Loss of GluN2D subunit results in social recognition deficit, social stress, 5-HT2C receptor dysfunction, and anhedonia in mice.

The N-methyl-d-aspartate (NMDA) receptor channel is involved in various physiological functions, including learning and memory. The GluN2D subunit of the NMDA receptor has low expression in the mature brain, and its role is not fully understood. In the present study, the effects of GluN2D subunit deficiency on emotional and cognitive function were investigated in GluN2D knockout (KO) mice. We found a reduction of motility (i.e., a depressive-like state) in the tail suspension test and a reduction of sucrose preference (i.e., an anhedonic state) in GluN2D KO mice that were group-housed with littermates. Despite apparently normal olfactory function and social interaction, GluN2D KO mice exhibited a decrease in preference for social novelty, suggesting a deficit in social recognition or memory. Golgi-Cox staining revealed a reduction of the complexity of dendritic trees in the accessory olfactory bulb in GluN2D KO mice, suggesting a deficit in pheromone processing pathway activation, which modulates social recognition. The deficit in social recognition may result in social stress in GluN2D KO mice. Isolation housing is a procedure that has been shown to reduce stress in mice. Interestingly, 3-week isolation and treatment with agomelatine or the 5-hydroxytryptamine-2C (5-HT2C) receptor antagonist SB242084 reversed the anhedonic-like state in GluN2D KO mice. In contrast, treatment with the 5-HT2C receptor agonist CP809101 induced depressive- and anhedonic-like states in isolated GluN2D KO mice. These results suggest that social stress that is caused by a deficit in social recognition desensitizes 5-HT2c receptors, followed by an anhedonic- and depressive-like state, in GluN2D KO mice. The GluN2D subunit of the NMDA receptor appears to be important for the recognition of individuals and development of normal emotionality in mice. 5-HT2C receptor antagonism may be a therapeutic target for treating social stress-induced anhedonia. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Involvement of the N-methyl-D-aspartate receptor GluN2D subunit in phencyclidine-induced motor impairment, gene expression, and increased Fos immunoreactivity.

BACKGROUND: Noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists evoke a behavioral and neurobiological syndrome in experimental animals. We previously reported that phencyclidine (PCP), an NMDA receptor antagonist, increased locomotor activity in wildtype (WT) mice but not GluN2D subunit knockout mice. Thus, the aim of the present study was to determine whether the GluN2D subunit is involved in PCP-induced motor impairment. RESULTS: PCP or UBP141 (a GluN2D antagonist) induced potent motor impairment in WT mice but not GluN2D KO mice. By contrast, CIQ, a GluN2C/2D potentiator, induced severe motor impairment in GluN2D KO mice but not WT mice, suggesting that the GluN2D subunit plays an essential role in the effects of PCP and UBP141, and an appropriate balance between GluN2C and GluN2D subunits might be needed for appropriate motor performance. The level of the GluN2D subunit in the mature mouse brain is very low and restricted. GluN2D subunits exist in brainstem structures, the globus pallidus, thalamus, and subthalamic nucleus. We found that the expression of the c-fos gene increased the most among PCP-dependent differentially expressed genes between WT and GluN2D KO mice, and the number of Fos-positive cells increased after PCP administration in the basal ganglia motor circuit in WT mice but not GluN2D KO mice. CONCLUSION: These results suggest that the GluN2D subunit within the motor circuitry is a key subunit for PCP-induced motor impairment, which requires an intricate balance between GluN2C- and GluN2D-mediated excitatory outputs.

Quantitative Detection of µ Opioid Receptor: Western Blot Analyses Using µ Opioid Receptor Knockout Mice.

Increasing evidence suggests that µ opioid receptor (MOP) expression is altered during the development of and withdrawal from substance dependence. Although anti-MOP antibodies have been hypothesized to be useful for estimating MOP expression levels, inconsistent MOP molecular weights (MWs) have been reported in studies using anti-MOP antibodies. In the present study, we generated a new anti-MOP antibody (N38) against the 1-38 amino acid sequence of the mouse MOP N-terminus and conducted Western blot analysis with wildtype and MOP knockout brain lysates to determine the MWs of intrinsic MOP. The N38 antibody detected migrating bands with relative MWs of 60-67 kDa in the plasma membrane fraction isolated from wildtype brain, but not from the MOP knockout brain. These migrating bands exhibited semi-linear density in the range of 3-30 µg membrane proteins/lane. The N38 antibody may be useful for quantitatively detecting MOP.

A region of N-type Ca(2+) channel critical for blockade by the dihydropyridine amlodipine.

