rats is attributed to the change of HVA Ca(2+) channel currents, and that increased HVA Ca(2+) channel function underlies the cerebellar dysfunction and ataxic phenotype of GRY rats.

We previously reported that slow depolarization of substantia gelatinosa neurons is evoked by repetitive stimulation of C-fibers of dorsal root in adult rat spinal cord transverse slices with the dorsal root attached, which was considered to be an event relevant to spinal nociception. In the present study, we investigated the effects of adenosine 5′-triphosphate (ATP) and its analogs on the slow depolarization. ATP (10–100 μM) significantly inhibited the amplitude of slow depolarization in a concentration-dependent manner. The inhibitory effect of ATP was not reversed by suramin, an antagonist for some P2-purinoceptors, and was mimicked by a P2Y selective agonist uridine 5′-triphosphate, but not a P2X selective agonist α,β-methylene ATP. These results suggest that ATP inhibits the slow depolarization of substantia gelatinosa neurons relevant to nociceptive transmission in the spinal dorsal horn, via suramin-insensitive P2Y purinoceptors.

To elucidate whether rat transient receptor potential (TRP-R), a rat TRP4 homolog, functions as a store-operated Ca 2+ channel (SOC), we have measured the Ca 2+ entry after thapsigargin treatment in Xenopus oocytes injected with mRNA for TRP-R. While non-injected oocytes elicited an SOC response, significantly larger responses were observed in the oocytes expressing TRP-R. The oocyte-native SOC response was inhibited by injection of antisense oligodeoxyribonucleotide for mammalian TRP1. When Ca 2+ concentration–SOC response curve was examined, the EC 50 value was much smaller in oocytes expressing TRP-R than that of non-injected oocytes. These results suggest that TRP-R functions as SOC having higher sensitivity to external Ca 2+ than amphibian TRP1 channel.

We had previously established a patch sensor system for detecting overflow of excitatory amino acids (EAA) from hippocampal slices induced by a single electric stimulus. We performed simultaneous recordings of patch sensor current and whole-cell current in rat hippocampal slices. Tetanus of mossy fibers produced long-term potentiation (LTP) of excitatory postsynaptic current (EPSC) recorded from a CA3 pyramidal cell with an increase in single electric stimulus-evoked patch current amplitude recorded with a patch sensor positioned in the stratum lucidum. In Schaffer collateral–CA1 synapses, however, LTP of EPSC was accompanied by an increase in AMPA-evoked whole-cell current, but not with change in patch current in the stratum radiatum. These results indicate that EAA release is increased during mossy fiber LTP but not CA1 LTP.

Properties of voltage-sensitive Ca2+ channels expressed in the Xenopus oocyte after injection of rat brain mRNA were investigated using the whole-cell voltage-clamp method as depolarization-induced Ba2+ current, IBa. The apparent decay profile of IBa was considered to be the sum of a transient current (tau approximately 0.4 s) and a more sustained current (tau approximately 4 s). The sustained component was isolated by a weak depolarization from -20 to 0 mV, only detected in the mRNA-injected cells, and rather sensitive to omega-conotoxin GVIA. Moreover, increases in cytosolic cyclic AMP caused potentiation of the long-lasting current. These results suggest that slow-inactivating states of transplanted Ca2+ channels are preferentially modulated by cyclic AMP-dependent protein kinase.

The relation between the habenula and central dopaminergic system was investigated by examining the inhibitory effects of methamphetamine on the firing rate of dopamine neurons in the substantia nigra of habenular lesioned and unlesioned rats. Electrolytic lesions of the habenula attenuated the methamphetamine-induced inhibition of dopamine neurons in the substantia nigra. This, along with other findings, indicates possibly that the habenula is involved in a feedback pathway from the striatum to the substantia nigra and regulates the activity of dopamine neurons in the substantia nigra.