NR2B Protein Levels Research Articles

Different trends in modulation of NMDAR1 and NMDAR2B gene expression in cultured cortical and hippocampal neurons after lead exposure

Exposure to heavy metal lead (Pb 2+) has been reported to cause problems in cognitive functions of the brain, e.g. memory loss and difficulties in mental development. N-Methyl- d-aspartate receptors (NRs) are important molecules that are known to be involved in mediation of learning and memory. In order to investigate the effects of Pb 2+ on the gene expression of NR1 and NR2B subunits in neurons, primary cell cultures of rat cortical and hippocampal neurons were employed. After treatments with different concentrations of Pb 2+ ions in culture medium (0, 5, 10, 25 and 50 μM), the cellular localization of Pb 2+ in neurons was evaluated by laser scan confocal microscopy by using a Pb 2+ ion specific fluorescence probe. In addition, the gene expression of NR1 and NR2B subunits was determined by reverse transcriptase-polymerase chain reaction, immunofluorescence and Western blotting. The results of the present study showed that both cortical and hippocampal neurons accumulated intracellular Pb 2+ in accordance with the concentrations of Pb 2+ ions present in the culture medium. After Pb 2+ treatments, levels of NR1 mRNA, immunoreactivity and protein were found to be unchanged but levels of NR2B mRNA, immunoreactivity and protein were found to be significantly increased in cortical neurons. In contrast, both NR1 and NR2B mRNAs, immunoreactivity and proteins were found to be significantly decreased in hippocampal neurons. The changes in gene expression were found to be dose dependent in accordance with the Pb 2+ concentrations. The present results indicate that Pb 2+ has a differential effect on the expression of NR1 and NR2B subunits in cortical and hippocampal neurons, respectively. It is likely that the toxic effects of Pb 2+ may cause differential damage to different types of memory that are mediated by cortical and hippocampal neurons, respectively.

Differential effects of enrichment on learning and memory function in NR2B transgenic mice

It has been known that environmental enrichment leads to better learning and memory in mice. However, the molecular mechanisms are not known. In this study, we used the 10th–12th of the NR2B transgenic (Tg) lines, in which the NMDA receptor function is enhanced via the NR2B subunit transgene in neurons of the forebrain, to test the hypothesis of the involvement of NMDA receptor function in enrichment-induced better learning and memory. Consistent with our previous results, both larger long-term potentiation (LTP) in the hippocampus and superior learning and memory were observed in naive NR2B Tg mice even after the 10th–12th generation of breeding. After enrichment, wild-type mice exhibited overall improvement in their performances in contextual and cued conditioning, fear extinctions, and novel object recognition tasks. Interestingly, the same enrichment procedures could not further increase the performance of NR2B Tg mice in contextual conditioning, cued conditioning, or fear extinction, thereby indicating that enhanced NMDA receptor function can occlude these enrichment effects. However, we found that in the novel object recognition task enriched NR2B Tg mice exhibited much longer recognition memory (up to 1 week), compared to that (up to 3 days) in naive NR2B Tg mice. Furthermore, our biochemical experiments showed that enrichment significantly increased protein levels of GluR1, NR2B, and NR2A subunits of glutamate receptors in both wild-type and NR2B Tg mice. Therefore, our results suggest an interactive nature of molecular pathways involved in both environmental and genetic NMDA receptor manipulations for enhancing learning and memory.

Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats.

The NMDA receptor (NMDAR) has been implicated in the induction of LTP at hippocampal synapses, and has been proposed to play a significant role in the involvement of the hippocampus with learning and memory. Aged rats are known to have deficits in LTP, learning and memory. We tested the hypothesis that aged rats might have deficits in expression of NMDAR subunits. Aged rats have significantly lower levels of NR2B mRNA and protein compared to young animals. This complements a recent report which showed improved learning and memory in mice which overexpress NR2B. No changes were seen in either the mRNA or the protein levels of the NMDAR subunit NR2A, nor in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate receptor (AMPAR) subunit GluR2. Our data support the hypothesis that age related alterations in the expression of the NMDAR NR2B subunit might underlie deficits in LTP and learning and memory in aged animals.

