Brain masculinization requires androgen receptor function

Testicular testosterone produced during a critical perinatal period is thought to masculinize and defeminize the male brain from the inherent feminization program and induce male-typical behaviors in the adult. These actions of testosterone appear to be exerted not through its androgenic activity, but rather through its conversion by brain aromatase into estrogen, with the consequent activation of estrogen receptor (ER)-mediated signaling. Thus, the role of androgen receptor (AR) in perinatal brain masculinization underlying the expression of male-typical behaviors remains unclear because of the conversion of testosterone into estrogen in the brain. Here, we report a null AR mutation in mice generated by the Cre-loxP system. The AR-null mutation in males (AR(L-/Y)) resulted in the ablation of male-typical sexual and aggressive behaviors, whereas female AR-null homozygote (AR(L-/L-)) mice exhibited normal female sexual behaviors. Treatment with nonaromatizable androgen (5alpha-dihydrotestosterone, DHT) was ineffective in restoring the impaired male sexual behaviors, but it partially rescued impaired male aggressive behaviors in AR(L-/Y) mice. Impaired male-typical behaviors in ERalpha(-/-) mice were restored on DHT treatment. The role of AR function in brain masculinization at a limited perinatal stage was studied in AR(L-/L-) mice. Perinatal DHT treatment of females led to adult females sensitive to both 17beta-estradiol and DHT in the induction of male-typical behaviors. However, this female brain masculinization was abolished by AR inactivation. Our results suggested that perinatal brain masculinization requires AR function and that expression of male-typical behaviors in adults is mediated by both AR-dependent and -independent androgen signaling.     

Nitrous oxide (N(2)O) requires the N-methyl-D-aspartate receptor for its action in Caenorhabditis elegans

Nitrous oxide (N(2)O, also known as laughing gas) and volatile anesthetics (VAs), the original and still most widely used general anesthetics, produce anesthesia by ill-defined mechanisms. Electrophysiological experiments in vertebrate neurons have suggested that N(2)O and VAs may act by distinct mechanisms; N(2)O antagonizes the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors, whereas VAs alter the function of a variety of other synaptic proteins. However, no genetic or pharmacological experiments have demonstrated that any of these in vitro actions are responsible for the behavioral effects of either class of anesthetics. By using genetic tools in Caenorhabditis elegans, we tested whether the action of N(2)O requires the NMDA receptor in vivo and whether its mechanism is shared by VAs. Distinct from the action of VAs, N(2)O produced behavioral defects highly specific and characteristic of that produced by loss-of-function mutations in both NMDA and non-NMDA glutamate receptors. A null mutant of nmr-1, which encodes a C. elegans NMDA receptor, was completely resistant to the behavioral effects of N(2)O, whereas a non-NMDA receptor-null mutant was normally sensitive. The N(2)O-resistant nmr-1(null) mutant was not resistant to VAs. Likewise, VA-resistant mutants had wild-type sensitivity to N(2)O. Thus, the behavioral effects of N(2)O require the NMDA receptor NMR-1, consistent with the hypothesis formed from vertebrate electrophysiological data that a major target of N(2)O is the NMDA receptor.     

Protein kinase C potentiation of N-methyl-D-aspartate receptor activity is not mediated by phosphorylation of N-methyl-D-aspartate receptor subunits

