N-methyl-D-aspartate Receptor Subunit 2B Research Articles

Long-term imipramine treatment increases N-methyl-d-aspartate receptor activity and expression via epigenetic mechanisms

Imipramine, a major antidepressant, is known to inhibit reuptake of serotonin and norepinephrine, which contributes to recovery from major depressive disorder. It has recently been reported that acute imipramine treatment inhibits N-methyl-d-aspartate (NMDA) receptor activity. However, the mechanisms underlying long-term effects of imipramine have not been identified. We tested these distinct effects in mouse cortical neurons and found that acute (30s) imipramine treatment decreased Ca2+ influx through NMDA receptors, whereas long-term treatment (48h) increased Ca2+ influx via the same receptors. Furthermore, long-term treatment increased NMDA receptor 2B (NR2B) subunit expression via epigenetic changes, including increased acetylation of histones H3K9 and H3K27 in the NR2B promoter and decreased activity of histone deacetylase 3 (HDAC3) and HDAC4. These results suggest that the long-term effects of imipramine on NMDA receptors are quite different from its acute effects. Furthermore, increased NR2B expression via epigenetic alterations might be a part of the mechanism responsible for this long-term effect.

Expression of immediate-early genes in the dorsal cochlear nucleus in salicylate-induced tinnitus.

Spontaneous neuronal activity in dorsal cochlear nucleus (DCN) may be involved in the physiological processes underlying salicylate-induced tinnitus. As a neuronal activity marker, immediate-early gene (IEG) expression, especially activity-dependent cytoskeletal protein (Arc/Arg3.1) and the early growth response gene-1 (Egr-1), appears to be highly correlated with sensory-evoked neuronal activity. However, their relationships with tinnitus induced by salicylate have rarely been reported in the DCN. In this study, we assessed the effect of acute and chronic salicylate treatment on the expression of N-methyl D-aspartate receptor subunit 2B (NR2B), Arg3.1, and Egr-1. We also observed ultrastructural alterations in the DCN synapses in an animal model of tinnitus. Levels of mRNA and protein expression of NR2B and Arg3.1 were increased in rats that were chronically administered salicylate (200 mg/kg, twice daily for 3, 7, or 14 days). These levels returned to baseline 14 days after cessation of treatment. However, no significant changes were observed in Egr-1 gene expression in any groups. Furthermore, rats subjected to long-term salicylate administration showed more presynaptic vesicles, thicker and longer postsynaptic densities, and increased synaptic interface curvature. Alterations of Arg3.1 and NR2B may be responsible for the changes in the synaptic ultrastructure. These changes confirm that salicylate can cause neural plasticity changes at the DCN level.

Caveolin-1 in the anterior cingulate cortex modulates chronic neuropathic pain via regulation of NMDA receptor 2B subunit.

Chronic pain is still a basic science and clinical challenge. Unraveling of the neurobiological mechanisms involved in chronic pain will offer novel targets for the development of therapeutic strategies. It is well known that central sensitization in the anterior cingulate cortex (ACC) plays a critical role in initiation, development, and maintenance of chronic pain. However, the underlying mechanisms still remain elusive. Here, we reported that caveolin-1 (Cav-1), a scaffolding protein in membrane rafts, was persistently upregulated and activated in the ACC neurons after chronic constriction injury (CCI) in mice. Knockdown or blocking of Cav-1 in the contralateral ACC to the injury side reversed CCI-induced pain behavioral and neuronal sensitization and overexpression of Cav-1 in the ipsilateral ACC-induced pain behavior in the unaffected hindpaw. Furthermore, we found that Cav-1 directly binding with NMDA receptor 2B subunit (NR2B) and promotion of NR2B surface levels in the ACC contributed to modulation of chronic neuropathic pain. Disrupting the interaction of Cav-1 and NR2B through microinjection of a short peptide derived from the C-terminal of NR2B into the ACC exhibited a significant anti-nociception effect associated with decrease of surface NR2B expression. Moreover, Cav-1 increased intracellular Ca(2+) concentration and activated the ERK/CREB signaling pathway in an NR2B-dependent manner in the ACC. Our findings implicate that Cav-1 in the ACC neurons modulates chronic neuropathic pain via regulation of NR2B and subsequent activation of ERK/CREB signaling, suggesting a possible caveolin-mediated process would participate in neuronal transmission pathways implicated in pain modulation.

