CGX-1007 prevents excitotoxic cell death via actions at multiple types of NMDA receptors

Glutamate induced excitotoxic injury through over-activation of N-methyl-D-aspartate receptors (NMDARs) plays a critical role in the development of many neurodegenerative diseases. The present study was undertaken to evaluate the role of CGX-1007 (Conantokin G) as a neuroprotective agent against NMDA-induced excitotoxicity. Conantokin G, a cone snail peptide isolated from Conus geographus is reported to selectively inhibit NR2B containing NMDARs with high specificity and is shown to have potent anticonvulsant and antinociceptive effects. CGX-1007 significantly reduced the excitotoxic cell death induced by NMDA in organotypic hippocampal brain slice cultures in a concentration-dependent manner. In contrast, ifenprodil, another NR2B specific antagonist failed to offer neuroprotection against NMDA-induced excitotoxicity. We further determined that the neuroprotection observed is likely due to the action of CGX-1007 at multiple NMDA receptor subtypes. In a series of electrophysiology experiments, CGX-1007 inhibited NMDA-gated currents in human embryonic kidney (HEK) 293 cells expressing NMDA receptors containing either NR1a/NR2B or NR1a/NR2A subunit combinations. CGX-1007 produced a weak inhibition at NR1a/NR2C receptors, whereas it had no effect on NR1a/NR2D receptors. Further, the inhibition of NMDA receptors by CGX-1007 was voltage-dependent with greater inhibition seen at hyperpolarized membrane potentials. The voltage-dependence of CGX-1007 activity was also observed in recordings of NMDA-gated currents evoked in native receptors expressed in cortical neurons in culture. Based on our results, we conclude that CGX-1007 is a potent neuroprotective agent that acts as an antagonist at both NR2A and NR2B containing receptors.     

Preferential inhibition by antidiarrheic 2‐methoxy‐4‐methylphenol of Ca2+ influx across acquired N‐methyl‐D‐aspartate receptor channels composed of NR1/NR2B subunit assembly

In our previous studies, particular phenolic ingredients, such as 2‐methoxy‐4‐methylphenol (2M4MP), of the antidiarrheic drug wood creosote significantly prevented cell death by both hydrogen peroxide and glutamate in cultured rat hippocampal neurons. In this study, we further evaluated the pharmacological properties of 2M4MP on Ca²⁺ influx across native and acquired N‐methyl‐D‐aspartate (NMDA) receptor (NMDAR) channels. The addition of 2M4MP significantly prevented the loss of cellular viability and the increase in intracellular free Ca²⁺ levels as determined by Fluo‐3 in cultured rat hippocampal neurons briefly exposed to NMDA. Brief exposure to NMDA also led to a marked increase in mitochondrial free Ca²⁺ levels determined by Rhod‐2, in addition to intracellular free Ca²⁺ levels, in HEK293 cells expressing either NR1/NR2A or NR1/NR2B subunit channels. The further addition of the general NMDAR channel blocker dizocilpine similarly inhibited the increase of intracellular Ca²⁺ levels by NMDA in both types of acquired NMDAR channels, whereas the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in HEK293 cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similarly, 2M4MP significantly and selectively inhibited the NMDA‐induced influx of Ca²⁺ across acquired NR1/NR2B channels in a concentration‐dependent manner. Moreover, prior daily oral administration of 2M4MP significantly reduced the infarct volume in the ipsilateral cerebral hemisphere in rats with middle cerebral artery occlusion 1 day after reperfusion. These results suggest that 2M4MP may protect neurons from excitotoxicity through preferential inhibition of Ca²⁺ influx across NMDAR channels composed of NR1/NR2B subunits. © 2010 Wiley‐Liss, Inc.     

