PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A

Our previous study has shown that the phases of circadian rhythms of ocular melatonin and dopamine are always opposite and intraocular melatonin injection suppresses dopamine release. Therefore, it is possible that dopamine rhythms result from inhibitory action of melatonin. We have examined this possibility in the following experiments. In the first experiment effects of continuous light on melatonin and dopamine release were examined. The data indicated that continuous light exposure resulted in loss of circadian rhythmicity of melatonin and dopamine by suppressing melatonin and enhancing dopamine levels throughout the day. To further examine the effects of light in the second experiment, 2 h light pulse was applied during the night, then temporal changes of melatonin and dopamine release were studied. The light pulse rapidly suppressed melatonin release, whereas it rapidly increased dopamine release. These changes occurred within 30 min in both melatonin and dopamine. However, the recovery after the cessation of the light stimulus was slower in melatonin than dopamine. In the third experiment it was tested if dopamine release was increased by lowering melatonin release with an intraocular injection of the D2 agonist, quinpirol. Although quinpirol strongly inhibited melatonin release independently of the time of injection, dopamine did not always increase by the inhibition of melatonin. These results indicate that ocular dopamine rhythms are not simply produced by melatonin inhibitory action.     

Neuronal high-affinity glutamate transport in the rat central nervous system

EAAC1 is a neuronal and epithelial high affinity glutamate transporter previously cloned from rabbit intestine. Here we report the isolation of EAAC 1 from rat brain* and its expression in the central nervous system based on in situ hybridization. Strong signals were detected in brain, spinal cord and retina. Expression of EAAC1 was particularly strong in pyramidal cells of the cerebral cortex, pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, various thalamic nuclei and cells of certain retinal layers. EAAC1 was also expressed in non-glutamatergic neurons such as GABAergic cerebellar Purkinje cells and alpha-motor neurons of the spinal cord. We propose that EAAC1 is not only involved in the sequestration of glutamate at glutamatergic synapses and in protecting neurons from glutamate excitotoxicity, but also in the cellular metabolism involving glutamate.     

Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).     

NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex

NMDA receptors play important roles in learning and memory and in sculpting neural connections during development. After the period of peak cortical plasticity, NMDA receptor-mediated EPSCs (NMDAR EPSCs) decrease in duration. A likely mechanism for this change in NMDA receptor properties is the molecular alteration of NMDA receptor structure by regulation of NMDA receptor subunit gene expression. The four modulatory NMDAR2A-D (NR2A-D) NMDA receptor subunits are known to alter NMDA receptor properties, and the expression of these subunits is regulated developmentally. It is unclear, however, how the four NR2 subunits are expressed in individual neurons and which NR2 subunits are important to the regulation of NMDA receptor properties during development in vivo. Analysis of NR2 subunit gene expression in single characterized neurons of postnatal neocortex revealed that cells expressing NR2A subunit mRNA had faster NMDAR EPSCs than cells not expressing this subunit, regardless of postnatal age. Expression of NR2A subunit mRNA in cortical neurons at even low levels seemed sufficient to alter the NMDA receptor time course. The proportion of cells expressing NR2A and displaying fast NMDAR EPSCs increased developmentally, thus providing a molecular basis for the developmental change in mean NMDAR EPSC duration.     

Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A

The NMDA (N-methyl-D-aspartate) subclass of glutamate receptor is essential for the synaptic plasticity thought to underlie learning and memory and for synaptic refinement during development. It is currently believed that the NMDA receptor (NMDAR) is a heteromultimeric channel comprising the ubiquitous NR1 subunit and at least one regionally localized NR2 subunit. Here we report the characterization of a regulatory NMDAR subunit, NR3A (formerly termed NMDAR-L or chi-1), which is expressed primarily during brain development. NR3A co-immunoprecipitates with receptor subunits NR1 and NR2 in cerebrocortical extracts. In single-channel recordings from Xenopus oocytes, addition of NR3A to NR1 and NR2 leads to the appearance of a smaller unitary conductance. Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cerebrocortical neurons. These data suggest that NR3A is involved in the development of synaptic elements by modulating NMDAR activity.     

Conditioned eyeblink response is impaired in mutant mice lacking NMDA receptor subunit NR2A

NMDA receptor channels, heteromeric assemblies of subunits with diverse subtypes, play critical roles in various kinds of synaptic plasticity underlying learning and memory. To elucidate the roles of subunits NR2A and NR2C in motor learning, we investigated acquisition of the classically conditioned eyeblink response in a delayed-conditioning paradigm by gene knockout mice. Mutant mice lacking NR2C exhibited no significant defect; however, early acquisition of the task was impaired in mutant mice lacking NR2A or both NR2A and NR2C. Based on the distribution of these subunits in brain, these results indicate that acquisition of the conditioned response does not depend on NMDA receptors in the cerebellar cortex, but that its early acquisition involves the hippocampus and/or cerebellar deep nuclei.     