Amlodipine, a dihydropyridine derivative, has been shown to block not only L-type but also N-type Ca(2+) channels. Aiming to understand the mechanism underlying such a selective blockade by amlodipine, the interaction of amlodipine with N-type channels was investigated using the Xenopus oocyte expression system together with the two-microelectrode voltage-clamp technique and the binding assay for [(3)H]amlodipine. When expressed as the alpha(1B)alpha(2/)delta(1)beta(1a) combination, the N-type channel formed a high affinity binding site for [(3)H]amlodipine (K(d), 3.08nM) and was profoundly blocked by amlodipine (IC(50), 2.7 microM at -60mV). By contrast, R-type (alpha(1E)alpha(2/)delta(1)beta(1a)) channels did not possess a high affinity binding site for [(3)H]amlodipine and their channel activities were not influenced by amlodipine. In comparison of amino acid sequences in the transmembrane regions IIIS5, IIIS6 and IVS6 of the alpha(1) subunit, which are involved in dihydropyridine binding in L-type channels, the two amino acid residues Lys(1287) (corresponding to Met(1295) in alpha1B) and Phe(1699) (corresponding to Leu(1697) in alpha(1B)) were unique in alpha(1E). An amino acid substitution of Lys1287Met in IIIS5 of alpha(1E) conferred a high affinity binding site for amlodipine (K(d), 13.1nM) and a sensitivity to amlodipine (IC(50), 11.3 microM). In N-type channel, reversely, an amino acid substitution of Met1295Lys in IIIS5 of alpha(1B) deprived a high affinity binding site for amlodipine and reduced the channel blockade by amlodipine (IC(50), 29.6 microM). The results indicate that Met(1295) in the region IIIS5 of alpha(1B) is critical for amlodipine to efficiently bind and block the N-type Ca(2+) channel.

Genetic deletion of vesicular monoamine transporter-2 (VMAT2) reduces dopamine transporter activity in mesencephalic neurons in primary culture.

Our aim was to investigate whether a defect in vesicular monoamine transporter-2 (VMAT2) activities would affect dopaminergic cell functions or not. We examined mesencephalon dopaminergic cultures prepared from VMAT2 wild-type, heterozygous or homozygous knockout (KO) 14-day-old mouse fetuses to determine the number of tyrosine hydroxylase (TH)-positive cells and dopamine transporter activity. The number of TH-positive cells remained unchanged in the VMAT2-KO cultures. Of interest, the dopamine transporter activity in the homozygous cells was significantly decreased, but not in the heterozygous cells, suggesting that complete deletion of VMAT2 inhibited dopamine transporter function. Furthermore, dopamine transporter activity was prominently decreased in the synaptosomal fraction of neonatal homozygous VMAT2-KO mice compared with that of wild-type/heterozygous VMAT2-KO ones, indicating that VMAT2 activity might be one of the factors regulating dopamine transporter activities. To test this possibility, we used reserpine, a VMAT2 inhibitor. Reserpine (1muM) decreased dopamine transporter activity (approx. 50%) in wild-type and heterozygous VMAT2-KO cultures but not in homozygous ones, indicating that blockade of VMAT2 activity reduced dopamine transporter activity. To investigate possible mechanisms underlying the decreased dopamine transporter activity in VMAT2-KO mice, we measured dopamine transporter activities after 24-48h exposure of primary cultures of mesencephalic neurons to dopamine receptor antagonists, PKC inhibitor, PI(3)K inhibitor, and l-DOPA. Among these drugs, l-DOPA slightly reduced the dopamine transporter activities of all genotypes, but the other drugs could not. Since the ratios of reduction in dopamine transporter activity of each genotype treated with l-DOPA were similar, substrate inhibition of dopamine transporters was not the main mechanism underlying the reduced dopamine transporter activity due to genetic deletion of VMAT2. Our results demonstrate that genetic deletion of VMAT2 did not induce immediate cell death but did markedly inhibit dopamine transporter activity.

Repeated methamphetamine administration alters expression of the NMDA receptor channel epsilon2 subunit and kinesins in the mouse brain.

Repeated amphetamine administration results in behavioral sensitization. Behavioral sensitization related to abuse and/or relapse may be associated with stable changes in gene expression. To explore the participating genes, we examined the changes in gene expression levels 24 h or 21 days (long-term withdrawal period) after chronic methamphetamine (METH) treatment for 2 weeks. The expression of several genes related to glutamatergic neural transmission was altered, although changes in the corresponding protein expression were not always consistent with the results for mRNA expression. Of interest, in the frontal cortex of mice treated with METH for 2 weeks, protein expression levels of KIF17 and the N-methyl-D-asparate (NMDA) receptor channel epsilon2 subunit (NRepsilon2) were concomitantly increased. The alteration in expression of these proteins, KIF17 and NRepsilon2, might be a part of the molecular basis of the behavioral sensitization to METH.

Methamphetamine modulation of gene expression in the brain: analysis using customized cDNA microarray system with the mouse homologues of KIAA genes.

Amphetamine abuse may be associated with adaptive changes in gene expression. In the present study, we used a newly developed cDNA array system comprising mouse KIAA (mKIAA) cDNA clones to examine changes in gene expression after chronic methamphetamine (MAP) treatment. Mice were daily treated with saline or MAP (2 mg/kg, ip) for 2 weeks. Approximately 800 mKIAA clones were blotted onto a nylon membrane and hybridized with 33P-labeled DNA derived from mRNAs from mouse whole brain. MAP-induced changes were found in several clones by using whole brain mRNA. Since gene expression of Per2, one of the period protein-related proteins, was the most affected by MAP treatment, its expression was further analyzed in pooled hippocampi from 20 mice that had been treated with saline or MAP (2 mg/kg, ip) for 2 weeks. The gene expression and protein expression of Per2 in the hippocampus were increased by MAP treatment. In the hippocampus, Per2 gene expression was under the regulation of circadian rhythm and increases in Per2 expression were due to the phase shift induced by chronic MAP treatment. These findings suggest that unique expression changes of period protein-related proteins in the hippocampus occur in MAP abuse.

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.