Agonist Sites Research Articles

Intra-hippocampal D-cycloserine rescues decreased social memory, spatial learning reversal, and synaptophysin levels in aged rats.

Aging is characterized by a decrease in N-methyl-D-aspartate receptors (NMDARs) in the hippocampus, which might be one of the factors involved in the age-dependent cognitive decline. D-Cycloserine (DCS), a partial agonist of the NMDAR glycine recognition site, could improve memory deficits associated to neurodegenerative disorders and cognitive deficits observed in normal aging. The aim of the present study was to explore whether DCS would reverse age-dependent memory deficits and decreases in NMDA receptor subunits (GluN1, GluN2A, and GluN2B) and the presynaptic protein synaptophysin in Wistar rats. We investigated the effects of pre-training infusions of DCS (10 μg/hemisphere) in the ventral hippocampus on two hippocampal-dependent learning tasks, the social transmission of food preference (STFP), and the Morris water maze (MWM). The results revealed that infusions of DCS administered before the acquisition sessions rescued deficits in the STFP retention and MWM reversal learning in old rats. DCS also significantly increased the hippocampal levels of synaptophysin in old rats, which correlated with STFP and MWM performance in all tests. Moreover, although the levels of the GluN1 subunit correlated with the MWM acquisition and reversal, DCS did not enhance the expression of such synaptic protein. The present behavioral results support the role of DCS as a cognitive enhancer and suggest that enhancing the function of NMDARs and synaptic plasticity in the hippocampus may be related to improvement in social memory and spatial learning reversal in aged animals.

N-methyl-d-aspartate-type glutamate receptor modulators and related medications for the enhancement of auditory system plasticity in schizophrenia

Deficits in N-methyl-d-aspartate-type (NMDAR) function contribute to cognitive deficits in schizophrenia, particularly dysfunction in neuroplasticity, defined as reduced learning during training on exercises that place implicit, increasing demands on early sensory (auditory and visual) information processing. Auditory mismatch negativity (MMN) can be both a target engagement biomarker for the NMDAR and a proxy measure of neurophysiological plasticity. This review covers the evidence for using NMDAR modulator and related compounds for enhancement of cognition, with a particular focus on early auditory processing/plasticity. Compounds covered include glycine site agonists, glycine and system A-type transporter inhibitors, d-amino acid oxidase inhibitors, memantine and nicotinic alpha-7 acetylcholine receptor agonists. As opposed to daily treatment studies focusing on schizophrenia in general, intermittent, non-daily treatment combining NMDAR modulators with neuroplasticity-based paradigms, using MMN as target-engagement biomarkers show promise as treatments to both remediate plasticity deficits and overall functional deficits.

Positive N-Methyl-D-Aspartate Receptor Modulation by Rapastinel Promotes Rapid and Sustained Antidepressant-Like Effects.

BackgroundModulation of glutamatergic synaptic transmission by N-methyl-D-aspartate receptors can produce rapid and sustained antidepressant effects. Rapastinel (GLYX-13), initially described as a N-methyl-D-aspartate receptor partial glycine site agonist, exhibits rapid antidepressant effect in rodents without the accompanying dissociative effects of N-methyl-D-aspartate receptor antagonists.MethodsThe relationship between rapastinel’s in vitro N-methyl-D-aspartate receptor pharmacology and antidepressant efficacy was determined by brain microdialysis and subsequent pharmacological characterization of therapeutic rapastinel concentrations in N-methyl-D-aspartate receptor-specific radioligand displacement, calcium mobilization, and medial prefrontal cortex electrophysiology assays.ResultsBrain rapastinel concentrations of 30 to 100 nM were associated with its antidepressant-like efficacy and enhancement of N-methyl-D-aspartate receptor-dependent neuronal intracellular calcium mobilization. Modulation of N-methyl-D-aspartate receptors by rapastinel was independent of D-serine concentrations, and glycine site antagonists did not block rapastinel’s effect. In rat medial prefrontal cortex slices, 100 nM rapastinel increased N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents and enhanced the magnitude of long-term potentiation without any effect on miniature EPSCs or paired-pulse facilitation responses, indicating postsynaptic action of rapastinel. A critical amino acid within the NR2 subunit was identified as necessary for rapastinel’s modulatory effect.ConclusionRapastinel brain concentrations associated with antidepressant-like activity directly enhance medial prefrontal cortex N-methyl-D-aspartate receptor activity and N-methyl-D-aspartate receptor-mediated synaptic plasticity in vitro. At therapeutic concentrations, rapastinel directly enhances N-methyl-D-aspartate receptor activity through a novel site independent of the glycine coagonist site. While both rapastinel and ketamine physically target N-methyl-D-aspartate receptors, the 2 molecules have opposing actions on N-methyl-D-aspartate receptors. Modest positive modulation of N-methyl-D-aspartate receptors by rapastinel represents a novel pharmacological approach to promote well-tolerated, rapid, and sustained improvements in mood disorders.

