N-Methyl-D-aspartate (NMDA) receptors are important targets for drugs of abuse such as ethanol, toluene, and ketamine. Ligand-gated ion channels assembled from the NR1 and NR3 subunits have functional and pharmacological properties that are distinct from those of conventional NMDA receptors containing NR2 subunits. In the present study we used voltageclamp electrophysiology to characterize excitatory glycine-activated receptors assembled from NR1, NR3A, and NR3B subunits expressed in human embryonic kidney (HEK) 293 cells. These glycine-activated receptors were not stimulated by glutamate or kainic acid and were resistant to magnesium block. A wide variety of NMDA receptor antagonists including D-2-amino-5-phosphonovaleric acid, ifenprodil, memantine, (5R,10S)-(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or acamprosate did not inhibit glycine-activated NR1/NR3A/NR3B receptors. Likewise, these receptors were not affected by antagonists of inhibitory glycine receptors or glycine transporters. The NMDA receptor glycine site agonist, D-serine, partially activated NR1/ NR3A/NR3B receptors, whereas the antagonist, 5,7-dichlorokynurenic acid, inhibited receptor currents. Conversely, the antagonist, 7-chlorokynurenic acid, and the partial agonist, R-(ϩ)-3-amino-1-hydroxy-2-pyrrolidinone (HA-966), potentiated glycine-stimulated currents of these receptors. NR1/ NR3A/NR3B receptor currents were inhibited by 10 to 21% by ethanol and toluene but were relatively insensitive to ketamine. Ethanol inhibition was enhanced in receptors expressing the NR1(L819A) mutant, whereas those containing NR1(F639A) or NR1(M813A) showed no change relative to the wild-type NR1. The results of this study indicate that coexpression of NR1, NR3A, and NR3B subunits in HEK 293 cells results in glycineactivated receptors with novel functional and pharmacological properties.N-Methyl-D-aspartate (NMDA) receptors are glutamateactivated ion channels that contain NR1 and NR2 subunits. A third class of NMDA receptor subunits has also been reported and consists of the NR3A and NR3B subunits (Ciabarra et al., 1995;Sun et al., 1998). NR3 subunits share structural features with the NR1 subunit, including the S1 and S2 agonist binding domains and some sequence similarity across transmembrane domains (Chatterton et al., 2002). Coexpression of the NR3A or NR3B subunits with NR1 and NR2 in recombinant expression systems decreases NMDA receptor current (Chatterton et al., 2002) and reduces calcium permeability (Matsuda et al., 2002). In NR3A-deficient mice, NMDA receptor currents are significantly enhanced (Das et al., 1998), suggesting that these subunits may be important modulators of NMDA receptor function.As for NR1 subunits, NR3 subunits do not form functional channels when expressed alone. However, coexpression of NR1 and NR3 subunits in oocytes has been reported to produce functional channels that are gated by glycine but not glutamate (Chatterton et al., 2002; but see Smothers and Woodward, 2003). In...

Interferon Alpha (IFNα) is a pleomorphic cytokine produced by nucleated cells in response to viral infection. In patients, treatment with IFNα has side effects including cognitive impairment resembling subcortical dementia, which is a hallmark of HIV associated dementia (HAD). IFNα is increased in the cerebrospinal fluid of HAD patients compared to HIV patients without dementia. In this study, blocking IFNα in a HIV encephalitis (HIVE) mouse model with intraperitoneal (i.p) injections of IFNα neutralizing antibodies (NAb) significantly improved cognitive function compared to untreated or control antibody treated HIVE mice during water radial arm maze behavioral testing. Treatment with IFNα NAb significantly decreased microgliosis and prevented loss of dendritic arborization in the brains of HIVE mice. Furthermore, treatment of primary neuron cultures with IFNα resulted in dose dependent loss of dendritic arborization that was blocked with IFNα NAb treatment and partially blocked with NMDA antagonists (AP5 and MK801) indicating glutamate signaling is involved in IFNα mediated neuronal damage. These results show that IFNα has a major role in the pathogenesis of HIVE in mice and is likely important in the development neurocognitive dysfunction in humans with HIV. Blocking IFNα could be important in improving cognitive and pathological developments in HAD patients and may be clinically important in other neuroinflammatory diseases as well.

Alcohols, inhaled anesthetics, and some injectable anesthetics inhibit the function of N-methyl-D-aspartate (NMDA) receptors, but the mechanisms responsible for this inhibition are not fully understood. Recently, it was shown that ethanol inhibition of NMDA receptors was reduced by mutation of residues in the transmembrane (TM) segment 3 of the NR1 subunit (F639A) or in TM4 of the NR2A subunit (A825W), suggesting putative ethanol binding sites. We hypothesized that the actions of other anesthetics might also require these amino acids and evaluated the effects of anesthetics on the NMDA receptors expressed in Xenopus oocytes with two-electrode voltage-clamp recording.

1 Toluene is a representative example of a class of industrial solvents that are voluntarily inhaled as drugs of abuse. Previous data from this lab and others has shown that toluene modulates the function of N-methyl-D-aspartate (NMDA), g-aminobutyric acid (GABA) and glycine receptors at concentrations that do not aect non-NMDA receptors. 2 We utilized two-electrode voltage-clamp and whole cell patch-clamp techniques to assess the eects of toluene on neuronal nicotinic acetylcholine receptors expressed in oocytes and cultured hippocampal neurons. Toluene (50 mM to 10 mM) produced a reversible, concentration-dependent inhibition of acetylcholine-induced current in Xenopus oocytes expressing various nicotinic receptor subtypes. The a4b2 and a3b2 subunit combinations were signi®cantly more sensitive to toluene inhibition than the a4b4, a3b4 and a7 receptors. 3 Receptors composed of a4 and b2(V253F) subunits showed a4b4-like toluene sensitivity while those containing a4 and b4(F255V) subunits showed a4b2-like sensitivity. 4 In hippocampal neurons, toluene (50 mM to 10 mM) dose-dependently inhibited ACh-mediated responses with an IC 50 of 1.1 mM. 5 Taken together, these results suggest that nicotinic receptors, like NMDA receptors, show a subunit-dependent sensitivity to toluene and may represent an important site of action for some of the neurobehavioural eects of toluene.

N-Methyl-D-aspartate (NMDA) receptors gate a slow and calciumrich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/ GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC 50 values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/ GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC 50 values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonistgated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits.