Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels.

Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.

High-resolution fast-tomography brain-imaging beamline at the Taiwan Photon Source.

The new Brain Imaging Beamline (BIB) of the Taiwan Photon Source (TPS) has been commissioned and opened to users. The BIB and in particular its endstation are designed to take advantage of bright unmonochromatized synchrotron X-rays and target fast 3D imaging, ∼1 ms exposure time plus very high ∼0.3 µm spatial resolution. A critical step in achieving the planned performances was the solution to the X-ray induced damaging problems of the detection system. High-energy photons were identified as their principal cause and were solved by combining tailored filters/attenuators and a high-energy cut-off mirror. This enabled the tomography acquisition throughput to reach >1 mm3 min-1, a critical performance for large-animal brain mapping and a vital mission of the beamline.

Cerebrospinal fluid of chronic osteoarthritic patients induced interleukin-6 release in human glial cell-line T98G.

BACKGROUND: Chronic osteoarthritic pain is not well understood in terms of its pathophysiological mechanism. Activated glial cells are thought to play a role in the maintenance of chronic pain. T98G glioblastoma cell line was previously observed to release higher amounts of interleukin-6 (IL-6) when treated with cerebrospinal fluid (CSF) from patients with another chronic pain condition, post-herpetic neuralgia. In this study, we investigated the ability of CSF from patients diagnosed with knee osteoarthritis suffering from chronic pain, to trigger the release of pro-inflammatory cytokines, IL-6, IL-1beta and tumour necrosis factor alpha (TNF-α) from T98G. Characterization of upstream signalling was also explored. METHODS: Fifteen osteoarthritis patients undergoing total knee replacement due to chronic knee pain and 15 patients without pain undergoing other surgeries with spinal anaesthesia were prospectively recruited. CSF was collected during anaesthesia. CSF were added to cultured T98G cells in the presence of lipopolysaccharide. IL-6, IL-1ß and TNF-α release from T98G cells were measured using enzyme immunoassay. Antibody array and western blotting were performed using CSF-triggered T98G cell lysates to identify possible signalling targets. Age, gender and pain scores were recorded. Mann-Whitney U test was used to compare IL-6 release and protein expression between groups. Association between IL-6 and pain score was analysed using linear regression. RESULTS: Significant higher levels of IL-6 were released by T98G cells when induced by osteoarthritis patients' CSF in the presence of LPS. The IL-6 levels showed positive association with pain score (adjusted B estimate = 10.1 (95% Confidence Interval 4.3-15.9); p = 0.001). Antibody array conducted with 6 pooled T98G cell lysate induced with osteoarthritis pain patient CSF identified greater than 2-fold proteins including STE20-related kinase adaptor protein and spleen tyrosine kinase. Further validation done using western blotting of individual CSF-triggered T98G cell lysate showed non-significant increase. CONCLUSION: Higher IL-6 release from T98G when triggered by OA-CSF, in the presence of LPS, suggest the presence of "unknown molecule" in CSF that may be crucial in the maintenance phase of chronic pain in our osteoarthritis population. Further studies on the signalling pathways involved in pain and relevance of IL-6 release from T98G cells in other pain models are needed.

GluN2D-Containing N-methyl-d-Aspartate Receptors Mediate Synaptic Transmission in Hippocampal Interneurons and Regulate Interneuron Activity.