The NMDA receptor subunit NR2B of neonatal rat brain: complex formation and enrichment in axonal growth cones

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors comprises a family of highly homologous subunits which assemble into oligomeric protein complexes. Alterations in subunit composition are developmentally regulated, leading to functionally distinct receptor populations. Here, the contribution of the subunit NR2B to NMDA receptor complex formation was analysed in neonatal rat brain, employing polyclonal antibodies raised against NR2B-specific synthetic peptides. By hydrodynamic size fractionation of the solubilized receptor protein and chemical cross-linking, NR2B antigen was found to be associated with several protein species of up to 690 kDa molecular weight. These observations show NR2B to be part of a multimeric receptor complex. Fractionation of cortex homogenates from E18 rat embryos on sucrose density gradients revealed NR2B polypeptide to be highly enriched in axonal growth cones. A similar distribution was found by fluorescence microscopy of immature hippocampal neurons, showing a preferential accumulation of NR2B antigen in axonal growth cones and varicosities. In mature cells, NR2B antigen displayed a punctated distribution pattern with redistribution to somato-dendritic spheres. The association of NR2B with axonal growth cones and processes of immature neurons suggests a role of NMDA receptors in the regulation of neurite outgrowth and migration.     

Light and electron microscope distribution of the NMDA receptor subunit NMDAR1 in the rat nervous system using a selective anti-peptide antibody

NMDA receptors play key roles in synaptic plasticity and neuronal development, and may be involved in learning, memory, and compensation following injury. A polyclonal antibody that recognizes four of seven splice variants of NMDAR1 was made using a C-terminus peptide (30 amino acid residues). NMDAR1 is the major NMDA receptor subunit, found in most or all NMDA receptor complexes. On immunoblots, this antibody labeled a single major band migrating at M(r) = 120,000. The antibody did not cross-react with extracts from transfected cells expressing other glutamate receptor subunits, nor did it label non-neuronal tissues. Immunostained vibratome sections of rat tissue showed labeling in many neurons in most structures in the brain, as well as in the cervical spinal cord, dorsal root and vestibular ganglia, and in pineal and pituitary glands. Staining was moderate to dense in the olfactory bulb, neocortex, striatum, some thalamic and hypothalamic nuclei, the colliculi, and many reticular, sensory, and motor neurons of the brainstem and spinal cord. The densest stained cells included the pyramidal and hilar neurons of the CA3 region of the hippocampus, Purkinje cells of the cerebellum, supraoptic and magnocellular paraventricular neurons of the hypothalamus, inferior olive, red nucleus, lateral reticular nucleus, peripheral dorsal cochlear nucleus, and motor nuclei of the lower brainstem and spinal cord. Ultrastructural localization of immunostaining was examined in the hippocampus, cerebral cortex, and cerebellar cortex. The major staining was in postsynaptic densities apposed by unstained presynaptic terminals with round or mainly round vesicles, and in associated dendrites. The pattern of staining matched that of previous in situ hybridization but differed somewhat from that of binding studies, implying that multiple types of NMDA receptors exist. Comparison with previous studies of localization of other glutamate receptor types revealed that NMDAR1 may colocalize with these other types in many neurons throughout the nervous system.     

Visualisation of non-glycosylated somatostatin receptor two (ngsst2) immunoreactivity in the rat central nervous system

The biological actions of the neuropeptides somatostatin-14 and -28 are receptor-mediated. To date, five G protein-coupled receptors sst1 to sst5 have been characterised pharmacologically and their genes have been cloned. In this study, we used an affinity-purified polyclonal antibody (AS-68) raised against a specific N-terminal peptide sequence of sst2 to localise N-terminal sst2-immunoreactive regions in the rat brain and the cervical spinal cord. The specificity of the antiserum was demonstrated by Western and slot blotting experiments using a N-terminal sst2 fusion protein. Further blotting experiments with a sst2(A)-transfected cell line and rat CNS membrane proteins showed that the antibody detected the non-glycosylated and/or non-sialated receptor. A strong signal using an sst2(A)-transfected CHO-K1 cell line was obtained only if the cells had been treated with N-Glycosidase F prior to the immunochemical detection. Two variants of sst2 (sst2(A) and sst2(B)) have been identified by cloning procedures and gene expression studies in the rodents. They differ in their carboxy-termini: AS-68 would, however, be able to recognise the non-glycosylated form of both these variants. We present here the central nervous system distribution of non-glycosylated sst2-immunoreactivity in the rat using this N-terminal antibody. The sst2 non-glycosylated N-terminal like immunoreactivity was distributed throughout the brain with cells and processes labelled in the cerebral cortex and the basal ganglia (neostriatum, substantia nigra), in the limbic system (hippocampal formation, amygdala), in the diencephalon (epithalamus, thalamus, hypothalamus), the superior colliculus, the periaqueductal grey matter and some of the reticular formation nuclei. The distribution of the non-glycosylated sst2-like immunoreactivity detected here was consistent with that predicted from the localisation of sst2 mRNA and SRIF-ligand binding studies.     