Monosialotetrahexosylganglioside Inhibits the Expression of p-CREB and NR2B in the Auditory Cortex in Rats with Salicylate-Induced Tinnitus.

This study investigated the effects of monosialotetrahexosylganglioside (GM1) on the expression of N-methyl-D-aspartate receptor subunit 2B (NR2B) and phosphorylated (p)-cyclic AMP response element-binding protein (CREB) in the auditory cortex of rats with tinnitus. Tinnitus-like behavior in rats was tested with the gap prepulse inhibition of acoustic startle paradigm. We then investigated the NR2B mRNA and protein and p-CREB protein levels in the auditory cortex of tinnitus rats compared with normal rats. Rats treated for 4 days with salicylate exhibited tinnitus. NR2B mRNA and protein and p-CREB protein levels were upregulated in these animals, with expression returning to normal levels 14 days after cessation of treatment; baseline levels of NR2B and p-CREB were also restored by GM1 administration. These data suggest that chronic salicylate administration induces tinnitus via upregulation of p-CREB and NR2B expression, and that GM1 can potentially be used to treat tinnitus.

Overexpression of NMDAR2B in an inflammatory model of Alzheimer's disease: modulation by NOS inhibitors.

Alzheimer's disease (AD) is a common form of age-related dementia, characterized by deposition of amyloid Aβ plaques, neuroinflammation and neurodegeneration. N-methyl-D-aspartate receptors (NMDAR) are postsynaptic glutamate receptors that play a role in memory formation and are targets for memantadine, an anti-AD drug. Nitric oxide (NO) signaling has been involved in both memory development through neuronal NO synthase (nNOS), and neuroinflammation through inducible NO synthase (iNOS) which mediates CNS inflammatory processes. To study the expression of the NMDAR2B subunit in an inflammatory model of AD before and after treatment with NO modulators. AD was induced in mice by a single dose of lipopolysaccharide (LPS). Behavioral tests for spatial and non-spatial memories and locomotor activity were performed to assess disease severity and progression. The effects of L-NAME (general NOS inhibitor), 1400W (iNOS inhibitor), diflunisal (systemic anti-inflammatory drug that does not cross the blood brain barrier), and L-arginine, the substrate for NOS was determined. Immunohistochemistry was done to confirm AD and brain lysates were tested for Aβ formation, levels of NMDAR2B subunits, and brain NO levels. Systemic LPS induced AD, as shown by cognitive impairment; increased levels of Aβ and concomitant increase in the brain NO concentrations. This was associated with overexpression of NMDAR2B. All tested drugs improved behavioral dysfunction, prevented Aβ formation and NMDAR overexpression, and lead to decrease in NO concentration in the brain. L-Arginine alone, however, did not produce similar improvements. NMDAR2B subunits are overexpressed in an inflammatory model of AD and NO inhibitors ameliorate this expression.

Effect of intraperitoneal WSLP/NR2B siRNA compound on neuropathic pain in rats

Objective To evaluate the effect of intraperitoneal water soluble lipopolymer (WSLP)/ N-methyl-D-aspartate receptor subunit 2B (NR2B) siRNA compound on the neuropathic pain (NP) in rats.Methods Eighty-four healthy male Sprague-Dawley rats,aged 6 weeks,weighing 180-220 g,were randomly divided into 7 groups (n =12 each) using a random number table:control group (group C),sham operation group (group S),NP group,WSLP/NR2B siRNA group (siWSLP group),WSLP/negative control siRNA group (ncWSLP group),PEI/NR2B siRNA group (PEI group) and WSLP group (WSLP group).NP was produced by ligation of the left L5 spinal nerve.In group S,the left L5 spinal nerve was only exposed,but not ligated.In group C,the rats underwent no treatment.Groups siWSLP,ncWSLP,PEI and WSLP received single intraperitoneal injection of WSLP/NR2B siRNA,WSLP/negative control siRNA,PEI/NR2B siRNA and WSLP compound 2 ml,respectively,at 10 days after NP.At 1 day before operation,7 days after operation,and 3,7,14 and 21 days after intraperitoneal injection,6 rats in each group were chosen randomly to measure mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL).At 3 days after intraperitoneal injection,the left 6 rats in each group were sacrificed and the spinal cord was removed for detection of NP2B mRNA expression (using PCR) and NR2B expression (by Western blot).Results Compared with group C,MWT was significantly decreased,and TWL was shortened on 7 days after operation and 3,7 and 21 days after intraperitoneal injection,and the expression of NR2B mRNA and protein was down-regulated on 3 days after administration in the other groups.Compared with group NP,MWT was significantly increased,and TWL was prolonged on day 3 and 7 after intraperitoneal injection,and the expression of NR2B mRNA and protein was down-regulated on day 3 after administration in siWSLP group.Conclusion Intraperitoneal WSLP/NR2B siRNA compound can effectively relieve the NP in rats. Key words: Gene therapy; Transfection ; RNA, small interfering; Receptors, N-methyl-D-aspartate ; Neuralgia; Injections,intraperitoneal