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamatergic receptors that have been implicated in learning, development, and neuropathological conditions. They are typically composed of GluN1 and GluN2A-D subunits. Whereas a great deal is known about the role of GluN2A- and GluN2B-containing NMDARs, much less is known about GluN2D-containing NMDARs. Here we explore the subunit composition of synaptic NMDARs on hippocampal interneurons. GluN2D mRNA was detected by single-cell PCR and in situ hybridization in diverse interneuron subtypes in the CA1 region of the hippocampus. The GluN2D subunit was detectable by immunoblotting and immunohistochemistry in all subfields of the hippocampus in young and adult mice. In whole-cell patch-clamp recordings from acute hippocampal slices, (+)-CIQ, the active enantiomer of the positive allosteric modulator CIQ, significantly enhanced the amplitude of the NMDAR component of miniature excitatory postsynaptic currents (mEPSCs) in CA1 interneurons but not in pyramidal cells. (+)-CIQ had no effect in slices from Grin2d-/- mice, suggesting that GluN2D-containing NMDARs participate in excitatory synaptic transmission onto hippocampal interneurons. The time course of the NMDAR component of the mEPSC was unaffected by (+)-CIQ potentiation and was not accelerated in slices from Grin2d-/- mice compared with wild-type, suggesting that GluN2D does not detectably slow the NMDAR EPSC time course at this age. (+)-CIQ increased the activity of CA1 interneurons as detected by the rate and net charge transfer of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal cells. These data provide evidence that interneurons contain synaptic NMDARs possessing a GluN2D subunit, which can influence interneuron function and signal processing.

Ontogenic Profile and Synaptic Distribution of GluN3 Proteins in the Rat Brain and Hippocampal Neurons.

N-Methyl-D-aspartate receptors are localized to synaptic and extrasynaptic sites of dendritic spines and shafts. Here, the ontogenic profiles of GluN3A and GluN3B subunits in the rat brain were determined. A developmental switch from GluN3A to GluN3B proteins was detected within the first two postnatal weeks of crude synaptosomes prepared from forebrain and midbrain. Further fractionation of crude synaptosomes revealed the preferential localization of GluN3B to synaptic regions from P7 onwards. Immunolabeling and biochemical fractionation of rat P7 cultured hippocampal neurons showed that GluN3B was predominantly at synaptic sites. Unlike GluN2A and GluN2B, both GluN3 subunits were mostly associated with peripheral components of the postsynaptic density (PSD) rather than its core. When considering the non-PSD fraction, the overall extrasynaptic/synaptic spatial profile of GluN3B differed from GluN3A. Heterologous expression of GluN3B with GluN1 in HEK293FT cells could not be co-immunoprecipitated with PSD-95 unless co-expressed with a PSD-95-interacting GluN2 subunit, suggesting that anchoring of GluN3B at synaptic sites may require co-assembly with another scaffold-interacting NMDAR subunit.

Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases.

The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases.

Cdk5/p25-induced cytosolic PLA2-mediated lysophosphatidylcholine production regulates neuroinflammation and triggers neurodegeneration.

The deregulation of cyclin-dependent kinase 5 (Cdk5) by p25 has been shown to contribute to the pathogenesis in a number of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). In particular, p25/Cdk5 has been shown to produce hyperphosphorylated tau, neurofibrillary tangles as well as aberrant amyloid precursor protein processing found in AD. Neuroinflammation has been observed alongside the pathogenic process in these neurodegenerative diseases, however the precise mechanism behind the induction of neuroinflammation and the significance in the AD pathogenesis has not been fully elucidated. In this report, we uncover a novel pathway for p25-induced neuroinflammation where p25 expression induces an early trigger of neuroinflammation in vivo in mice. Lipidomic mass spectrometry, in vitro coculture and conditioned media transfer experiments show that the soluble lipid mediator lysophosphatidylcholine (LPC) is released by p25 overexpressing neurons to initiate astrogliosis, neuroinflammation and subsequent neurodegeneration. Reverse transcriptase PCR and gene silencing experiments show that cytosolic phospholipase 2 (cPLA2) is the key enzyme mediating the p25-induced LPC production and cPLA2 upregulation is critical in triggering the p25-mediated inflammatory and neurodegenerative process. Together, our findings delineate a potential therapeutic target for the reduction of neuroinflammation in neurodegenerative diseases including AD.

Cleavage of the NR2B subunit amino terminus of N-methyl-D-aspartate (NMDA) receptor by tissue plasminogen activator: identification of the cleavage site and characterization of ifenprodil and glycine affinities on truncated NMDA receptor.

Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicates tPA as an important neuromodulator of the N-methyl-d-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg(67)). This cleavage is tPA-specific, plasmin-independent, and removes a predicted ~4-kDa fragment (Arg(27)-Arg(67)) from the amino-terminal domain of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg(67) to Ala(67) impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg(27)-Arg(67)-truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln(29)-Arg(67) deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC(50) with no change in glutamate EC(50). Our results confirmed NR2B as a novel proteolytic substrate of tPA, where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.

The new X-ray/visible microscopy MAXWELL technique for fast three-dimensional nanoimaging with isotropic resolution.

Microscopy by Achromatic X-rays With Emission of Laminar Light (MAXWELL) is a new X-ray/visible technique with attractive characteristics including isotropic resolution in all directions, large-volume imaging and high throughput. An ultrathin, laminar X-ray beam produced by a Wolter type I mirror irradiates the sample stimulating the emission of visible light by scintillating nanoparticles, captured by an optical system. Three-dimensional (3D) images are obtained by scanning the specimen with respect to the laminar beam. We implemented and tested the technique with a high-brightness undulator at SPring-8, demonstrating its validity for a variety of specimens. This work was performed under the Synchrotrons for Neuroscience-an Asia-Pacific Strategic Enterprise (SYNAPSE) collaboration.

New insights into the not-so-new NR3 subunits of N-methyl-D-aspartate receptor: localization, structure, and function.

The NR3 subunits (NR3A and NR3B) are new players in a well established field of N-methyl-d-aspartate (NMDA) receptors, previously involving the NR1 and NR2 subunits. Their incorporation into conventional NMDA receptors forms glutamate-activated NR1/NR2/NR3 triheteromers, whereas the omission of the glutamate-binding NR2 subunits results in excitatory glycine-activated NR1/NR3 diheteromers. These NR3-containing NMDA receptors exhibit several differences in receptor properties compared with the conventional NR1/NR2 receptors. This review highlights the major landmarks that have been achieved in the past decade or so involving NR3 subunit research in four key areas: the spatiotemporal mapping of NR3 protein, the structural elucidation of NR3 domains, pharmacological characterization of NR3-containing receptors, and the successful generation of NR3 knockout/transgenic animals. It is expected that further characterization of their functional roles coupled with the identification of endogenous and exogenous ligands will eventually advance the understanding of the basic pharmacology and the complex role of NMDA receptors in higher brain functions and neurological disorders.

Nicotinic receptor mechanism in supramammillary nucleus mediates physiological regulation of neural activity in dorsal hippocampal field CA1 of anaesthetized rat.

Evidence suggests that stimulation of the region of the rostral pontine oralis (RPO) nucleus and the peripheral application of a noxious stimulus activates an ascending system that also modulates hippocampal neural responses during behavioral arousal. Indeed, the two stimuli and behavioral arousal elicit theta activation and the suppression of population spikes (PS) in dorsal hippocampus field CA1. Interestingly, such neural responses in CA1 are also elicited by microinjection of the cholinergic agonist carbachol into the hypothalamic supramammillary nucleus (SuM). In the present in vivo electrophysiological study, we tested the hypothesis that cholinergic neural elements in the SuM modulate the neural drive to CA1 on RPO stimulation or the peripheral application of a noxious stimulus. Pharmacological investigation showed that intra-SuM microinjection of either a muscarinic or a nicotinic receptor antagonist attenuated the SuM carbachol-induced neural effects in CA1, namely, theta activation and PS suppression. However, neither antagonist attenuated the CA1 effects of intra-SuM microinjection of the excitatory neurotransmitter glutamate. Subsequent investigations revealed that microinjection of only the nicotinic antagonist, mecamylamine, into the lateral SuM selectively attenuated the responses elicited in CA1 by stimulation of the RPO or on nociceptive stimulation with hind paw injection of formalin (5%, 0.05 ml); whereas, microinjection of mecamylamine into the medial SuM did not affect the hippocampal responses elicited by either type of stimulation. Furthermore, application of mecamylamine into the lateral SuM attenuated the CA1 responses induced by injection of formalin into the contralateral, but not the ipsilateral hind paw. The lateralization of drug effect is consistent with the predominant unilateral anatomical connections between the SuM and the septohippocampal region. These findings provide novel evidence that nicotinic cholinoceptive neurons in the lateral SuM are common elements of the neural drive(s) to the hippocampus on RPO activation and noxious stimulation.

