Hideko Yamamoto

Regional Differences in Extracellular Dopamine and Serotonin Assessed by In Vivo Microdialysis in Mice Lacking Dopamine and/or Serotonin Transporters

Cocaine conditioned place preference (CPP) is intact in dopamine transporter (DAT) knockout (KO) mice and enhanced in serotonin transporter (SERT) KO mice. However, cocaine CPP is eliminated in double-KO mice with no DAT and either no or one SERT gene copy. To help determine mechanisms underlying these effects, we now report examination of baselines and drug-induced changes of extracellular dopamine (DAex) and serotonin (5-HTex) levels in microdialysates from nucleus accumbens (NAc), caudate putamen (CPu), and prefrontal cortex (PFc) of wild-type, homozygous DAT- or SERT-KO and heterozygous or homozygous DAT/SERT double-KO mice, which are differentially rewarded by cocaine. Cocaine fails to increase DAex in NAc of DAT-KO mice. By contrast, systemic cocaine enhances DAex in both CPu and PFc of DAT-KO mice though local cocaine fails to affect DAex in CPu. Adding SERT to DAT deletion attenuates the cocaine-induced DAex increases found in CPu, but not those found in PFc. The selective SERT blocker fluoxetine increases DAex in CPu of DAT-KO mice, while cocaine and the selective DAT blocker GBR12909 increase 5-HTex in CPu of SERT-KO mice. These data provide evidence that (a) cocaine increases DAex in PFc independently of DAT and that (b), in the absence of SERT, CPu levels of 5-HTex can be increased by blocking DAT. Cocaine-induced alterations in CPu DA levels in DAT-, SERT-, and DAT/SERT double-KO mice appear to provide better correlations with cocaine CPP than cocaine-induced DA level alterations in NAc or PFc.

Localization of 5-HT2A Receptor in rat cerebral cortex and olfactory system revealed by immunohistochemistry using two antibodies raised in rabbit and chicken

Serotonin 2A receptor (5-HT2A receptor) is widely distributed in the central nervous system, and has been suggested to be involved in a variety of behavioral conditions and neuropsychiatric disorders. Two polyclonal antibodies were raised against the N-terminus peptide of rat 5-HT2A receptor in chickens (5-HT2A-N) and a glutathione S-transferase fusion protein that contained the C-terminus of the mouse 5-HT2A receptor in rabbits (5-HT2A-C). Affinity-purified 5-HT2A-N and -C antibodies reacted strongly with a single band of 77-78 kDa in postsynaptic density proteins prepared from the rat cortex. The distribution pattern of immunoreactive structures in the rat brain was virtually the same for the two antibodies. The highest levels of immunoreactivity were observed in the olfactory bulb, neocortex, claustrum, piriform cortex, mamillary bodies, pontine nuclei, red nucleus and cranial motor nuclei. In the olfactory bulb, mitral cells were intensely labeled. In the neocortex, many immunoreactive neurons were found in layers II-VI. In layer IV of the neocortex, strong neuropil labeling was observed. In a double-labeling study using chicken 5-HT2A-N and rabbit anti-glial fibrillary acidic protein (GFAP) antibody, a considerable number of GFAP positive cells also showed 5-HT2A immunoreactivity. By using an immunoelectron microscopic technique, 5-HT2A receptor immunoreaction was shown to be localized just beneath the postsynaptic membrane thickening of asymmetric synapses.

Protocadherin-α Family Is Required for Serotonergic Projections to Appropriately Innervate Target Brain Areas

Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain, where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-α (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles, we examined serotonergic fibers in a mouse mutant (PcdhaΔCR/ΔCR) lacking the Pcdha cytoplasmic region-encoding exons, which are common to the gene cluster. In the first week after birth, the distribution pattern of serotonergic fibers in PcdhaΔCR/ΔCR mice was similar to wild-type, but by 3 weeks of age, when the serotonergic axonal termini complete their arborizations, the distribution of the projections was abnormal. In some target regions, notably the globus pallidus and substantia nigra, the normally even distribution of serotonin axonal terminals was, in the mutants, dense at the periphery of each region, but sparse in the center. In the stratum lacunosum-moleculare of the hippocampus, the mutants showed denser serotonergic innervation than in wild-type, and in the dentate gyrus of the hippocampus and the caudate-putamen, the innervation was sparser. Together, the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections.

Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding

The type 1 sigma receptor expressed in Xenopus oocytes showed binding abilities for the sigma‐1 ligands, [3H](+)pentazocine and [3H]NE‐100, with similar kinetic properties as observed in native tissue membranes. Amino acid substitutions (Ser99Ala, Tyr103Phe and di‐Leu105,106di‐Ala) in the transmembrane domain did not alter the expression levels of the type 1 sigma receptor as determined by immunoblot analysis using an anti‐type 1 sigma receptor antiserum. By contrast, ligand binding was significantly suppressed by the substitutions. These findings provide evidence that the transmembrane domain of the type 1 sigma receptor plays a critical role in ligand binding of this receptor.

Caspase-independent cell death by low concentrations of nitric oxide in PC12 cells: Involvement of cytochrome C oxidase inhibition and the production of reactive oxygen species in mitochondria

We reported previously that low levels of nitric oxide (NO) induced cell death with properties of apoptosis, including chromatin fragmentation and condensation in undifferentiated PC12 pheochromocytoma cells. The present study demonstrates that cytotoxicity of low concentrations of NO is mediated by inhibition of mitochondrial cytochrome c oxidase and generation of reactive oxygen species (ROS). An NO donor, (±)‐(E)‐4‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexenamide (NOR3) induced cell death even at low concentrations (10–100 μM), whereas peroxynitrite and a peroxynitrite generator, 3‐(4‐morpholinyl)‐sydnonimine (SIN‐1), did not have a significant effect on cell viability up to a concentration of 0.5 mM. The NOR3‐induced cell death was unaffected by pretreatment with superoxide dismutase (SOD) or its mimetic peroxynitrite scavenger, manganese(III) tetrakis(benzoic acid)porphyrin chloride (Mn‐TBAP), or with uric acid. These findings indicate that peroxynitrite does not contribute to this cell death. Furthermore, neither the release of cytochrome c from mitochondrial membranes, the cleavage of poly‐ADP ribose polymerase (PARP), nor the activation of caspase‐3‐like activities was observed. Inhibitors of PARP, benzamide, and aminobenzamide, had no effect on the NOR3‐induced cell death. In addition, pretreatment with general or selective caspase inhibitors, benzyloxy‐carbonyl‐Val‐Ala‐Asp‐fluoromethylketone (Z‐VAD‐fmk), N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde (Ac‐DEVD‐CHO), and benzyloxycarbonyl‐Asp‐2,6‐dichlorobenzoyloxymethylketone (Z‐Asp‐Ch2‐DCB) did not prevent NOR3‐induced cell death. Taken together, these findings suggest that cell death induced by NOR3 occurs by a caspase‐independent mechanism. In contrast, we found an early increase in mitochondrial H2O2 production during NOR3 exposure using the fluorescent dye 2′,7′‐dichlorofluorescin‐diacetate (DCFH‐DA) and dihydrorohdamine123 (DHR123), and these events were accompanied by strong inhibition of cytochrome c oxidase activity in the cells. Furthermore, we observed that several antioxidants, such as ascorbate, glutathione (GSH), cysteine, tetrahydrobiopterin, and dithiothreitol (DTT), all effectively prevented the NOR3‐induced cell death. NOR3 treatment decreased the level of total intracellular GSH, but did not affect the activities of antioxidant enzymes SOD, GSH‐peroxidase (GPX), and catalase. These results suggest that cell death induced at physiologically low concentrations of NO is mediated by ROS production in mitochondria, most likely resulting from the inhibition of cytochrome c oxidase, with ROS acting as an initiator of caspase‐independent cell death.