The assessment of the T102C polymorphism of the 5HT2a receptor gene, 3723G/A polymorphism of the NMDA receptor 3A subunit gene (GRIN3A) and 421C/A polymorphism of the NMDA receptor 2B subunit gene (GRIN2B) among cardiac surgery patients with and without delirium

ObjectiveThe studies regarding the role of genes polymorphism in development of postoperative delirium are extremely rare. Therefore, we investigated the potential association of polymorphism in 5HT2a receptor gene and N-methyl-d-aspartate (NMDA) receptor 3A and 2B subunits genes with postoperative delirium. MethodWe conducted a prospective, nested, case–control study. For analysis, 3723G/A (rs3739722) polymorphism in the GRIN3A gene, 421C/A (rs3764028) polymorphism in the GRIN2B gene and T102C (rs6313) polymorphism in the 5HT2A gene were selected. ResultsGenetic analysis confirmed that there were significant differences in genotype frequencies for 3723G/A between delirium patients and controls. No other significant associations were observed. Moreover, according to the multivariate conditional logistic regression analysis the presence of AG haplotype of GRIN3A gene was independently associated with postoperative delirium. ConclusionsThese findings suggest that the genetic variations of NR3A subunit of NMDA receptor may be a predisposing factor to delirium among the Polish population of cardiac surgery patients.

Intrathecal injection of the peptide myr-NR2B9c attenuates bone cancer pain via perturbing N-methyl-D-aspartate receptor-PSD-95 protein interactions in mice.

N-methyl-D-aspartate receptor (NMDARs)-dependent central sensitization plays an important role in cancer pain. Binding of NMDAR subunit 2B (NR2B) by postsynaptic density protein-95 (PSD-95) can couple NMDAR activity to intracellular enzymes, such as neuronal nitric oxide synthase (nNOS), facilitate downstream signaling pathways, and modulate NMDAR stability, contributing to synaptic plasticity. In this study, we investigated whether perturbing the specific interaction between spinal NR2B-containing NMDAR and PSD-95, using a peptide-mimetic strategy, could attenuate bone cancer-related pain behaviors. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer-related pain behaviors. Western blotting was applied to examine the expression of spinal phospho-Tyr1472 NR2B, nNOS, and PSD-95. We further investigated the effects of intrathecal injection of the mimetic peptide Myr-NR2B9c, which competitively disrupts the interaction between PSD-95 and NR2B, on nociceptive behaviors and on the upregulation of phospho-Tyr1472 NR2B, nNOS, and PSD-95 associated with bone cancer pain in the spinal cord. Inoculation of osteosarcoma cells induced progressive bone cancer pain and resulted in a significant upregulation of phospho-Tyr1472 NR2B, nNOS, and PSD-95. Intrathecal administration of Myr-NR2B9c attenuated bone cancer-evoked mechanical allodynia, thermal hyperalgesia, and reduced spinal phospho-Tyr1472 NR2B, nNOS, and PSD-95 expression. Intrathecal administration of Myr-NR2B9c reduced bone cancer pain. Internalization of spinal NR2B and dissociation NR2B-containing NMDARs activation from downstream nNOS signaling may contribute to the analgesic effects of Myr-NR2B9c. This approach may circumvent the negative consequences associated with blocking NMDARs, and may be a novel strategy for the treatment of bone cancer pain.

Intrathecal administration of roscovitine prevents remifentanil-induced postoperative hyperalgesia and decreases the phosphorylation of N-methyl-D-aspartate receptor and metabotropic glutamate receptor 5 in spinal cord.