N-methyl-D-aspartate receptors (NMDARs) are Ca(2+)-permeable glutamate-gated ion channels whose physiological properties in neurons are modulated by protein kinase C (PKC). The present study was undertaken to determine the role in PKC-induced potentiation of the NR1 and NR2A C-terminal tails, which serve as targets of PKC phosphorylation [Tingley, W. G., Ehlers, M. D., Kameyama, K., Doherty, C., Ptak, J. B., Riley, C. T. & Huganir, R. L. (1997) J. Biol. Chem. 272, 5157-5166]. Serine residue 890 in the C1 cassette is a primary target of PKC phosphorylation and a critical residue in receptor clustering at the membrane. We report herein that the presence of the C1 cassette reduces PKC potentiation and that mutation of Ser-890 significantly restores PKC potentiation. Splicing out or deletion of other C-terminal cassettes singly or in combination had little or no effect on PKC potentiation. Moreover, experiments involving truncation mutants reveal the unexpected finding that NMDARs assembled from subunits lacking all known sites of PKC phosphorylation can show PKC potentiation. These results indicate that PKC-induced potentiation of NMDAR activity does not occur by direct phosphorylation of the receptor protein but rather of associated targeting, anchoring, or signaling protein(s). PKC potentiation of NMDAR function is likely to be an important mode of NMDAR regulation in vivo and may play a role in NMDA-dependent long-term potentiation.     

谷氨酸及其N-甲基-D-天冬氨酸受体在肺损伤中的作用

谷氨酸是哺乳动物中枢神经系统兴奋性氨基酸神经递质,N-甲基-D-天冬氨酸（N—methyl—D-aspartate,NMDA）受体是谷氨酸重要的离子型受体。多种外周非神经组织也发现NMDA受体在维持组织正常的生理功能、参与组织细胞的损伤与修复等方面发挥重要作用。近年来发现在肺组织同样存在NMDA受体的表达,后者参与多种肺部疾病的发生与发展。     

N-methyl-D-aspartate receptor antagonist desegregates eye-specific stripes.

The optic tecta of surgically produced three-eyed tadpoles were chronically exposed to the N-methyl-D-aspartate (NMDA) receptor antagonist aminophosphonovaleric acid (APV), or to NMDA itself, to assess the influence of NMDA receptor/channels on the eye-specific segregation of retinal ganglion cell (RGC) terminals that occurs whenever two retinas innervate one tectal lobe. Exposure of the tectum to the active isomer of APV produces desegregation of the RGC terminals without blocking electrical activity in the afferents or altering their terminal arbor morphology. Exposure to the inactive isomer of APV causes no perturbation of the normal stripe pattern. APV-induced desegregation is completely reversible within 2 weeks of removal of the APV. In addition, exposure of the optic tectum to NMDA results in stripes with sharper borders and fewer forks and fusions than untreated animals. These results suggest that the NMDA receptor/channel plays a role in eye-specific segregation in the three-eyed tadpole.     

An N-methyl-D-aspartate receptor channel blocker with neuroprotective activity

Excitotoxicity, resulting from sustained activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype, is considered to play a causative role in the etiology of ischemic stroke and several neurodegenerative diseases. The NMDA receptor is therefore a target for the development of neuroprotective agents. Here, we identify an N-benzylated triamine (denoted as NBTA) as a highly selective and potent NMDA-receptor channel blocker selected by screening a reduced dipeptidomimetic synthetic combinatorial library. NBTA blocks recombinant NMDA receptors expressed in Xenopus laevis oocytes with a mean IC(50) of 80 nM; in contrast, it does not block GluR1, a glutamate receptor of the non-NMDA subtype. The blocking activity of NBTA on NMDA receptors exhibits the characteristics of an open-channel blocker: (i) no competition with agonists, (ii) voltage dependence, and (iii) use dependence. Significantly, NBTA protects rodent hippocampal neurons from NMDA receptor, but not kainate receptor-mediated excitotoxic cell death, in agreement with its selective action on the corresponding recombinant receptors. Mutagenesis data indicate that the N site, a key asparagine on the M2 transmembrane segment of the NR1 subunit, is the main determinant of the blocker action. The results highlight the potential of this compound as a neuroprotectant.     