Kinetic characterization of the nitric oxide toxicity for PC12 cells: effect of half-life time of NO release

We investigated the effects of low concentrations of nitric oxide (NO) on cell viability using NO donors, (+/-)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hex enamid e (NOR1), (+/-)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR2), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3) and (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1- yl]-3-pyr idine (NOR4). The half-life times of the NO release from these four NOR analogs, NOR1, NOR2, NOR3 and NOR4, were determined (6.5, 84, 105 and 340 min, respectively) by using 4,5-diaminofluorescein (DAF-2), a newly developed indicator of NO. Exposure of undifferentiated PC12 cells to low concentrations of NO donors, NOR2 or NOR3 (1-100 microM), but not NOR1 nor NOR4, resulted in cell death in a dose- and time-dependent manner, as determined from cell viability assessed by 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium (MTT) assay. After 24 h exposure to 50 microM NOR2 or NOR3, more than 90% of PC12 cells had died. Furthermore, while the toxic effect of NOR3 was attenuated by replacing the medium at 20 min, 1 or 2 h after drug addition, it was continued by replacing the medium at 3 h or later after drug addition. The cell death was characterized by DNA degradation, nuclear condensation and fragmentation, suggesting apoptosis-like cell death. Pretreatment with an antioxidant ascorbic acid (0.1-0.5 mM) completely prevented the cell death caused by NOR3, while glutathione (0.1-0.2 mM) and cysteine (0.2-0.4 mM) provided partial protection. These findings suggest that the cell toxicity induced by NO at low concentrations strongly depends upon the duration of expose to NO from NO donors, and these toxic effects are effectively prevented by the antioxidant, ascorbic acid.

Down‐regulation of protocadherin‐α A isoforms in mice changes contextual fear conditioning and spatial working memory

Diverse protocadherins (Pcdhs), which are encoded as a large cluster (composed of α, β and γ clusters) in the genome, are localized to axons and synapses. The Pcdhs have been proposed to contribute to the generation of sophisticated neural networks and to regulate brain function. To address the molecular roles of Pcdhs in regulating individual behavior, here we generated knockdown mice of Pcdh‐α proteins and examined their behavioral abnormalities. There are two alternative splicing variants of the Pcdh‐α constant region, Pcdh‐α A and B isoforms, with different cytoplasmic tails. Pcdh‐αΔBneo/ΔBneo mice, in which the Pcdh‐α B splicing variant was absent and the Pcdh‐α A isoforms were down‐regulated to approximately 20% of the wild‐type level, exhibited enhanced contextual fear conditioning and disparities in an eight‐arm radial maze. Similar abnormalities were found in Pcdh‐αΔAneo/ΔAneo mice, which lacked 57 amino acids of the Pcdh‐α A cytoplasmic tail. These learning abnormalities were, however, not seen in Pcdh‐αΔB/ΔB mice [in which the neomycin‐resistance (neo) gene cassette was removed from the Pcdh‐αΔBneo/ΔBneo alleles], in which the expression level of the Pcdh‐α A isoforms was recovered, although the Pcdh‐α B isoforms were still completely missing in the brain. In addition, the amount of 5‐hydroxytryptamine increased in the hippocampus of the hypomorphic Pcdh‐α A mutant mice but not in recovery Pcdh‐αΔB/ΔB. These results suggested that the level of Pcdh‐α A isoforms in the brain has an important role in regulating learning and memory functions and the amount of 5‐hydroxytryptamine in the hippocampus.

Functional identification of ASCT1 neutral amino acid transporter as the predominant system for the uptake of l-serine in rat neurons in primary culture