N-methyl-D-aspartate receptor (NMDAR) and metabotropic glutamate receptor 5 (mGluR5) play an important role in nociceptive processing and central sensitization. Our previous study showed that tyrosine phosphorylation of NMDAR subunit 2B (NR2B) at Tyr1472 in spinal dorsal horn contributes to the postoperative hyperalgesia induced by remifentanil. Cyclin-dependent kinase 5 (Cdk5) has been implicated in synaptic plasticity, learning, memory and pain signaling via regulating the phosphorylation of NMDAR and mGluR5. In the present study, a rat model of postoperative pain was used to investigate the role of Cdk5 in spinal dorsal horn in remifentanil-induced hyperalgesia and the intervention of pretreatment with Cdk5 inhibitor roscovitine. Intraoperative infusion of remifentanil (0.04 mg/kg, subcutaneous) significantly enhanced mechanical allodynia and thermal hyperalgesia induced by plantar incision during the postoperative period (each lasting between 2 h and 48 h), which were attenuated by pretreatment with roscovitine. Correlated with the pain behavior changes, Western blotting revealed that there was a significant increase in the expression of Cdk5 and its activator p35/p25, and further the kinase activity of Cdk5 in spinal dorsal horn after intraoperative infusion of remifentanil. The phosphorylation of NR2A at Ser1232, the phosphorylation of NR2B at Tyr1472 and the phosphorylation of mGluR5 at Ser1167 were also significantly up-regulated. Furthermore, these increases were attenuated by pretreatment with roscovitine. These results suggested that Cdk5 may contribute to remifentanil-induced postoperative hyperalgesia via regulating the phosphorylation of NMDAR and mGluR5 in spinal dorsal horn. These findings provide experimental evidence for the further application of Cdk5 inhibitor in preventing remifentanil-induced hyperalgesia.

Effects of olfactory bulb lesion on neural stem cells proliferation and expression of NMDA-receptor subunit 2B in the subventricular zone of rats

Objective To investigate the effects of olfactory bulb(OB) lesion on neural stem cells proliferation and expression of NMDA receptor subunit 2B in subventricular zone(SVZ) of rats. Methods Sixty adult female SD rats were randomly divided into normal group, saline group and OB lesion group. OB lesion was induced by N-methyl-D-aspartic acid (NMDA) injection. Each group was respectively divided into four time points including 3 d, 7 d, 14 d and 28 d. Immunohistochemistry staining was used to detect the number of Nestin, Ki67 and NR2B-positive cells in the SVZ. Results (1)Nestin positive cells in the SVZ were shown at the different time of three groups.Seven days after OB lesion, IOD value of nestin-positive cells began to increase((29601±1788)/0.01 mm2, P<0.05), reached the maximum at 14 d((49800±3701)/0.01 mm2, P<0.05) and still sustained a high level at 28 day((27600±3209)/0.01 mm2, P<0.05). (2)Ki67 positive cells in the SVZ were shown at the different time of three groups.The number of Ki67-positive cells was increased significantly at 7 d, 14 d and 28 d after OB lesion compared to normal group and saline group (P<0.05). (3)NR2B immune expression in the SVZ was shown at the different time of three groups. The NR2B-positive cells increased at 3 d after OB lesion(58.80±2.95, P<0.05), reached the maximum at 14 d(68.40±4.04, P<0.05). At 28 d of OB lesion, the number of positive cells was reduced, but still sustained a high level(62.20±3.56, P<0.05). (4)The positive cells of NR2B and Ki67 were highly positive correlation at different time after OB lesion(r=0.968, P<0.05). Conclusions OB lesion can stimulate neural stem cell proliferation and increases the expression of NR2B. The increased mode of NR2B is in accordance with the schedule of the neural stem cells increase induced by OB lesion. Therefore, it indicates that the NMDA receptor subunit 2B may be involved in neural stem cell proliferation. Key words: Olfactory bulb; Subventricular zone; Neural stem cell; NMDA receptor

Role of endogenous hydrogen sulfide of the spinal cord in the maintenance of diabetic neuropathic pain in rats