Изменение специфического связывания [N-метил-<sup>3</sup>H]флунитразепама при воздействии анксиогенами

Изучено влияние лекарственных препаратов и фармакологических соединений, обладающих анксиогенными свойствами: йохимбин, кофеин, производное β-карболинов — соединение β-ССВ на специфическое связывание полного агониста бензодиазепинового участка ГАМКА рецептора [N-метил-3H]флунитразепама. Кофеин в дозе 100 мг/кг, n-бутил-β-карболин-3 карбоксилат (β-ССВ) в дозе 3 мг/кг и йохимбин в дозе 5 мг/кг вызывали анксиогенный эффект у мышей линии Balb/c и C57Bl/6. Анксиогенная активность кофеина, β-ССВ и йохимбина сопряжена с падением уровня бензодиазепиновой рецепции.

The fifth subunit of the (α4β2)2 β2 nicotinic ACh receptor modulates maximal ACh responses.

The fifth subunit in the (α4β2)2 α4 nicotinic ACh receptor (nAChR) plays a determining role in the pharmacology of this nAChR type. Here, we have examined the role of the fifth subunit in the ACh responses of the (α4β2)2 β2 nAChR type. The role of the fifth subunit in receptor function was explored using two-electrode voltage clamp electrophysiology, along with subunit-targeted mutagenesis and the substituted cysteine scanning method applied to fully linked (α4β2)2 β2 receptors. Covalent modification of the cysteine-substituted fifth subunit with a thiol-reactive agent (MTS) caused irreversible inhibition of receptor function. ACh reduced the rate of the reaction to MTS, but the competitive inhibitor dihydro-β-erythroidine had no effect. Alanine substitution of conserved residues that line the core of the agonist sites on α4(+)/β2(-) interfaces did not impair receptor function. However, impairment of agonist binding to α4(+)/β2(-) agonist sites by mutagenesis modified the effect of ACh on the rate of the reaction to MTS. The extent of this effect was dependent on the position of the agonist site relative to the fifth subunit. The fifth subunit in the (α4β2)2 β2 receptor isoform modulates maximal ACh responses. This effect appears to be driven by a modulatory, and asymmetric, association with the α4(+)/β2(-) agonist sites. This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Identification of AICP as a GluN2C-Selective N-Methyl-d-Aspartate Receptor Superagonist at the GluN1 Glycine Site.