Mapping the binding site of the neuroprotectant ifenprodil on NMDA receptors

Ifenprodil is a noncompetitive antagonist of NMDA receptors highly selective for the NMDA receptor 2B (NR2B) subunit. It is widely used as a pharmacological tool to discriminate subpopulations of NMDA receptors, and derivatives are currently being developed as candidate neuroprotectants. Despite numerous studies on the mechanism of action of ifenprodil on NMDA receptors, the structural determinants responsible for the subunit selectivity have not been identified. By combining functional studies on recombinant NMDA receptors and biochemical studies on isolated domains, we now show that ifenprodil binds to the N-terminal leucine/isoleucine/valine-binding protein (LIVBP)-like domain of NR2B. In this domain, several residues, both hydrophilic and hydrophobic, were found to control ifenprodil inhibition. Their location in a modeled three-dimensional structure suggests that ifenprodil binds in the cleft of the LIVBP-like domain of NR2B by a mechanism (Venus-flytrap) resembling that of the binding of Zn on the LIVBP-like domain of NR2A. These results reinforce the proposal that the LIVBP-like domains of NMDA receptors, and possibly of other ionotropic glutamate receptors, bind modulatory ligands. Moreover, they identify the LIVBP-like domain of the NR2B subunit as a promising therapeutic target and provide a framework for designing structurally novel NR2B-selective antagonists.     

The Neuroprotective Peptide Poly-Arginine-12 (R12) Reduces Cell Surface Levels of NMDA NR2B Receptor Subunit in Cortical Neurons; Investigation into the Involvement of Endocytic Mechanisms

We have previously reported that cationic poly-arginine and arginine-rich cell-penetrating peptides display high-level neuroprotection and reduce calcium influx following in vitro excitotoxicity, as well as reduce brain injury in animal stroke models. Using the neuroprotective peptides poly-arginine R12 (R12) and the NR2B9c peptide fused to the arginine-rich carrier peptide TAT (TAT-NR2B9c; also known as NA-1), we investigated the mechanisms whereby poly-arginine and arginine-rich peptides reduce glutamate-induced excitotoxic calcium influx. Using cell surface biotin protein labeling and western blot analysis, we demonstrated that R12 and TAT-NR2B9c significantly reduced cortical neuronal cell surface expression of the NMDA receptor subunit NR2B. Chemical endocytic inhibitors used individually or in combination prior to glutamate excitotoxicity did not significantly affect R12 peptide neuroprotective efficacy. Similarly, pretreatment of neurons with enzymes to degrade anionic cell surface proteoglycans, heparan sulfate proteoglycan (HSPG), and chondroitin sulfate proteoglycan (CSPG), as well as sialic acid residues, did not significantly affect peptide neuroprotective efficacy. While the exact mechanisms responsible for R12 peptide-mediated NMDA receptor NR2B subunit cell surface downregulation were not identified, an endocytic process could not be ruled out. The study supports our hypothesis that arginine-rich peptides reduce excitotoxic calcium influx by reducing the levels of cell surface ion channels.     

Toll‐like receptor 4 differentially regulates adult hippocampal neurogenesis in an age‐ and sex‐dependent manner

Toll‐like receptor 4 (TLR4) is primarily responsible for initiating an immune response following pathogen recognition. However, TLR4 is also expressed on neural progenitor cells and has been reported to regulate hippocampal neurogenesis as young male TLR4 knockout mice show increases in cell proliferation and doublecortin positive cells. Whether these effects occur in both sexes and are sustained with normal aging is currently unknown. The present study evaluated whether TLR4 deficiency alters adult hippocampal neurogenesis in young (3–4 months) and aged (18–20 months), male and female, TLR4 deficient (TLR4?/?; B6.B10ScN‐Tlr4lps‐del/JthJ) and wild type (WT) mice. Additionally, neurogenesis within the dorsal and the ventral hippocampal subdivisions was evaluated to determine if TLR4 has differential effects across the hippocampus. Bromodeoxyuridine (BrdU) was administered to quantify new cell survival as well as cell differentiation. Ki‐67 was measured to evaluate cell proliferation. Results show that young TLR4?/? females had higher rates of proliferation and neuronal differentiation in both the dorsal and ventral hippocampus relative to WT females. Young TLR4?/? males show elevated proliferation and neuronal differentiation mainly in the ventral hippocampus. While young TLR4?/? mice show enhanced neurogenesis compared to young WT mice, the increase was not apparent in the aged TLR4?/? mice. Both aged WT and TLR4?/? mice showed a decrease in proliferation, new cell survival, and neuronal differentiation compared to young WT and TLR4?/? mice. The data collectively indicate that TLR4 regulates hippocampal neurogenesis in young adults, but that these effects are region‐specific in males and that females show broader changes in neurogenesis throughout the hippocampus.     

NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice

Previous research has shown that peripheral inflammation and peripheral nerve injury alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices. Peripheral nerve injury-induced changes in the pharmacological and electrophysiological properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 microM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation.     

Hepatocyte growth factor improves synaptic localization of the NMDA receptor and intracellular signaling after excitotoxic injury in cultured hippocampal neurons

To examine the effects of HGF on synaptic densities under excitotoxic conditions, we investigated changes in the number of puncta detected by double immunostaining with NMDA receptor subunits and presynaptic markers in cultured hippocampal neurons. Exposure of hippocampal neurons to excitotoxic NMDA (100 muM) decreased the synaptic localization of NMDA receptor subunit NR2B, whereas synaptic NR1 and NR2A clusters were not altered. Colocalization of PSD-95, a scaffolding protein of the receptor, with the presynaptic protein synapsin I was also decreased after excitotoxicity. Treatment with HGF attenuated these decreases in number. The decrease in the levels of surface NR2B subunits following the addition of the excitotoxic NMDA was also attenuated by the HGF treatment. The decrease in CREB phosphorylation in response to depolarization-evoked NMDA receptor activation was prevented by the HGF treatment. These results suggest that HGF not only prevented neuronal cell death but also attenuated the decrease in synaptic localization of NMDA receptor subunits and prevented intracellular signaling through the NMDA receptor.     

神经干细胞分化的神经元离子型谷氨酸受体NMDA亚单位的mRNA和蛋白表达

目的在体外研究由大鼠神经干细胞(NSCs)分化而来神经元细胞中离子型谷氨酸NMDA受体表达。方法分离培养孕14～16d胎鼠皮质和海马神经干细胞，对NSCs进行nestin和分化鉴定。通过RT—PCR、Western blot免疫印迹和免疫组化检测NSCs分化的神经元细胞中离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B的mRNA和蛋白表达。结果从孕14～16d胎鼠大脑中分离培养出NSCs，NSCs分化后的神经元可以表达离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B。结论由NSCs分化而来的神经元能表达离子型谷氨酸NMDA受体。     

Expression of NMDA Receptor mRNAs in Rat Motoneurons is Down-regulated after Axotomy

The cytotoxic effects of glutamate via the N -methyl-D-aspartate (NMDA) receptor have been suggested to take part in the events leading to death of motoneurons after neonatal axotomy. By the use of in situ hybridization and immunohistochemistry we have investigated motoneuron mRNA expression of the NMDA receptor subunits NR1, NR2B and NR2D and of the NR1 subunit protein in two lesion models leading to partial motoneuron death: sciatic nerve transection early postnatally in the rat and ventral root avulsion in the adult rat. The results were compared with a lesion model with no subsequent death of motoneurons, i.e. sciatic nerve transection in the adult rat. All lesions were followed by down-regulation of the mRNAs for all studied subunits in severed motoneuron populations; down-regulation was detectable already at early stages postoperatively before any significant death had taken place. The strongest down-regulation was in fact seen in the lesion with the largest loss of motoneurons (ventral root avulsion). The reduction in the expression of NR1 mRNA was paralleled by a decrease in NR1 subunit protein. We conclude that down-regulation of NMDA receptor subunit expression is part of the acute response to axonal injury in motoneurons, whether or not neuronal death follows, and that the susceptibility of lesioned motoneurons to excitotoxic effects should be highest early after axonal injury.     

NR2B subunit of the NMDA receptor in the basolateral amygdala is necessary for the acquisition of conditioned defeat in Syrian hamsters

Reversible inactivation of the basolateral amygdala (BLA) disrupts the acquisition and expression of conditioned defeat (CD), an ethological model of conditioned fear, suggesting that the BLA may be a critical component of the neural circuit mediating behavioral plasticity associated with the experience of social defeat. We have also shown that this effect is N-methyl-d-aspartic acid (NMDA) receptor-dependent, because infusion of d,l-2-amino-5-phosphovalerate (APV) into the BLA also impairs the acquisition of CD. APV is a non-selective NMDA antagonist, however, thus it disrupts the entire heteromeric receptor complex, making it difficult to distinguish the relative contributions of either the NR2A or NR2B receptor subtypes on the acquisition of CD. There is ample evidence, however, that the NR2B subunit of the NMDA receptor in the amygdala is critical for mediating long-term potentiation and plasticity related to fear learning. The purpose of the present experiment was to determine whether infusion of ifenprodil, a selective antagonist of the NR2B subunit, into the BLA would block the acquisition (but not expression) of CD. In Experiment 1, infusion of ifenprodil immediately before defeat training significantly decreased submissive behaviors and restored territorial aggression when hamsters were later paired with a non-aggressive intruder (NAI). Conversely, infusion of ifenprodil immediately before CD testing failed to inhibit the expression of submissive behaviors in previously defeated hamsters. These results support the hypothesis that the BLA is a critical site for the plasticity underlying social defeat-induced changes in behavior.     

Postnatal switching of NMDA receptor subunits from NR2B to NR2A in rat facial motor neurons

The subunit composition of N-methyl-D-aspartate (NMDA) receptors affects their function under normal and pathological conditions. Functional NMDA receptors are expressed in lower motor neurons, but their subunit composition has not been defined. Here, we employed electrophysiology, quantitative PCR, and immunohistochemistry to investigate the subunit composition of NMDA receptors in postnatal motor neurons of the Wistar rat facial nucleus (FN). Whole-cell patch clamp recordings of acutely dissociated motor neurons from postnatal days 3 and 4 (P3-P4) showed that ifenprodil, a specific antagonist of the NMDA receptor 2B (NR2B) subunit, inhibited 91.62%+/-2.02% of NMDA-induced current, whereas NVP-AAM007, a specific antagonist of the NMDA receptor 2A (NR2A) subunit, inhibited much less of the current (16.69%+/-3.28%). Starting from P5, the inhibitory effects of ifenprodil and NVP-AAM007 gradually decreased and increased, respectively, such that the effect of NVP-AAM007 exceeded that of ifenprodil by P10. At P14, most of the NMDA-induced current was inhibited by NVP-AAM007 (84.59%+/-3.35%). Consistent with this, NR2B mRNA and protein were expressed highly at P3 and then gradually decreased by more than 75% by P14 in FN motor neurons, while NR1 was expressed stably over the same ages. However, NR2A mRNA and protein showed relatively constant levels between P3-P10 and decreased to 45% and 75% of the P3 level, respectively, by P14. Thus, analysis of functional NMDA receptors is critical to revealing subunit switching, which may be an important step in postnatal development of FN motor neurons.     

Forebrain NR2B overexpression enhancing fear acquisition and long‐term potentiation in the lateral amygdala

N‐methyl‐d ‐aspartic acid (NMDA) receptor‐dependent long‐term potentiation (LTP) at the thalamus–lateral amygdala (T‐LA) synapses is the basis for acquisition of auditory fear memory. However, the role of the NMDA receptor NR2B subunit in synaptic plasticity at T‐LA synapses remains speculative. In the present study, using transgenic mice with forebrain‐specific overexpression of the NR2B subunit, we have observed that forebrain NR2B overexpression results in enhanced LTP but does not alter long‐term depression (LTD) at the T‐LA synapses in transgenic mice. To elucidate the cellular mechanisms underlying enhanced LTP at T‐LA synapses in these transgenic mice, AMPA and NMDA receptor‐mediated postsynaptic currents have been measured. The data show a marked increasing in the amplitude and decay time of NMDA receptor‐mediated currents in these transgenic mice. Consistent with enhanced LTP at T‐LA synapses, NR2B‐transgenic mice exhibit better performance in the acquisition of auditory fear memory than wild‐type littermates. Our results demonstrate that up‐regulation of NR2B expression facilitates acquisition of auditory cued fear memory and enhances LTP at T‐LA synapses.     