Objective To evaluate the role of endogenous hydrogen sulfide in the maintenance of diabetic neuropathic pain (DNP) in rats.Methods The DNP models were established by intraperitoneal injection of stretopzin (STZ) in 24 male SD rats aged 4 weeks (150-170 g),and other 24 rats were intraperitoneally injected with the same volume of normal saline as normal group.All the rats were implanted intrathecal catheter.The rats with DNP were randomly divided into 2 subgroups (n =12 each):saline group (group D) and methemoglobin group (group DM).The rats in normal group were randomly divided into 2 subgroups (n =12 each):saline group (group C) and methemoglobin group (group CM).Three weeks after intraperitoneal injection,the rats were separately intrathecally administered methemoglobin 10 μ mol/L (10 μ1) (group DM),saline 10 μl (group D),methemoglobin 10 μmol/L (10 μl) (group CM) and saline 10 μl (group C),respectively,once a day for 4 days.The paw withdrawal mechanical threshold (PWT) of the rats were measured before and after injection at 1 (T1),2 (T2),3 (T3),4 (T4) days,then the rats were sacrificed at T4 point after the measurement and the lumbar segments of the spinal cord were removed for determination of N-methyl-D-aspartate receptor subunit 2B (NR2B) protein expression (by Western blotting) and NR2B mRNA expression [by reverse transcriptase-polymerase chain reaction (RT-PCR)].Results As compared with group C and group CM,the PWT in group D and group DM was significantly reduced (P ＜ 0.05),while the expression of NR2B protein and mRNA was significantly increased (P ＜ 0.05).As Compared with group D [NR2B mRNA (0.89 ± 0.09),NR2B protein (1.28 ± 0.17)],the PWT in group DM was significantly increased at T2-T4 point before the injection and T1-T4 point after the injection,while the expression of NR2B mRNA (0.69 ±0.11) and NR2B protein (0.98 ± 0.14) in group DM was significantly decreased (P ＜ 0.05).As compared with the point before the injection (3.8 ± 0.9 and 6.1 ± 1.4),the PWT in group DM were significantly increased at T1-T2 point after the injection (7.5 ± 1.8 and 8.7 ± 1.6) (P ＜ 0.05).Conclusion Endogenous hydrogen sulfide in the spinal cord is involved in the maintenance of DNP in rats and the possible mechanisms were related with up-regulation of NR2B in the spinal dorsal horn. Key words: Hydrogen sulfide; N-methyl-D-aspartate receptor subunit 2B; Spinal dorsal horn; Diabete ; Pain

Effects of dezocine on diabetic neuropathic pain and expression of NMDA receptor subunit 2B in spinal dorsal horns of rats

Objective To evaluate the effects of dezocine on diabetic neuropathic pain （DNP ） and expression of NMDA receptor subunit 2B （NR2B） in the spinal dorsal horns of rats .Methods Forty-eight male Sprague-Dawley rats , aged 4 weeks , weighing 150-170 g , with DNP induced by intraperitoneal injection of streptozocin （STZ） 50 mg/kg （successful induction of diabetes was defined as blood glucose 〉16.7 mmol/L） , were randomly divided into 2 groups （ n=24 each） using a random number table：DNP group and dezocine group （group D） .Twenty-four normal rats were chosen and served as normal control group （group C） .In group D , dezocine 2.52 mg/kg was injected intramuscularly once a day for 7 consecutive days starting from 2nd week after STZ injection ,while the rats in DNP and C groups received the equal volume of normal saline .Paw withdrawl threshold （PWT） to mechanical stimulation was measured before dezocine injection （T0 ） ,and on 1st ,3rd ,5th and 7th days after dezocine injection （T1-4 ） and on 7th day after the end of dezocine injection （T5 ） .Twelve rats in each group were sacrificed after measurement of PWT at T4 ,and T5 .The lumbar segments of the spinal cord were removed for determination of NR2B protein expression （by immuno-histochemistry and Western blot ） and NR2B＆nbsp;mRNA expression （by RT-PCR ） in the spinal dorsal horns .Results Compared with group C ,the PWT at T0-5 in group DNP and at T0 and T5 in group D was significantly decreased , and the expression of NR2B protein and mRNA at T4 ,5 in DNP group and at T5 in D group was up-regulated （ P0.05） . The PWT was significantly lower at T0 and T5 ,and the expression of NR2B protein and mRNA was higher at T5 than at T4 in group D （ P〈0.05 ） .Conclusion Dezocine can effectively relieve DNP in rats and inhibition of NR2B expression in the spinal dorsal horns is involved in the mechanism . Key words: Analgesics,opioid; Neuralgia; Diabetes mellitus; Spinal cord; Receptors,N-methyl-D-aspartate

Expression of immediate-early genes in the inferior colliculus and auditory cortex in salicylate-induced tinnitus in rat