N-methyl-d-aspartate (NMDA)-type ionotropic glutamate receptors mediate excitatory neurotransmission in the central nervous system and are critically involved in brain function. NMDA receptors are also implicated in psychiatric and neurological disorders and have received considerable attention as therapeutic targets. In this regard, administration of d-cycloserine (DCS), which is a glycine site NMDA receptor agonist, can enhance extinction of conditioned fear responses. The intriguing behavioral effects of DCS have been linked to its unique pharmacological profile among NMDA receptor subtypes (GluN1/2A-D), in which DCS is a superagonist at GluN2C-containing receptors compared with glycine and a partial agonist at GluN2B-containing receptors. Here, we identify (R)-2-amino-3-(4-(2-ethylphenyl)-1H-indole-2-carboxamido)propanoic acid (AICP) as a glycine site agonist with unique GluN2-dependent differences in agonist efficacy at recombinant NMDA receptor subtypes. AICP is a full agonist at GluN1/2A (100% response compared with glycine), a partial agonist at GluN1/2B and GluN1/2D (10% and 27%, respectively), and a highly efficacious superagonist at GluN1/2C receptors (353%). Furthermore, AICP potencies are enhanced compared with DCS with EC50 values in the low nanomolar range (1.7 nM at GluN1/2C). We show that GluN1/2C superagonism of AICP and DCS is mediated by overlapping but distinct mechanisms and that AICP selectively enhances responses from recombinant GluN1/2C receptors in the presence of physiological glycine concentrations. This functional selectivity of AICP for GluN2C-containing NMDA receptors is more pronounced compared with DCS, suggesting that AICP can be a useful tool compound for uncovering the roles of GluN2C subunits in neuronal circuit function and in the development of new therapeutic strategies.

Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors.

This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

The Role of Dopamine D1 and D3 Receptors in N-Methyl-D-Aspartate (NMDA)/GlycineB Site-Regulated Complex Cognitive Behaviors following Repeated Morphine Administration.

Background:Opiate addiction is associated with complex cognitive impairment, which contributes to the development of compulsive drug use and relapses. Dopamine and N-methyl-D-aspartate receptors play critical roles in opiate-induced cognitive deficits. However, the roles of D1 and D3 receptors in the N-methyl-D-aspartate/glycineB receptor-regulated cognitive behaviors induced by morphine remain unknown.Methods:The 5-choice serial reaction time task was used to investigate the cognitive profiles associated with repeated morphine administration in D1 (D1-/-)- and D3 (D3-/-)-receptor knockout mice. The expression of phosphorylated NR1, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element-binding protein (CREB) in the brain was examined by western blotting. D1-/- and D3-/- mice were treated with the N-methyl-D-aspartate/glycineB site agonist l-aminocyclopropanecarboxylic acid and the antagonist L-701,324 to chronically disrupt N-methyl-D-aspartate receptor function and investigate their effects on morphine-induced cognitive changes.Results:Repeated morphine administration impaired attentional function and caused impulsive and compulsive behaviors. D1-/- mice exhibited hardly any premature nosepokes. D3-/- mice showed robustly increased morphine-induced impulsive behavior. The numbers of premature responses were decreased by L-701,324 administration and increased by ACPC administration; these effects were completely abolished in D1-/- mice due to their inability to perform reward-based tasks. In contrast, the inhibitory effects of L-701,324 on impulsive behavior were significantly augmented in D3-/- mice.Conclusions:N-methyl-D-aspartate/glycineB site functions may contribute to morphine-induced cognitive deficits, especially those related to impulsive behavior. D1 and D3 receptors may have contrasting effects with respect to modulating impulsive behavior. D3 receptors have inhibitory effects on impulsive behaviors, and these effects are clearly mediated by N-methyl-D-aspartate/glycineB receptor and μ-opioid receptor interactions.

Uncommon Features of a Novel γ-Proteobacterial Pentameric Ligand-Gated Ion Channel Revealed by its Crystal Structure

Pentameric ligand-gated ion channels (pLGICs) control synaptic neurotransmission in higher eukaryotes via an allosteric mechanism, where agonist binding in the extra-cellular domain triggers the opening of an ion-conducting pore in the transmembrane domain. Here we present the x-ray structure at 2.6 Å resolution of a novel bacterial homologue bound to an agonist, an amino acid analogue, in an apparently open conformation. This new structure presents unique features compared to the other pLGICs structures described to date: the presence of a constriction ring formed by 5 arginines that partially obstruct the vestibule; and an open channel pore with an extra large diameter. Moreover, the agonist doesn’t bind the normal agonist site located under loop C at the interface of two subunits. Instead, it is found to bind at a site previously described as an allosteric site for other bacterial receptors. In addition, the structure clearly shows several monovalent cations in the ion channel pore. Overall, this new structure is most closely related to ELIC structure, also from a gamma-proteobacteria, but its open pore differs markedly from the one seen in ELIC. We will discuss the implications of this new structure for the evolution and function of bacterial and eukaryotic pLGICs.