The uptake of L-serine, a nonessential amino acid known to be transported by the neutral amino acid transporter system ASC, was studied in primary cultures of rat neurons and astrocytes, and compared with that in human embryonic kidney (HEK293) cells transfected with rat ASCT1 cDNA. We first cloned neutral amino acid transporter ASCT1 from rat neurons in primary culture as a transporter candidate for L-serine uptake in the brain. The predicted amino acid sequence from rat ASCT1 exhibited significant homology with mouse and human ASCT1s. The amino acid sequence of rat ASCT1 was 92 and 84% identical to that of mouse and of human ASCT1, respectively. HEK293 cells expressing the rat ASCT1 cDNA showed a saturable dose-dependent and Na(+)-dependent increase in L-[(3)H] serine uptake by high affinity ( K(m) = 67 microM). The substrate selectivity of rat ASCT1 was the same as those of the mouse and human transporter. Northern blot analysis revealed that ASCT1 mRNA was ubiquitously expressed in the brain, with its highest concentration in the striatum and hippocampus. When the uptake of L -[(3)H] serine into rat primary neurons or astrocytes was compared with that of HEK293 cells expressing rat ASCT1 or rat ASCT2 cDNA, the inhibition profile of amino acids for the rat neurons quite resembled that for HEK293 cells expressing rat ASCT1. In contrast, the profile for rat astrocytes was a mixture of that for HEK293 cells expressing rat ASCT1 and that for the cells expressing rat ASCT2. Furthermore, L-[(3)H] serine uptake in neurons was fully Na(+)-dependent. ASCT1 mRNA was expressed in both primary neurons and astrocytes, whereas ASCT2 mRNA was expressed only in astrocytes, as determined by using RT-PCR with primers specific for the rat ASCT1 or rat ASCT2 transporter. Taken together, these findings indicate that ASCT1 predominantly contributes to the uptake of L-serine in primary neurons.

Characterization of rapid and high-affinity uptake of L-serine in neurons and astrocytes in primary culture

The non‐essential amino acid L‐serine was shown to be required to support the survival of rat cerebellar Purkinje neurons because of lack of the expression of the L‐serine biosynthesis enzyme 3‐phosphoglycerate dehydrogenase in them. In the present study, we investigated L‐[3H]serine uptake in primary cultures of neurons and astrocytes from the rat telencephalon. In both neurons and astrocytes, L‐[3H]serine uptake was dependent on temperature and Na+ ions, and exhibited a single component of high‐affinity uptake sites (K m=15.0 and 17.2 μM for neurons and astrocytes, respectively). Kinetic analysis of L‐[3H]serine uptake also revealed that the uptake into neurons was faster than that into astrocytes. The selectivity of inhibition by amino acids of the L‐[3H]serine uptake resembled that of the system ASC transporters ASCT1 and ASCT2. Neutral amino acids L‐alanine, L‐serine, L‐cysteine, and L‐threonine strongly inhibited the uptake by both cell types. Furthermore, in astrocytes, but not in neurons, L‐valine and L‐proline also inhibited L‐[3H]serine uptake. Neither α‐methyl aminoisobutyric acid (a system A‐specific substrate) nor 2‐aminobicyclo(2,2,1)heptane‐2‐carboxylic acid (a system L‐specific substrate) inhibited the uptake of L‐[3H]serine in both neurons and astrocytes. Expression of ASCT transporters in both neurons and astrocytes was examined by use of reverse transcriptase polymerase chain reaction and immunoblot analysis. Whereas transcripts (mRNAs) of both ASCT1 and ASCT2 transporters were detected in astrocytes, only the mRNA of the former subtype was detected in neurons. Immunoblot analysis confirmed the presence of ASCT1 in both neurons and astrocytes. These findings indicate that neurons accumulate a high level of L‐serine by using a Na+‐dependent, high‐affinity transport system, operating predominantly through the ASCT1 transporter subtype.

Endothelin 1 Stimulates Na+,K+‐ATPase and Na+‐K+‐Cl− Cotransport Through ETA Receptors and Protein Kinase C‐Dependent Pathway in Cerebral Capillary Endothelium

Abstract: The effect of endothelins (ET‐1 and ET‐3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET‐1 or ET‐3 dose‐dependently enhanced K+ uptake (EC50 = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo‐d‐Trp‐d‐Asp‐Pro‐d‐Val‐Leu). Neither the selective ETB agonists IRL 1620 [N‐succinyl‐(Glu9,‐Ala11,15)‐ET‐1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)‐ET‐1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K+‐ATPase) and bumetanide (inhibitor of Na+‐K+‐Cl− cotransport) reduced (up to 40% and up to 70%, respectively) the ET‐1‐stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12‐myristate 13‐acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K+‐ATPase and Na+‐K+‐Cl− cotransport. ET‐1‐but not PMA‐stimulated K+ uptake was inhibited by 5‐(N‐ethyl‐N‐isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET‐1‐stimulated Na+,K+‐ATPase and Na+‐K+‐Cl− cotransport activity that is not mediated by PKC.