A genetically modified mouse model probing the selective action of ifenprodil at the N-methyl-D-aspartate type 2B receptor

Selective antagonism of N-methyl-d-aspartate (NMDA) 2B subunit containing receptors has been suggested to have potential therapeutic application for multiple CNS disorders. The amino terminal NR2B residues 1 to 282 were found to be both necessary and sufficient for the binding and function of highly NR2B subunit specific antagonists like ifenprodil and CP-101,606. Using a genetic approach in mice, we successfully replaced the murine NR2B gene function by "knocking-in" (KI) a chimeric human NR2A/B cDNA containing the minimal domain abolishing ifenprodil binding into the endogenous NR2B locus. Patch-clamp recording from hippocampal cultures of the NR2B KI mice demonstrated that their NMDA receptors have reduced sensitivity to both ifenprodil and CP-101,606, as predicted, but also have a lower affinity for glycine. The NR2B KI mice exhibited normal locomotor activity making this ifenprodil-insensitive mouse model a valuable tool to test the specificity of NR2B selective antagonists in vivo.     

Alcohol inhibition of neurogenesis: A mechanism of hippocampal neurodegeneration in an adolescent alcohol abuse model

Adolescents diagnosed with an alcohol use disorder show neurodegeneration in the hippocampus, a region important for learning, memory, and mood regulation. This study examines a potential mechanism by which excessive alcohol intake, characteristic of an alcohol use disorder, produces neurodegeneration. As hippocampal neural stem cells underlie ongoing neurogenesis, a phenomenon that contributes to hippocampal structure and function, we investigated aspects of cell death and cell birth in an adolescent rat model of an alcohol use disorder. Immunohistochemistry of various markers along with Bromo‐deoxy‐Uridine (BrdU) injections were used to examine different aspects of neurogenesis. After 4 days of binge alcohol exposure, neurogenesis was decreased by 33 and 28% at 0 and 2 days after the last dose according to doublecortin expression. To determine whether this decrease in neurogenesis was due to effects on neural stem cell proliferation, quantification of BrdU‐labeled cells revealed a 21% decrease in the dentate gyrus of alcohol‐exposed brains. Cell survival and phenotype of BrdU‐labeled cells were assessed 28 days after alcohol exposure and revealed a significant, 50% decrease in the number of surviving cells in the alcohol‐exposed group. Reduced survival was supported by significant increases in the number of pyknotic‐, FluoroJade B positive‐, and TUNEL‐positive cells. However, so few cells were TUNEL‐positive that cell death is likely necrotic in this model. Although alcohol decreased the number of newborn cells, it did not affect the percentage of cells that matured into neurons (differentiation). Thus, our data support that in a model of an adolescent alcohol use disorder, neurogenesis is impaired by two mechanisms: alcohol‐inhibition of neural stem cell proliferation and alcohol effects on new cell survival. Remarkably, alcohol inhibition of neurogenesis may outweigh the few dying cells per section, which implies that alcohol inhibition of neurogenesis contributes to hippocampal neurodegeneration in alcohol use disorders. © 2009 Wiley‐Liss, Inc.     

Glutamate enhances proliferation and neurogenesis in human neural progenitor cell cultures derived from the fetal cortex