Role of N-methyl-D-aspartate receptor in hyperoxia-induced lung injury

Glutamate (Glu) N-methyl-D-aspartate (NMDA) receptor is present in the lungs, and NMDA receptor antagonist MK-801 attenuates oxidant lung injury. We hypothesized that Glu excitotoxicity may participate in the pathogenesis of hyperoxia-induced lung injury. To determine possible pulmonary protective effects, we administered 0.05 ml/kg MK-801 or saline intraperitoneally daily to neonatal rats exposed to more than 95% oxygen in air. After 7 days, MK-801 decreased the hyperoxia-associated elevation of wet-to-dry lung weight, total leukocyte and neutrophil counts, total protein and lactate dehydroase in BAL fluid, total myeloperoxidase activity, and lung pathological injury. MK-801 inhibited hyperoxia-associated increments in reactive oxygen species production and NF-kappaB production. Hence, NMDA receptor antagonist MK-801 ameliorates hyperoxia-induced lung injury in neonatal rats, and is associated with decreased reactive oxygen species and NF-kappaB. We conclude that Glu may play an important role in hyperoxia-induced lung injury by activation of NMDA receptor.     

The promise of N-methyl-D-aspartate receptor antagonists in fibromyalgia

There is strong evidence that intravenous administration of ketamine following a standardized protocol could be used as a diagnostic test for a central sensitization in the central nervous system in patients with FM. The combination of a weak opioid and an NMDA-receptor antagonist with few side effects is presently a promise for treatment of pain in a subgroup of patients with FM. The response to intravenously administered ketamine may help select patients for this treatment modality.     

N-methyl-D-aspartate receptor plasticity in kindling: quantitative and qualitative alterations in the N-methyl-D-aspartate receptor-channel complex.

Kindling is an animal model of epilepsy and neuronal plasticity produced by periodic electrical stimulation of the brain. Electrophysiologic studies indicate that this phenomenon is associated with increased participation of N-methyl-D-aspartate (NMDA) receptors in excitatory synaptic transmission. Biochemical studies suggest that a change intrinsic to the NMDA receptor-channel complex may contribute to the increase in NMDA receptor-mediated synaptic transmission. We tested this idea by measuring the binding of 3-[(+)-2-(carboxypiperazin-4-yl)][1,2-3H]propyl-1-phosphonic acid ([3H]CPP), [3H]glycine, and tritiated N-[(1-thienyl)cyclohexyl]piperidine [( 3H]TCP) to rat hippocampal membranes. In this preparation these ligands are selective for the NMDA receptor, the strychnine-insensitive glycine receptor, and the NMDA receptor-gated ion channel, respectively. Kindling increased the density of CPP, glycine, and TCP binding sites in hippocampal membranes by 47%, 42%, and 25%, respectively. No significant changes were detected in the affinity of these binding sites. Surprisingly, alterations in the glycine binding site were detected in animals sacrificed 1 month but not 1 day after the final kindling stimulation. Thus, delayed upregulation of the NMDA receptor-channel complex may be one molecular mechanism that maintains the long-lasting hyperexcitability of hippocampal neurons in kindled animals.     

Blockade of N-methyl-D-aspartate receptor activation suppresses learning-induced synaptic elimination

Auditory filial imprinting in the domestic chicken is accompanied by a dramatic loss of spine synapses in two higher associative forebrain areas, the mediorostral neostriatum/hyperstriatum ventrale (MNH) and the dorsocaudal neostriatum (Ndc). The cellular mechanisms that underlie this learning-induced synaptic reorganization are unclear. We found that local pharmacological blockade of N-methyl-D-aspartate (NMDA) receptors in the MNH, a manipulation that has been shown previously to impair auditory imprinting, suppresses the learning-induced spine reduction in this region. Chicks treated with the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) during the behavioral training for imprinting (postnatal day 0-2) displayed similar spine frequencies at postnatal day 7 as naive control animals, which, in both groups, were significantly higher than in imprinted animals. Because the average dendritic length did not differ between the experimental groups, the reduced spine frequency can be interpreted as a reduction of the total number of spine synapses per neuron. In the Ndc, which is reciprocally connected with the MNH and not directly influenced by the injected drug, learning-induced spine elimination was partly suppressed. Spine frequencies of the APV-treated, behaviorally trained but nonimprinted animals were higher than in the imprinted animals but lower than in the naive animals. These results provide evidence that NMDA receptor activation is required for the learning-induced selective reduction of spine synapses, which may serve as a mechanism of information storage specific for juvenile emotional learning events.     