Osmotic activation of the hypothalamo-neurohypophysial system reversibly downregulates the NMDA receptor subunit, NR2B, in the supraoptic nucleus of the hypothalamus

NMDA receptor activation produces a characteristic pattern of neuronal firing in magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus of the hypothalamus (SON) which has been associated with greater hormone release in vivo and in vitro. In addition, i.c.v. administered NMDA receptor blockers suppress the dehydration-induced rise in plasma vasopressin and drinking. To investigate the role of NMDA receptor subunits in the neuroendocrine functions of the magnocellular neuroendocrine cells of the hypothalamus, we examined the effects of osmotic stimulation on the protein expression of the NMDA receptor subunits, NR1 and NR2B, important in binding glycine and glutamate, respectively. Homogenates of SON, paraventricular nucleus of the hypothalamus (PVN), cortex and lateral hypothalamus from control rats and rats given 2% saline water to drink for 4–10 days were subjected to SDS–PAGE and Western blot analysis. This saline water drinking regimen produced a significant rise in plasma osmolality levels. NR1 and NR2B immunoreactivity was detected in SON, PVN, lateral hypothalamus and cortex but not in liver homogenates using subunit-specific polyclonal antibodies and quantified using computer-assisted densitometry. Mean NR2B immunoreactivity was significantly lower in SON (29%) and PVN homogenates (23%) from saline-treated rats than in those from control rats. In addition, the effect of dehydration on NR2B was regionally specific since no significant changes in NR2B expression were observed in homogenates of cortex and lateral hypothalamus. Rehydration allowed recovery of plasma osmolality as well as NR2B protein levels in the SON. These results suggest that changes in NMDA receptor subunit expression contribute to the plasticity manifested by in magnocellular neuroendocrine cells in response to osmotic activation of the hypothalamo-neurohypophysial system. In addition, our results indicate that NMDA receptors on SON and PVN MNCs may contribute to neuroendocrinological functions associated with body fluid homeostasis.

Biphasic changes in the levels of N-methyl- d-aspartate receptor-2 subunits correlate with the induction and persistence of long-term potentiation

N-Methyl- d-aspartate glutamate receptors (NMDAR) form ion channels made up of polypeptides from two classes of subunits; NR1 is obligatory for function whereas members of the NR2 class regulate the properties of the channel. Long-term potentiation (LTP) of synaptic transmission is an event largely dependent on NMDAR activation, and is studied as the primary cellular model of memory in the mammalian brain. While there has been a focus on non-NMDARs in mediating the expression of LTP, we report here biochemical evidence for plasticity of the NMDAR that is associated with LTP persistence in awake animals. Following the establishment of LTP in perforant path synapses of the dentate gyrus, we observed a rise in NR2B protein levels 48 h post-tetanus which was dependent upon activation of NMDARs during the tetanization, and which strongly correlated with the degree of LTP measured at this time-point. We also observed a transient increase in both NR2B and NR2A protein levels 20 min post-tetanus that returned to control levels by 4 h. These early increases were not observed in anaesthetized animals which do not sustain persistent LTP. Our data demonstrate a marked plasticity of NMDAR subunit expression, which may affect LTP persistence, as well as the subsequent ability to induce LTP at previously activated synapses.

Developmental plasticity of NR1 and NR2B subunit expression in the supraoptic nucleus of the rat hypothalamus

Vasopressin and oxytocin neuroendocrine cells within the supraoptic nucleus of the adult hypothalamus (SON) display mRNA expression for the NMDA receptor subunits, NR1 and NR2B, NR2C and NR2D. The NR2B subunit confers slow decay kinetics (relative to NR1/NR2A receptors) and high magnesium sensitivity to NMDA receptor responses—properties which may contribute to the NMDA receptor-mediated bursting manifested by these cells. Therefore, we examined NR2B protein expression and its developmental profile in the SON and compared it to that in the cortex and cerebellum—areas which have been studied previously. We performed Western blot analysis on SON homogenates from embryonic, postnatal (PN7, 14, 21), and adult rats using an NR2B-specific antibody. Adult NR2B levels in the SON and PVN were similar but low relative to those of cortex. SON NR2B protein levels rose in the first postnatal week, remained high through PN21, and later declined to significantly lower levels in the adult. A similar profile was observed in cerebellum, where NR2B expression displayed a sharp peak at PN14 and later declined to minimal or undetectable levels in the adult. In contrast, NR2B continued to be overexpressed through adulthood in the cortex. The ontogenic pattern for NR1 expression, which included upregulation during early postnatal life and adulthood, was similar in the SON and cortex. A different pattern was observed for the cerebellum, where NR1 levels increased gradually after ED17 to reach significantly greater adult levels. Of all three areas studied, the SON displayed the earliest developmental rise in NR1 levels. SON explant cultures proved to be a useful preparation, since they contained neurons which synthesized NR1 and NR2B subunits in quantities similar to those of ED17 SON. Our findings suggest that NMDA receptors on SON neuroendocrine cells are assembled using NR1 and NR2B subunits, and that their plastic expression in early postnatal life may play a role during development.

Characterization of NMDA Receptor Subunit‐Specific Antibodies: Distribution of NR2A and NR2B Receptor Subunits in Rat Brain and Ontogenic Profile in the Cerebellum

Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at approximately 175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, mid-brain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and mid-brain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [3H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth.(ABSTRACT TRUNCATED AT 250 WORDS)