Excitatory amino acids such as glutamate play important roles in the central nervous system. We previously demonstrated that a neurosteroid, dehydroepiandrosterone (DHEA), has powerful effects on the cell proliferation of human neural progenitor cells (hNPC) derived from the fetal cortex, and this effect is modulated through NMDA receptor signaling. Here, we show that glutamate can significantly increase the proliferation rates of hNPC. The increased proliferation could be blocked by specific NMDA receptor antagonists, but not other glutamate antagonists for kainate-AMPA or metabotropic receptors. The NR1 subunit of the NMDA receptor was detectable in elongated bipolar or unipolar cells with small cell bodies. These NR1-positive cells were colocalized with GFAP immunoreactivity. Detection of the phosphorylation of cAMP response element-binding protein (pCREB) revealed that a subset of NR1-positive hNPC could respond to glutamate. Furthermore, we hypothesized that glutamate treatment may affect mainly the hNPC with a radial morphology and found that glutamate as well as DHEA selectively affected elongated hNPC; these elongated cells may be a type of radial glial cell. Finally we asked whether the glutamate-responsive hNPC had an increased potential for neurogenesis and found that glutamate-treated hNPC produced significantly more neurons following differentiation. Together these data suggest that glutamate stimulates the division of human progenitor cells with neurogenic potential.     

Role of NMDA receptor subtypes in the induction of catalepsy and increase in Fos protein expression after administration of haloperidol

The increase of Fos expression in the striatum induced by haloperidol, an antagonist of the dopamine D2 receptor, might be related to the activation of glutamatergic neurotransmission, especially that of N-methyl-D-aspartate (NMDA) receptors. In this study, using behavioral and immunohistochemical techniques, we examined the effects of a noncompetitive NMDA antagonist, (+)-MK-801, and an NMDA receptor NR2B subunit antagonist, ifenprodil, on catalepsy, an extrapyramidal symptom; in this context, we also considered the expression of Fos protein in the forebrain after the administration of haloperidol. Catalepsy in mice, induced by the administration of haloperidol (1 mg/kg), was inhibited by pretreatment with (+)-MK-801 (0.2 mg/kg) or ifenprodil (10 mg/kg). Furthermore, pretreatment with (+)-MK-801 (0.2 mg/kg) significantly attenuated the induction of Fos-immunoreactive (IR) cells in the dorsomedial, dorsolateral, and ventrolateral striatum, but not in the shell region of the nucleus accumbens after the administration of haloperidol, whereas pretreatment with ifenprodil (10 mg/kg) significantly attenuated the induction of Fos-IR cells in all of these areas. It is known that ifenprodil binds sigma receptors and alpha-1 adrenergic receptors with high affinity. Pretreatment with the sigma receptor antagonist BD-1407 (3 mg/kg) or the alpha-1 adrenergic receptor antagonist prazosin (3 mg/kg) affected neither catalepsy nor the expression of Fos-IR cells after the administration of haloperidol. However, pretreatment with CP-101,606 (1 mg/kg), a selective antagonist for the NR2B subunit of the NMDA receptor, significantly attenuated catalepsy and the expression of Fos-IR cells in the forebrain after the administration of haloperidol. These results suggest that the NMDA receptor antagonists attenuated the induction of catalepsy and Fos-IR cells in forebrain after the administration of haloperidol. It was also suggested that haloperidol-induced expression of Fos-IR cells in the shell region of the nucleus accumbens might be differentially regulated by NMDA receptor subunits. Therefore, it appears that selective antagonists for the NR2B subunit of the NMDA receptor (e.g., CP-101,606) might be useful drugs for the treatment of extrapyramidal side effects (EPS) associated with the chronic use of typical antipsychotics such as haloperidol.     

Possible protection by notoginsenoside R1 against glutamate neurotoxicity mediated by N‐methyl‐D‐aspartate receptors composed of an NR1/NR2B subunit assembly

Notoginsenoside R1 (NTR1) is the main active ingredient in Panax notoginseng, a herbal medicine widely used in Asia for years. The purpose of this study was to investigate pharmacological properties of NTR1 on neurotoxicity of glutamate (Glu) in primary cultured mouse cortical neurons along with its possible mechanism of action. Wefound that NTR1 significantly protected neurons from the loss of cellular viability caused by brief exposure to 10 μM Glu for 1 hr in a dose‐dependent manner at concentrations from 0.1 to 10 μM, without affecting the viability alone. NTR1 significantly inhibited the increased number of cells positive to propidium iodide (PI) staining, increase of intracellular free Ca²⁺ ions, overproduction of intracellular reactive oxygen species, and depolarization of mitochondrial membrane potential in cultured neurons exposed to Glu, in addition to blocking decreased Bcl‐2 and increased Bax expression levels. We further evaluated the target site at which NTR1 protects neurons from Glu toxicity by using the acquired expression strategy of N‐methyl‐D ‐aspartate (NMDA) receptor subunits in human embryonic kidney 293 cells. We found that 10 μM NTR1 protected NR1/NR2B subunit expressing cells from cell death by 100 μM NMDA, but not cells expressing NR1/NR2A subunits, when determined by PI staining. These results suggest that NTR1 may preferentially protect neurons from Glu excitotoxicity mediated by NMDA receptor composed of an NR1/NR2B subunit assembly in the brain. © 2009 Wiley‐Liss, Inc.     