N-甲基-D-天冬氨酸受体NR2B亚基与神经元凋亡

神经元凋亡是缺血性卒中神经元死亡的重要形式之一.在神经元凋亡过程中,民N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体介导的兴奋性氨基酸毒性起着重要作用,是神经元凋亡的启动者和执行者.NR2B是该受体的主要调节亚基,其跨膜片段M2是一个面向胞质向膜内反折的膜襻区,形成离子通道的内壁,决定了NMDA受体离子通道对ca2+的通透性.谷氨酸主要通过钙超载介导细胞损伤,因此,NR2B亚基在缺血性卒中神经元凋亡中起着至关重要的作用.     

Nonpsychotropic cannabinoid acts as a functional N-methyl-D-aspartate receptor blocker.

Binding studies using the enantiomers of the synthetic cannabinoid 7-hydroxy-delta 6-tetrahydrocannabinol 1,1-dimethylheptyl homolog in preparations of rat brain cortical membranes reveal that the (+)-(3S,4S) enantiomer HU-211 blocks N-methyl-D-aspartate (NMDA) receptors in a stereospecific manner and that the interaction occurs at binding sites distinct from those of other noncompetitive NMDA antagonists or of glutamate and glycine. Moreover, HU-211 induces stereotype and locomotor hyperactivity in mice and tachycardia in rat, effects typically caused by NMDA receptor antagonists. HU-211 is also a potent blocker of NMDA-induced tremor, seizures, and lethality in mice. This compound may therefore prove useful as a nonpsychoactive drug that protects against NMDA-receptor-mediated neurotoxicity.     

Behavioral stress modifies hippocampal plasticity through N-methyl-D-aspartate receptor activation.

Behavioral stress has detrimental effects on subsequent cognitive performance in many species, including humans. For example, humans exposed to stressful situations typically exhibit marked deficits in various learning and memory tasks. However, the underlying neural mechanisms by which stress exerts its effects on learning and memory are unknown. We now report that in adult male rats, stress (i.e., restraint plus tailshock) impairs long-term potentiation (LTP) but enhances long-term depression (LTD) in the CA1 area of the hippocampus, a structure implicated in learning and memory processes. These effects on LTP and LTD are prevented when the animals were given CGP39551 (the carboxyethylester of CGP 37849; DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, before experiencing stress. In contrast, the anxiolytic drug diazepam did not block the stress effects on hippocampal plasticity. Thus, the effects of stress on subsequent LTP and LTD appear to be mediated through the activation of the NMDA subtype of glutamate receptors. Such modifications in hippocampal plasticity may contribute to learning and memory impairments associated with stress.     

N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-D-aspartate receptor activity

The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G protein-coupled receptors is well documented but less is known about its biologically active metabolite, N-desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of N-desmethylclozapine on cloned human M1-M5 muscarinic receptors. N-desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC50 of 55 nM and was a more potent partial agonist (EC50, 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that N-desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of N-methyl-d-aspartate (NMDA) receptor-driven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of N-desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices. N-desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite N-desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that N-desmethylclozapine contributes to clozapine's clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.     