Distinct roles of GRIN2A and GRIN2B variants in neurological conditions.

Rapid advances in sequencing technology have led to an explosive increase in the number of genetic variants identified in patients with neurological disease and have also enabled the assembly of a robust database of variants in healthy individuals. A surprising number of variants in the GRIN genes that encode N-methyl-D-aspartate (NMDA) glutamatergic receptor subunits have been found in patients with various neuropsychiatric disorders, including autism spectrum disorders, epilepsy, intellectual disability, attention-deficit/hyperactivity disorder, and schizophrenia. This review compares and contrasts the available information describing the clinical and functional consequences of genetic variations in GRIN2A and GRIN2B. Comparison of clinical phenotypes shows that GRIN2A variants are commonly associated with an epileptic phenotype but that GRIN2B variants are commonly found in patients with neurodevelopmental disorders. These observations emphasize the distinct roles that the gene products serve in circuit function and suggest that functional analysis of GRIN2A and GRIN2B variation may provide insight into the molecular mechanisms, which will allow more accurate subclassification of clinical phenotypes. Furthermore, characterization of the pharmacological properties of variant receptors could provide the first opportunity for translational therapeutic strategies for these GRIN-related neurological and psychiatric disorders.

Forebrain medial septum sustains experimental neuropathic pain.

The present study explored the role of the medial septal region (MS) in experimental neuropathic pain. For the first time, we found that the MS sustains nociceptive behaviors in rodent models of neuropathic pain, especially in the chronic constriction injury (CCI) model and the paclitaxel model of chemotherapy-induced neuropathic pain. For example, inactivation of the MS with intraseptal muscimol (2 µg/µl, 0.5 µl), a GABA mimetic, reversed peripheral hypersensitivity (PH) in the CCI model and induced place preference in a conditioned place preference task, a surrogate measure of spontaneous nociception. The effect of intraseptal muscimol on PH was comparable to that seen with microinjection of the local anesthetic, lidocaine, into rostral ventromedial medulla which is implicated in facilitating experimental chronic nociception. Cellular analysis in the CCI model showed that the MS region sustains nociceptive gain with CCI by facilitating basal nociceptive processing and the amplification of stimulus-evoked neural processing. Indeed, consistent with the idea that excitatory transmission through MS facilitates chronic experimental pain, intraseptal microinjection of antagonists acting at AMPA and NMDA glutamate receptors attenuated CCI-induced PH. We propose that the MS is a central monitor of bodily nociception which sustains molecular plasticity triggered by persistent noxious insult.

Hydrogen sulphide reduces insulin secretion from HIT-T15 cells by a KATP channel-dependent pathway.

Hydrogen sulphide (H(2)S), a naturally occurring gas exerts physiological effects by opening K(ATP) channels. Anti-diabetic drugs (e.g. glibenclamide) block K(ATP) channels and abrogate H(2)S-mediated physiological responses which suggest that H(2)S may also regulate insulin secretion by pancreatic beta-cells. To investigate this hypothesis, insulin-secreting (HIT-T15) cells were exposed to NaHS (100 microM) and the K(ATP) channel-driven pathway of insulin secretion was tracked with various fluorescent probes. The concentration of insulin released from HIT-T15 cells decreased significantly after NaHS exposure and this effect was reversed by the addition of glibenclamide (10 microM). Cell viability and intracellular ATP and glutathione (GSH) levels remained unchanged, suggesting that changes in insulin secretion were not ATP linked or redox dependent. Through fluorescence imaging studies, it was found that K(+) efflux occurs in cells exposed to NaHS. The hyperpolarised cell membrane, a result of K(+) leaving the cell, prevents the opening of voltage-gated Ca(2+) channels. This subsequently prevents Ca(2+) influx and the release of insulin from HIT-T15 cells. This data suggest that H(2)S reduces insulin secretion by a K(ATP) channel-dependent pathway in HIT-T15 cells. This study reports the molecular mechanism by which H(2)S reduces insulin secretion and provides further insight into a recent observation of increased pancreatic H(2)S production in streptozotocin-diabetic rats.