Quantitative analysis of the generation of different striatal neuronal subtypes in the adult brain following excitotoxic injury

Recent findings in adult rodents have provided evidence for the formation of new striatal neurons from subventricular zone (SVZ) precursors following stroke. Little is known about which factors determine the magnitude of striatal neurogenesis in the damaged brain. Here we studied striatal neurogenesis following an excitotoxic lesion to the adult rat striatum induced by intrastriatal quinolinic acid (QA) infusion. New cells were labeled with the thymidine-analogue 5-bromo-2'-deoxyuridine (BrdU) and their identity was determined immunocytochemically with various phenotypic markers. The unilateral lesion gave rise to increased cell proliferation mainly in the ipsilateral SVZ. At 2 weeks following the insult, there was a pronounced increase of the number of new neurons co-expressing BrdU and a marker of migrating neuroblasts, doublecortin, in the ipsilateral striatum, particularly its non-damaged medial parts. About 80% of the new neurons survived up to 6 weeks, when they expressed the mature neuronal marker NeuN and were preferentially located in the outer parts of the damaged area. Lesion-generated neurons expressed phenotypic markers of striatal medium spiny neurons (DARPP-32) and interneurons (parvalbumin or neuropeptide Y). The magnitude of neurogenesis correlated to the size of the striatal damage. Our data show for the first time that an excitotoxic lesion to the striatum can trigger the formation of new striatal neurons with phenotypes of both projection neurons and interneurons.     

Effects of Chronic NMDA‐NR2b Inhibition in the Median Eminence of the Reproductive Senescent Female Rat

Gonadotrophin‐releasing hormone (GnRH) neurones of the hypothalamic‐pituitary‐gonadal (HPG) axis drive reproductive function and undergo age‐related decreases in activation during the transition to reproductive senescence. Decreased GnRH secretion from the median eminence (ME) partially arises from attenuated glutamatergic signalling via the NMDA receptor (NMDAR) and may be a result of changing NMDAR stoichiometry to favour NR2b over NR2a subunit expression with ageing. We have previously shown that the systemic inhibition of NR2b‐containing receptors with ifenprodil, an NR2b‐specific antagonist, stimulates parameters of luteinising hormone (used as a proxy for GnRH) release in both young and middle‐aged females. In the present study, we chronically administered ifenprodil, an NR2b‐specific antagonist, at the site of GnRH terminals in the ME or at GnRH perikarya in the preoptic area, in reproductively senescent middle‐aged female rats, aiming to determine whether NR2b antagonism could restore aspects of reproductive functionality. Effects on oestrous cyclicity, serum hormones, and protein expression of GnRH, NR2b and phosphorylated NR2b (Tyr‐1472) in the ME were measured. Chronic ifenprodil treatment in the ME (but not the preoptic area) altered oestrous cyclicity by increasing the percentage of days spent in pro‐oestrus. This was accompanied by increased GnRH fluorescence intensity in the external ME zone and a greater proportion of GnRH terminals that co‐labelled with pNR2b with treatment. We also observed changes in the relationships between protein immunofluorescence, serum hormone levels and other aspects of reproductive physiology in acyclic females, as revealed by bionetwork analysis. Together, these data support the hypothesis that NMDAR‐NR2b expression and phosphorylation state play a role in reproductive senescence and highlight the ME as a major player in reproductive ageing.