Preservation of N-methyl-D-aspartate receptor binding sites with age in rat neocortex

This study used [3H]dizocilpine ([3H]MK-801) binding to examine glycine, polyamine, and zinc subsites of the N-methyl-D-aspartate (NMDA) receptor in well-washed membranes derived from the neocortex of Fischer 344/Norwegian brown rats aged 3, 12, 24 and 37 months. [3H]dizocilpine binding in the presence of 100 microM glutamate was enhanced by the addition of 30 microM glycine. Binding in the presence of both glutamate and glutamate plus glycine were unaffected by age. The competitive polyamine site antagonist arcaine inhibited [3H]dizocilpine binding in a dose-dependent fashion and 50 microM spermidine caused a rightward shift in this dose response curve. IC50 values derived from these plots were not significantly affected by age. Similarly, zinc inhibited binding in a dose-dependent fashion and was also unaffected by age. These data indicate that the NMDA receptor is spared in aging.     

A mutation that alters magnesium block of N-methyl-D-aspartate receptor channels.

N-Methyl-D-aspartate (NMDA) receptors are blocked at hyperpolarizing potentials by extracellular Mg ions. Here we present a detailed kinetic analysis of the Mg block in recombinant wild-type and mutant NMDA receptors. We find that the Mg binding site is the same in the wild-type and native hippocampal NMDA receptor channels. In the mutant channels, however, Mg ions bind with a 10-fold lower affinity. On the basis of these results, we propose that the energy well at the Mg binding site in the mutants is shallow and the binding is unstable because of an increase in the rate of dissociation. We postulate that the dipole formed by the amide group of asparagine 614 of the epsilon 1 subunit contributes to the structure of the binding site but predict that additional ligands will be involved in coordinating Mg ions.     

Alcohol inhibition of the NMDA receptor function, long-term potentiation, and fear learning requires striatal-enriched protein tyrosine phosphatase

Alcohol's deleterious effects on memory are well known. Acute alcohol-induced memory loss is thought to occur via inhibition of NMDA receptor (NMDAR)-dependent long-term potentiation in the hippocampus. We reported previously that ethanol inhibition of NMDAR function and long-term potentiation is correlated with a reduction in the phosphorylation of Tyr(1472) on the NR2B subunit and ethanol's inhibition of the NMDAR field excitatory postsynaptic potential was attenuated by a broad spectrum tyrosine phosphatase inhibitor. These data suggested that ethanol's inhibitory effect may involve protein tyrosine phosphatases. Here we demonstrate that the loss of striatal-enriched protein tyrosine phosphatase (STEP) renders NMDAR function, phosphorylation, and long-term potentiation, as well as fear conditioning, less sensitive to ethanol inhibition. Moreover, the ethanol inhibition was "rescued" when the active STEP protein was reintroduced into the cells. Taken together, our data suggest that STEP contributes to ethanol inhibition of NMDAR function via dephosphorylation of tyrosine sites on NR2B receptors and lend support to the hypothesis that STEP may be required for ethanol's amnesic effects.     

Liposome reconstitution and modulation of recombinant N-methyl-D-aspartate receptor channels by membrane stretch

In this study, the heteromeric N-methyl-D-aspartate (NMDA) receptor channels composed of NR1a and NR2A subunits were expressed, purified, reconstituted into liposomes, and characterized by using the patch clamp technique. The protein exhibited the expected electrophysiological profile of activation by glutamate and glycine and internal Mg2+ blockade. We demonstrated that the mechanical energy transmitted to membrane-bound NMDA receptor channels can be exerted directly by tension developed in the lipid bilayer. Membrane stretch and application of arachidonic acid potentiated currents through NMDA receptor channels in the presence of intracellular Mg2+. The correlation of membrane tension induced by either mechanical or chemical stimuli with the physiological Mg2+ block of the channel suggests that the synaptic transmission can be altered if NMDA receptor complexes experience local changes in bilayer thickness caused by dynamic targeting to lipid microdomains, electrocompression, or chemical modification of the cell membranes. The ability to study gating properties of NMDA receptor channels in artificial bilayers should prove useful in further study of structure-function relationships and facilitate discoveries of new therapeutic agents for treatment of glutamate-mediated excitotoxicity or analgesic therapies.