Low Expression in Xenopus Oocytes and Unusual Functional Properties of α1β2γ2 GABAA Receptors with Non-Conventional Subunit Arrangement.

The major subunit isoform of GABAA receptors is α1β2γ2. The subunits are thought to surround an ion pore with the counterclockwise arrangement α1γ2β2α1β2 as seen from the outside of the neuron. These receptors have two agonist sites and one high affinity drug binding site specific for benzodiazepines. Recently, this receptor was postulated to assume alternative subunit stoichiometries and arrangements resulting in only one agonist site and one or even two sites for benzodiazepines. In order to force a defined subunit arrangement we expressed a combination of triple and dual concatenated subunits. Here we report that these unconventional receptors express only small current amplitudes in Xenopus oocytes. We determined agonist properties and modulation by diazepam of two of these receptors that resulted in currents large enough for a characterization, that is, β2-α1-γ2/α1-γ2 and β2-α1-γ2/β2-γ2. The first pentamer predicted to have two benzodiazepine binding sites shows similar response to diazepam as the standard receptor. As expected for both receptors with a single predicted agonist site the concentration response curves for GABA were characterized by a Hill coefficient < 1. β2-α1-γ2/β2-γ2 displayed a mM apparent GABA affinity for channel opening instead of the expected μM affinity. Based on their subunit and binding site stoichiometry, that contradicts all previous observations, their unusual functional properties and their very low expression levels in oocytes, we consider it unlikely that these unconventional receptors are expressed in neurons to an appreciable extent.

Activation and modulation of the nicotinic receptor

Nicotinic acetylcholine receptors (AChRs) are the best characterized ion channels representing the Cys-loop receptor superfamily. AChRs have all the machinery to recognize the neurotransmitter ACh and other agonists such as nicotine, and to transduce the agonist-induced conformational changes into the opening of the intrinsic cation channel. The gating machinery couples ligand binding, located at the extracellular portion, to the opening of the ion channel, located at the transmembrane region. The interface between the extracellular and the transmembrane domains is considered one of the most important structural and functional features for the process of gating. And finally, in the prolonged presence of agonists, the AChR becomes desensitized. Several drugs affect the functioning of these receptors. Among them, positive allosteric modulators (PAM) have acquired importance since are novel drugs for several neurological diseases. PAMs do not bind to the orthostetic binding sites but allosterically enhance the activity elicited by agonists by increasing the gating process and/or by decreasing desensitization. Instead, negative allosteric modulators (NAMs) produce the opposite effects. Interestingly, this negative effect is similar to that found for another class of allosteric drugs, i.e. non competitive antagonists (NCAs). However, the main difference between both categories of drugs is based on their distinct binding site locations. Although both NAMs and NCAs do not bind to the agonist sites, NACs bind to sites located in the ion channel, whereas NAMs bind to nonluminal sites. Interestingly, PAMs and NAMs might be developed as potential medications for the treatment of several diseases involving AChRs including, dementia-, skin-, and immunological-related diseases, drug addiction, and cancer. More exciting is the potential combination of specific agonists with PAMs. However, we are still in the beginning of understanding how these compounds act and how these drugs can be used therapeutically.