Status spongiosus of rat central nervous system induced by actinomycin D

The effect on central myelin of Actinomycin D, an RNA--and, secondarily, a protein-synthesis inhibitor, has been studied by light and electron microscopy. The intracranial injection of this drug produced an extensive status spongiosus of the white matter in the cerebrum, cerebellum, brain stem and optic nerve within 48 h. The status spongiosus was due to vacuole formation within the myelin sheath and to enlargement of the extracellular space. Three types of vacuoles were observed: (a) the most common varieties formed between the inner tongue and the remainder of the myelin sheath; (b) a second variety formed by enlargement of the periaxonal space with separation of the axon from its myelin sheath, and (c) a less common type of vacuolization was due to splitting of the myelin lamellae at the interperiod line to form large intramyelinic vacuoles. Myelinic vacuoles were preceded by nuclear and cytoplasmic changes in oligodendrocytes, which included nucleolar segregation, disaggregation, and diminution in number of ribosomes. These changes were similar to those previously reported in a variety of cells exposed to Actinomycin D. It is suggested that myelin vacuoles result secondarily from the Actinomycin D inhibitory effect on oligodendroglial RNA--and protein-synthesis, rather than from a direct effect of this drug on the myelin sheath.     

1,25-Dihydroxyvitamin D3 receptors in the central nervous system of the rat embryo

The major aim was to identify predictors of the large age differences that exist in eyeblink classical conditioning. Eyeblink conditioning was assessed in 190 participants over the age range of 20-89 years, with 150 trained in the paired condition and 40 trained in the explicitly unpaired control condition. Timed-interval tapping was used to assess cerebellar function. Blink reaction time and explicit learning and memory were also assessed. Stepwise multiple regression indicated that the effect of age accounted for the largest proportion of the variance, but the cerebellar measure also predicted eyeblink conditioning at a significant level. Reaction time and explicit memory measures did not account for a significant amount of the variance in eyeblink conditioning. Age-related effects in the cerebellum apparently affect timing and learning in normal adults.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

Subcellular and subsynaptic distribution of the NR1 subunit of the NMDA receptor in the neostriatum and globus pallidus of the rat: co-localization at synapses with the GluR2/3 subunit of the AMPA receptor

Glutamatergic neurotransmission in the neostriatum and the globus pallidus is mediated through NMDA-type as well as other glutamate receptors and is critical in the expression of basal ganglia function. In order to characterize the cellular, subcellular and subsynaptic localization of NMDA receptors in the neostriatum and globus pallidus, multiple immunocytochemical techniques were applied using antibodies that recognize the NR1 subunit of the NMDA receptor. In order to determine the spatial relationship between NMDA receptors and AMPA receptors, double labelling was performed with the NR1 antibodies and an antibody that recognizes the GluR2 and 3 subunits of the AMPA receptor. In the neostriatum all neurons with characteristics of spiny projection neurons, some interneurons and many dendrites and spines were immunoreactive for NR1. In the globus pallidus most perikarya and many dendritic processes were immunopositive. Immunogold methods revealed that most NR1 labelling is associated with asymmetrical synapses and, like the labelling for GluR2/3, is evenly spread across the synapse. Double immunolabelling revealed that in neostriatum, over 80% of NR1-positive axospinous synapses are also positive for GluR2/3. In the globus pallidus most NR1-positive synapses are positive for GluR2/3. In both regions many synapses labelled only for GluR2/3 were also detected. These results, together with previous data, suggest that NMDA and AMPA receptor subunits are expressed by the same neurons in the neostriatum and globus pallidus and that NMDA and AMPA receptors are, at least in part, colocalized at individual asymmetrical synapses. The synaptic responses to glutamate in these regions are thus likely be mediated by both AMPA and NMDA receptors at the level of individual synapses.     