Tinnitus could be associated with neuronal hyperactivity in the auditory center. As a neuronal activity marker, immediate-early gene (IEG) expression is considered part of a general neuronal response to natural stimuli. Some IEGs, especially the activity-dependent cytoskeletal protein (Arc) and the early growth response gene-1 (Egr-1), appear to be highly correlated with sensory-evoked neuronal activity. We hypothesize, therefore, an increase of Arc and Egr-1 will be observed in a tinnitus model. In our study, we used the gap prepulse inhibition of acoustic startle (GPIAS) paradigm to confirm that salicylate induces tinnitus-like behavior in rats. However, expression of the Arc gene and Egr-1 gene were decreased in the inferior colliculus (IC) and auditory cortex (AC), in contradiction of our hypothesis. Expression of N-methyl D-aspartate receptor subunit 2B (NR2B) was increased and all of these changes returned to normal 14 days after treatment with salicylate ceased. These data revealed long-time administration of salicylate induced tinnitus markedly but reversibly and caused neural plasticity changes in the IC and the AC. Decreased expression of Arc and Egr-1 might be involved with instability of synaptic plasticity in tinnitus.

Activation of GRs–Akt–nNOs–NR2B signaling pathway by second dose GR agonist contributes to exacerbated hyperalgesia in a rat model of radicular pain

Central Akt, neuronal nitric oxide synthase (nNOS) and N-methyl-D-aspartate receptor subunit 2B (NR2B) play key roles in the development of neuropathic pain. Here we investigate the effects of glucocorticoid receptors (GRs) on the expression and activation of spinal Akt, nNOS and NR2B after chronic compression of dorsal root ganglia (CCD). Thermal hyperalgesia test and mechanical allodynia test were used to measure rats after intrathecal injection of GR antagonist mifepristone or GR agonist dexamethasone for 21 days postoperatively. Expression of spinal Akt, nNOS, NR2B and their phosphorylation state after CCD was examined by western blot. The effects of intrathecal treatment with dexamethasone or mifepristone on nociceptive behaviors and the corresponding expression of Akt, nNOS and NR2B in spinal cord were also investigated. Intrathecal injection of mifepristone or dexamethasone inhibited PWMT and PWTL in CCD rats. However, hyperalgesia was induced by intrathecal injection of dexamethasone on days 12 to 14 after surgery. Treatment of dexamethasone increased the expression and phosphorylation levels of spinal Akt, nNOS, GR and NR2B time dependently, whereas administration of mifepristone downregulated the expression of these proteins significantly. GRs activated spinal Akt-nNOS/NR2B pathway play important roles in the development of neuropathic pain in a time-dependent manner.

Atomoxetine affects transcription/translation of the NMDA receptor and the norepinephrine transporter in the rat brain &amp;ndash; an in vivo study

Attention-deficit/hyperactivity disorder (ADHD) is the most frequently diagnosed neurodevelopmental disorder. The norepinephrine transporter (NET) inhibitor atomoxetine, the first nonstimulant drug licensed for ADHD treatment, also acts as an N-methyl-D-aspartate receptor (NMDAR) antagonist. The compound’s effects on gene expression and protein levels of NET and NMDAR subunits (1, 2A, and 2B) are unknown. Therefore, adolescent Sprague Dawley rats were treated with atomoxetine (3 mg/kg, intraperitoneal injection [ip]) or saline (0.9%, ip) for 21 consecutive days on postnatal days (PND) 21–41. In humans, atomoxetine’s earliest clinical therapeutic effects emerge after 2–3 weeks. Material from prefrontal cortex, striatum (STR), mesencephalon (MES), and hippocampus (HC) was analyzed either directly after treatment (PND 42) or 2 months after termination of treatment (PND 101) to assess the compound’s long-term effects. In rat brains analyzed immediately after treatment, protein analysis exhibited decreased levels of the NET in HC, and NMDAR subunit 2B in both STR and HC; the transcript levels were unaltered. In rat brains probed 2 months after final atomoxetine exposure, messenger RNA analysis also revealed significantly reduced levels of genes coding for NMDAR subunits in MES and STR. NMDAR protein levels were reduced in STR and HC. Furthermore, the levels of two SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, synaptophysin and synaptosomal-associated protein 25, were also significantly altered in both treatment groups. This in vivo study detected atomoxetine’s effects beyond NET inhibition. Taken together, these data reveal that atomoxetine seems to decrease glutamatergic transmission in a brain region-specific manner. Long-term data show that the compound’s impact is not due to an acute pharmacological effect but lasts or even amplifies after a drug-free period of 2 months, leading to altered development of synaptic composition. These alterations might contribute to atomoxetine’s clinical effects in the treatment of ADHD, a neurodevelopmental disorder in which synaptic processes and especially a dysregulated glutamatergic metabolism seem to be involved.