The clobazam metabolite N-desmethyl clobazam is an α2 preferring benzodiazepine with an improved therapeutic window for antihyperalgesia

Data from genetically modified mice suggest that benzodiazepine (BDZ)-site agonists with improved selectivity for α2-subtype GABAA receptors (α2GABAAR) are potentially useful for the treatment of neuropathic pain. Subtype-selective compounds available for preclinical tests in rodents support this concept but have not been approved for human use, hindering proof-of-concept studies in patients. We recently proposed that N-desmethyl clobazam (NDMC), the main metabolite of the licensed BDZ clobazam (CBZ), is responsible for most of the antihyperalgesia observed in mice after CBZ administration. In order to assess a potentially favorable pharmacological profile of NDMC, we analyzed differences in the GABAAR subtype specificity of CBZ, NDMC and diazepam (DZP) in recombinant receptors. DZP and CBZ potentiated sedating α1GABAARs and antihyperalgesic α2GABAARs with similar efficacies, whereas NDMC preferred α2GABAARs over α1GABAARs across a wide concentration range. In vivo, DZP and NDMC reduced neuropathic pain at doses between 3 and 30 mg/kg. At these doses, DZP had strong locomotor sedating effects while NDMC caused no or only weak sedation. Sedative effects of NDMC became apparent when the action of NDMC was restricted to α1GABAARs. However, when GABAAR point-mutated mice were studied that allow the analysis of antihyperalgesia and sedation in isolation, we found that, compared to DZP, NDMC had a significantly improved therapeutic window, consistent with its more favorable α2/α1 in vitro activity ratio. Given that NDMC should share the safety profile of its parent compound CBZ, it should be well-suited for proof-of-concept studies in human volunteers or patients.

PM432. Evaluation of the extrapyramidal side effects (EPS) liability of NMDA receptor glycine-binding site agonists

PM432. Evaluation of the extrapyramidal side effects (EPS) liability of NMDA receptor glycine-binding site agonists

Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors.

BackgroundPentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range.MethodsUsing oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep “notch” approach, and used these results to correct steady-state direct activation for inhibition.ResultsCombining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA.ConclusionsOur novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites.

Sensing Allosteric Modulator Binding to AMPA Receptors at the Glutamate-Binding Site

Positive allosteric modulators of AMPA receptors (GluAs) stabilize the activated GluA structure by binding to the glutamate-binding domain (LBD) dimer interface. By adding contacts across the dimer interface, allosteric modulators strengthen the LBD dimer and inhibit desensitization-linked dimer disruption. Structurally diverse molecules can occupy the interface to fill various pockets and act as LBD dimerizers. Functionally, allosteric modulators exert a primary allosteric effect by stabilizing the open channel; however, allosteric modulators with different dimer interface binding patterns lead to differing impacts on agonist site binding affinity. Here, we examine the secondary allosteric effects on the glutamate-binding site. Using nuclear magnetic resonance (NMR) spectroscopy, we observed the global backbone changes on the isolated GluA2 LBD as a result of allosteric modulator binding. Spectral changes at the dimer interface correlate well with allosteric modulator binding sites on bound X-ray crystal structures. The backbone residues near the agonist-binding site are also affected differently depending on the orientation of allosteric modulator binding. Further, allosteric modulator binding to the LBD induces local environmental changes at the agonist site as reported by NMR active nuclei on the LBD-bound agonist. By improving the molecular understanding of GluA allosteric modulators in the context of secondary agonist-site effects, our results provide further guidance for rational drug design.

A Nonrewarding NMDA Receptor Antagonist Impairs the Acquisition, Consolidation, and Expression of Morphine Conditioned Place Preference in Mice.