Attenuation of focal ischemic brain injury in mice deficient in the epsilon1 (NR2A) subunit of NMDA receptor

The role of glutamate neurotoxicity in cerebral ischemia has long been advocated but still remains controversial, because various glutamate receptor (GluR) antagonists showed inconsistent protective efficacy in brain ischemia models. To address this central issue of ischemic brain damage more directly, we used mutant mice deficient in the GluRepsilon1 (NR2A) subunit of NMDA receptor with or without additional heterozygous mutation in the GluRepsilon2 (NR2B) subunit. Those mutant mice, as well as their littermates, were subjected to focal cerebral ischemia by introducing a 6-0 nylon suture from left common carotid artery. Brain injury volumes after 2 hr of suture insertion, as evaluated by 2,3,5-triphenyltetrazolium chloride staining at 24 hr after ischemia, revealed significantly smaller injury size in GluRepsilon1 subunit knock-out mice compared with their wild-type littermates. The reduction in injury volume was not attributable to differences in body temperature or in blood flow during ischemia. Additional heterozygous GluRepsilon2 subunit disruption did not result in further reduction in injury volume. These data directly demonstrate relevance of NMDA receptor-mediated tissue injury after brain ischemia and provide evidence that GluRepsilon1 subunit is involved in these injurious mechanisms.     

Neurotrophin regulation of energy homeostasis in the central nervous system

Our hypothesis is that one cause of neuronal cell death and shrinkage in the aged central nervous system is an inability of neurons to maintain oxidant homeostasis in the face of increased levels of reactive oxygen species, decreased endogenous antioxidants, and impaired energy metabolism associated with physiological senescence, Alzheimer's, and Parkinson's diseases. Since treatment with nerve growth factor (NGF) reverses behavioral impairments in aged rats and stimulates cholinergic activity in the basal forebrain, while brain-derived neurotrophic factor appears to play a similar role in the striatum, we propose that neurotrophin-mediated cell-sparing reflects effects on oxidant homeostasis. Neurotrophins may play a similar cell-sparing role in hypoxic/ischemic injury to the nervous system, which also is mediated in part by reactive oxygen species. The degradation of one such species, H2O2, is catalyzed by catalase and glutathione peroxidase (GSH Px). The activity of the latter enzyme is dependent on glutathione reductase and the availability of NADPH for regeneration of reduced GSH. The GSH redox cycle is also regulated by enzymes of the hexose monophosphate shunt. NGF protects PC12 cells from H2O2 injury by stimulating the synthesis of antioxidant enzymes including catalase, GSH Px, glucose-6-phosphate dehydrogenase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. NGF also enhances recovery from the NAD+ losses occurring as a consequence of H2O2 treatment.     

Developmental failures of blood vessels within central nervous system

Improved prevention and detection methods, as well as advances in medical treatment have resulted in a trend of increasing numbers of cancer survivors. Today, there are over 8 million cancer survivors, 5 million of whom were diagnosed 5 or more years ago. Cancer, once thought of as an acute disease, is increasingly being classified as a chronic disease. This article reviews physical, psychological, and social aspects of cancer survivorship, as well as other issues related to a diagnosis of cancer.     

Microperoxisome distribution in the central nervous system of the rat

The distribution of microperoxisomes was studied in areas of the central nervous system having high concentrations of catecholaminergic neurons and in areas lacking this neuron type, using the alkaline DAB cytochemical method for catalase. Substantial numbers of microperoxisomes are found in neurons in the locus coeruleus and in nucleus A1 of the medulla, as well as in the substantia nigra, whereas few catalase-reactive bodies are seen in neurons of the cerebrum and cerebellum. The number of catalase-reactive microperoxisomes per unit area in the catecholaminergic neurons of the CNS is comparable to the number seen previously in neurons of the peripheral cervical sympathetic ganglia. Some spinal cord neurons also contain reactive microperoxisomes. Catalase-reactive microperoxisomes are numerous in oligodendrocytes of all areas studied, and in ependymal cells bordering the third and fourth ventricles. Astrocytes contain few reactive structures in the cytoplasm near the nucleus, but they are readily found in astrocytic processes and end-feet.     

Adriamycin-induced oxidative stress in rat central nervous system

Adriamycin elicited a stimulation of rat central nervous system lipid peroxidation, both in vivo and in vitro, as evidenced by the increase in the content of thiobarbituric acid reactants, which was found to be NADPH-dependent. The antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were seen to decrease on exposure to adriamycin (1 mg/kg for a period of 7 days), together with a significant decrement in the GSH/GSSG ratio, thus contributing to the oxidative insult to the tissue. The in vitro addition of GSH or vitamin E to brain homogenates offered protection against adriamycin-induced lipid peroxidation, suggesting that supplementation with these antioxidants could improve the therapeutic value of the drug.