Medial Septum Modulates Cellular Response Induced in Hippocampus on Microinjection of Cholinergic Agonists into Hypothalamic Lateral Supramammillary Nucleus.

Cholinergic mechanisms in supramammillary nucleus (SuM), especially the lateral SuM (lSuM) modulates septo-hippocampal neural activity. The lSuM, as compared to the contiguous medial SuM (mSuM) has relatively dense projections to hippocampus and cingulate cortex (Cg). In the present study, we have investigated whether the effects of cholinergic activation of SuM on hippocampal and cortical neural activities involve a cooperative interaction with the medial septum (MS). Microinjection of the broad-spectrum cholinergic agonist, carbachol, or the cholinergic-nicotinic receptor agonist, nicotine, into the lSuM and the mSuM in urethane anesthetized rat evoked a similar pattern of hippocampal theta rhythm. Despite that, only the lSuM microinjections resulted in an increase in expression of c-Fos-like immunoreactivity (c-Fos-ir) in neurons, including interneurons, of the ipsilateral hippocampus with a very dense expression in dentate gyrus. Likewise, a robust induction of c-Fos-ir was also observed in the ipsilateral Cg. Inhibition of the MS with muscimol pre-treatment attenuated both carbachol-evoked c-Fos-ir and theta activation. The findings indicate that cholinergic-nicotinic mechanisms in lSuM evoke not only neural activation via the ascending synchronizing pathway but also an MS-modulated expression of the plasticity-related molecule c-Fos in cortical regions that are strongly innervated by the lSuM.

Fluorogenic probes to monitor cytosolic phospholipase A2 activity.

Arachidonic acid derivatives equipped with either one or two fluorescent groups attached to the tip of the alkyl chains were synthesized and shown to function as inhibitor and substrate probes of cPLA2. The inhibitor probe was demonstrated to perform dual functions of inhibition and imaging while the substrate probe could be used for activity assay.

A New Generation of Arachidonic Acid Analogues as Potential Neurological Agent Targeting Cytosolic Phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) is an enzyme that releases arachidonic acid (AA) for the synthesis of eicosanoids and lysophospholipids which play critical roles in the initiation and modulation of oxidative stress and neuroinflammation. In the central nervous system, cPLA2 activation is implicated in the pathogenesis of various neurodegenerative diseases that involves neuroinflammation, thus making it an important pharmacological target. In this paper, a new class of arachidonic acid (AA) analogues was synthesized and evaluated for their ability to inhibit cPLA2. Several compounds were found to inhibit cPLA2 more strongly than arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor that is commonly used in the study of cPLA2-related neurodegenerative diseases. Subsequent experiments concluded that one of the inhibitors was found to be cPLA2-selective, non-cytotoxic, cell and brain penetrant and capable of reducing reactive oxygen species (ROS) and nitric oxide (NO) production in stimulated microglial cells. Computational studies were employed to understand how the compound interacts with cPLA2.

Curcumin Ameliorates Neuroinflammation, Neurodegeneration, and Memory Deficits in p25 Transgenic Mouse Model that Bears Hallmarks of Alzheimer's Disease.