N-methyl-D-aspartate (NMDA) receptor antagonists block morphine-induced conditioned place preference (CPP). Although polyamines are endogenous modulators of the NMDA receptor, it is not known whether polyaminergic agents induce CPP or modulate morphine-induced CPP. Here, we examined whether polyamine ligands modify morphine CPP acquisition, consolidation, and expression. Adult male albino Swiss mice received saline (0.9% NaCl, intraperitoneally (i.p.)) or morphine (5mg/kg, i.p.) and were respectively confined to a black or a white compartment for 30min for four consecutive days for CPP induction. The effect of arcaine (3mg/kg, i.p.) or spermidine (30mg/kg, i.p.), respectively, an antagonist and an agonist of the polyamine-binding site at the NMDA receptor, on the acquisition, consolidation, and expression of morphine CPP was studied. In those experiments designed to investigate whether spermidine prevented or reversed the effect of arcaine, spermidine (30mg/kg, i.p.) was administered 15min before or 15min after arcaine, respectively. Arcaine and spermidine did not induce CPP or aversion per se. Arcaine (3mg/kg, i.p.) impaired the acquisition, consolidation, and expression of morphine CPP. Spermidine prevented the impairing effect of arcaine on the acquisition of morphine CPP but not the impairing effect of arcaine on consolidation or expression of morphine CPP. These results suggest that arcaine may impair morphine CPP acquisition by modulating the polyamine-binding site at the NMDA receptor. However, the arcaine-induced impairment of consolidation and expression of morphine CPP seems to involve other mechanisms.

Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

Synergistic antidepressant-like effect of the joint administration of caffeine and NMDA receptor ligands in the forced swim test in mice.

The optimal treatment of depressed patients remains one of the most important challenges concerning depression. The identification of the best treatment strategies and development of new, safer, and more effective agents are crucial. The glutamatergic system seems to be a promising drug target, and consequently the use of the NMDA receptor ligands, particularly in co-administration with other substances exerting the antidepressant activity, has emerged among the new ideas. The objective of this study was to examine the effect of caffeine on the performance of mice treated with various NMDA modulators in the forced swim test. We demonstrated a significant interaction between caffeine (5 mg/kg) and the following NMDA receptor ligands: MK-801 (an antagonist binding in the ion channel, 0.05 mg/kg), CGP 37849 (an antagonist of the glutamate site, 0.312 mg/kg), L-701,324 (an antagonist of the glycine site, 1 mg/kg), and d-cycloserine (a high-efficacy partial agonist of the glycine site, 2.5 mg/kg), while the interaction between caffeine and the inorganic modulators, i.e., Zn2+ (2.5 mg/kg) and Mg2+ (10 mg/kg), was not considered as significant. Based on the obtained results, the simultaneous blockage of the adenosine and NMDA receptors may be a promising target in the development of new antidepressants.

Modal affinities of endplate acetylcholine receptors caused by loop C mutations.

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability ("modes"). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5-β5' linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.

D-Cycloserine reduces context specificity of sexual extinction learning

Backgroundd-Cycloserine (DCS) enhances extinction processes in animals. Although classical conditioning is hypothesized to play a pivotal role in the aetiology of appetitive motivation problems, no research has been conducted on the effect of DCS on the reduction of context specificity of extinction in human appetitive learning, while facilitation hereof is relevant in the context of treatment of problematic reward-seeking behaviors. MethodsFemale participants were presented with two conditioned stimuli (CSs) that either predicted (CS+) or did not predict (CS−) a potential sexual reward (unconditioned stimulus (US); genital vibrostimulation). Conditioning took place in context A and extinction in context B. Subjects received DCS (125mg) or placebo directly after the experiment on day 1 in a randomized, double-blind, between-subject fashion (Placebo n=31; DCS n=31). Subsequent testing for CS-evoked conditioned responses (CRs) in both the conditioning (A) and the extinction context (B) took place 24h later on day 2. Drug effects on consolidation were then assessed by comparing the recall of sexual extinction memories between the DCS and the placebo groups. ResultsPost learning administration of DCS facilitates sexual extinction memory consolidation and affects extinction’s fundamental context specificity, evidenced by reduced conditioned genital and subjective sexual responses, relative to placebo, for presentations of the reward predicting cue 24h later outside the extinction context. ConclusionsDCS makes appetitive extinction memories context-independent and prevents the return of conditioned response. NMDA receptor glycine site agonists may be potential pharmacotherapies for the prevention of relapse of appetitive motivation disorders with a learned component.