The Dyslexia-Associated Gene Dcdc2 Is Required for Spike-Timing Precision in Mouse Neocortex

Variants in dyslexia-associated genes, including DCDC2, have been linked to altered neocortical activation, suggesting that dyslexia associated genes might play as yet unspecified roles in neuronal physiology. Whole-cell patch clamp recordings were used to compare the electrophysiological properties of regular spiking pyramidal neurons of neocortex in Dcdc2 knockout (KO) and wild-type mice. Ribonucleic acid sequencing and reverse transcriptase polymerase chain reaction were performed to identify and characterize changes in gene expression in Dcdc2 KOs. Neurons in KOs showed increased excitability and decreased temporal precision in action potential firing. The RNA sequencing screen revealed that the N-methyl-D-aspartate receptor (NMDAR) subunit Grin2B was elevated in Dcdc2 KOs, and an electrophysiological assessment confirmed a functional increase in spontaneous NMDAR-mediated activity. Remarkably, the decreased action potential temporal precision could be restored in mutants by treatment with either the NMDAR antagonist (2R)-amino-5-phosphonovaleric acid or the NMDAR 2B subunit-specific antagonist Ro 25-6981. These results link the function of the dyslexia-associated gene Dcdc2 to spike timing through activity of NMDAR.

Antinociception and prevention of hyperalgesia by intrathecal administration of Ro 25-6981, a highly selective antagonist of the 2B subunit of N-methyl-d-aspartate receptor

BackgroundNR2B subunits (NMDA receptor 2B subunit) play an important role in generation of pain and forming central sensitization of pain. Ro 25-6981, a highly selective NR2B antagonist, gained much attention in recent years. In this study, we used a rat model of incisional pain to investigate effects of postoperative analgesia and changes of postoperative hyperalgesia induced by remifentanil through the pretreatment of intrathecal administration with Ro 25-6981. MethodsThe behavioral changes of rats have been evaluated by the paw withdrawal mechanical threshold and paw withdrawal thermal latency after intrathecal injection of Ro 25-6981. The expression of NR2B with tyrosine phosphorylation in the spinal dorsal horn was analyzed by Western blotting. ResultsIntrathecal injection of Ro 25-6981 significantly enhanced the paw withdrawal mechanical threshold and paw withdrawal thermal latency after the operation. Significant change has been observed after intrathecal injection of 800.0μg of Ro 25-6981 and at 2h after operation in the oblique pull test degree and BBB rating score. Pretreatment of Ro 25-6981 decreased the high level expression of NR2B with tyrosine phosphorylation in spinal dorsal horn of the rat model after the operation. ConclusionsIntrathecal injection of Ro 25-6981 had significant analgesic effects on incision pain in rats and effectively attenuated postoperative hyperalgesia induced by remifentanil.

Neuronal firing activity and gene expression changes in the subthalamic nucleus after transplantation of dopamine neurons in hemiparkinsonian rats

Dopamine (DA) depletion in the nigrostriatal system leads to basal ganglia dysfunction both in Parkinson's disease (PD) and in 6-hydroxy dopamine (6-OHDA)-lesioned rats with neuronal hyperactivity in the subthalamic nucleus (STN), i.e. increased firing rate and burst activity, together with enhanced beta oscillatory activity. Moreover, intrastriatal transplantation of DA neurons has been shown to functionally re-innervate the host striatum and restore DA input.However, the effects of those transplanted cells on the STN are not well characterized. Therefore, we transplanted cells, derived from the ventral mesencephalon of E12 rat embryos, intrastriatally in the unilateral 6-OHDA-lesioned rat model of PD. We combined behavioral and histological findings with electrophysiological extracellular recordings in the STN, as well as qRT-PCR analyses of dopaminergic, GABAergic, and glutamatergic transporter and receptor genes in the striatum and the STN. Transplanted animals displayed improved rotational behavior after amphetamine injection by 50% in rats with small grafts (586±109 SEM dopamine cells), or even overcompensation by 116% in rats with large grafts (3486±548 SEM dopamine cells). Electrophysiological measurements revealed, that in rats with large grafts burst activity was not affected, while STN neuronal firing rate, as well as beta oscillatory activity was alleviated, whereas small grafts had less impact. Interestingly, both behavioral and electrophysiological measures were dependent on the number of surviving tyrosine hydroxylase positive cells. Although grafted rats displayed restored expression of the GABA synthesizing enzymes Gad65 and Gad67 in the striatum compared to naive rats, the grafts induced a decreased mRNA expression of dopamine receptor Drd2, glutamate receptors AMPA3, NMDA2A, and NMDA2B, and glutamate transporter Eaat3. Interestingly, the NMDA receptor subunit 2B and glutamate transporter Eaat3 were also less expressed in the STN of grafted animals compared to naive rats.In summary, DA grafts restore functional deficits and cause partial improvement of subthalamic neuronal activity. Incomplete recovery, however, may be due to decreased receptor gene expression induced by DA grafts in the striatum and in the STN.