Several studies have indicated that neuroinflammation is indeed associated with neurodegenerative disease pathology. However, failures of recent clinical trials of anti-inflammatory agents in neurodegenerative disorders have emphasized the need to better understand the complexity of the neuroinflammatory process in order to unravel its link with neurodegeneration. Deregulation of Cyclin-dependent kinase 5 (Cdk5) activity by production of its hyperactivator p25 is involved in the formation of tau and amyloid pathology reminiscent of Alzheimer's disease (AD). Recent studies show an association between p25/Cdk5 hyperactivation and robust neuroinflammation. In addition, we recently reported the novel link between the p25/Cdk5 hyperactivation-induced inflammatory responses and neurodegenerative changes using a transgenic mouse that overexpresses p25 (p25Tg). In this study, we aimed to understand the effects of early intervention with a potent natural anti-inflammatory agent, curcumin, on p25-mediated neuroinflammation and the progression of neurodegeneration in p25Tg mice. The results from this study showed that curcumin effectively counteracted the p25-mediated glial activation and pro-inflammatory chemokines/cytokines production in p25Tg mice. Moreover, this curcumin-mediated suppression of neuroinflammation reduced the progression of p25-induced tau/amyloid pathology and in turn ameliorated the p25-induced cognitive impairments. It is widely acknowledged that to treat AD, one must target the early-stage of pathological changes to protect neurons from irreversible damage. In line with this, our results demonstrated that early intervention of inflammation could reduce the progression of AD-like pathological outcomes. Moreover, our data provide a rationale for the potential use of curcuminoids in the treatment of inflammation associated neurodegenerative diseases.

Expression and characterization of soluble amino-terminal domain of NR2B subunit of N-methyl-D-aspartate receptor.

N-methyl-D-aspartate (NMDA) receptors are involved in mediating excitatory synaptic transmissions in the brain and have been implicated in numerous neurologic disorders. The proximal amino-terminal domains (ATDs) of NMDA receptors constitute many modulatory binding sites that may serve as potential drug targets. There are few biochemical and structural data on the ATDs of NMDA receptors, as it is difficult to produce the functional proteins. Here an optimized method was established to reconstitute the insoluble recombinant ATD of NMDA receptor NR2B subunit (ATD2B) through productive refolding of 6xHis-ATD2B protein from inclusion bodies. Circular dichroism and dynamic light scattering characterizations revealed that the solubilized and refolded 6xHis-ATD2B adopted well-defined secondary structures and monodispersity. More significantly, the soluble 6xHis-ATD2B specifically bound ifenprodil to saturation. Ifenprodil bound to 6xHis-ATD2B with a dissociation constant (KD) of 127.5+/-45 nM, which was within the range of the IC50 determined electrophysiologically. This is the first report on a functional recombinant ATD2B with a characterized KD.

GABAergic neurons of the medial septum play a nodal role in facilitation of nociception-induced affect.

The present study explored the functional details of the influence of medial septal region (MSDB) on spectrum of nociceptive behaviours by manipulating intraseptal GABAergic mechanisms. Results showed that formalin-induced acute nociception was not affected by intraseptal microinjection of bicuculline, a GABAA receptor antagonist, or on selective lesion of septal GABAergic neurons. Indeed, the acute nociceptive responses were dissociated from the regulation of sensorimotor behaviour and generation of theta-rhythm by the GABAergic mechanisms in MSDB. The GABAergic lesion attenuated formalin-induced unconditioned cellular response in the anterior cingulate cortex (ACC) and blocked formalin-induced conditioned place avoidance (F-CPA), and as well as the contextual fear induced on conditioning with brief footshock. The effects of lesion on nociceptive-conditioned cellular responses were, however, variable. Interestingly, the lesion attenuated the conditioned representation of experimental context in dorsal hippocampus field CA1 in the F-CPA task. Collectively, the preceding suggests that the MSDB is a nodal centre wherein the GABAergic neurons mediate nociceptive affect-motivation by regulating cellular mechanisms in ACC that confer an aversive value to the noxious stimulus. Further, in conjunction with a modulatory influence on hippocampal contextual processing, MSDB may integrate affect with context as part of associative learning in the F-CPA task.