Effects of C-phycocyanin and Spirulina on Salicylate-Induced Tinnitus, Expression of NMDA Receptor and Inflammatory Genes

Effects of C-phycocyanin (C-PC), the active component of Spirulina platensis water extract on the expressions of N-methyl D-aspartate receptor subunit 2B (NR2B), tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β), and cyclooxygenase type 2 (COX-2) genes in the cochlea and inferior colliculus (IC) of mice were evaluated after tinnitus was induced by intraperitoneal injection of salicylate. The results showed that 4-day salicylate treatment (unlike 4-day saline treatment) caused a significant increase in NR2B, TNF-α, and IL-1β mRNAs expression in the cochlea and IC. On the other hand, dietary supplementation with C-PC or Spirulina platensis water extract significantly reduced the salicylate-induced tinnitus and down-regulated the mRNAs expression of NR2B, TNF-α, IL-1β mRNAs, and COX-2 genes in the cochlea and IC of mice. The changes of protein expression levels were generally correlated with those of mRNAs expression levels in the IC for above genes.

Expression of N-methyl-D-aspartate receptor subunit 2B containing N-methyl-D-aspartate receptors in the spinal dorsal horn of rats with hydrogen sulfide-induced pain

Objective To investigate the role of N-methyl-D-aspartate receptor subunit 2B (NR2B) containing N-methyl-D-aspartate (NMDA) receptors in the spinal dorsal horn of rats with hydrogen sulfide-induced pain.Methods Thirty male SD rats aged 7 weeks were randomly divided into 3 groups (n =10 each):control group (group C),H2S group (group S),and ketamine group (group K).Intraplantar administration of NaHS (H2S donor) 1 nmol (100 μl) for 5 days in group S and group K,and the equal volum (100 μl) of nodal saline (NS) in group C (once each day) was done.Then rats in group K were injected with ketamine 10 mg/kg (0.2 ml) intramuscularly for 7 days,and the equal volum of NS was injected in group C and group S (once each day).The paw withdrawal threshold (PWT) to mechanical stimulation was measured at first day before the first administration of NaHS/NS (Tc),20 min after the first administration of NaHS/NS (T1),20 min after the fifth administration of NaHS/NS (T2),and 20 min after the seventh administration of ketamine/NS (T3).The rats were sacrificed after the last PWT measurement.The lumber segment of the spinal cord was removed for determination of the expressions of NR2B subunit by using RT-PCR and Western blotting.Results The PWT at T1,T2 and T3 was (2.72 ± 1.17),(1.48 ± 0.57) and (2.40 ± 0.89) g respectively in group S,which was significantly lower than in group C,and the expressions of NR2B mRNA and protein in spinal dorsal horn was (0.75 ±0.07) and (1.36 ± 0.08) respectively,which was significantly higher than in group C.The PWT at T3 was (8.89 ±1.09)g in group K,which was higher than in group S,and the expression of NR2B mRNA and protein in spinal dorsal horn was (0.55 ± 0.05) and (1.02 ± 0.06) respectively,which was lower than in group S.Conclusion The upregnlation of NR2B subunit in spinal dorsal horn was one of meachanisms in hydrogen sulfide-induced pain and inhibition of NR2B expression may contribute to the antinociceptive effects of ketamine. Key words: Hydrogen sulfide; Ketamine; N-methyl-D-aspartate receptor subunit 2B; Spinal dorsal horn; Pain