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Abstract Non-selective benzodiazepines, such as diazepam, interact with equivalent affinity and agonist efficacy at GAB[A.sub.A] receptors containing either an α1, α2, α3 or α5 subunit. However, which of these particular [...]

Non-selective benzodiazepines, such as diazepam, interact with equivalent affinity and agonist efficacy at GABAA receptors containing either an α1, α2, α3 or α5 subunit. However, which of these particular subtypes are responsible for the anticonvulsant effects of diazepam remains uncertain. In the present study, we examined the ability of diazepam to reduce pentylenetetrazole (PTZ)-induced and maximal electroshock (MES)-induced seizures in mice containing point mutations in single (α1H101R, α2H101R or α5H105R) or multiple (α125H→R) α subunits that render the resulting GABAA receptors diazepam-insensitive. Furthermore, the anticonvulsant properties of diazepam, the α1- and α3-selective compounds zolpidem and TP003, respectively, and the α2/α3 preferring compound TP13 were studied against PTZ-induced seizures. In the transgenic mice, no single subtype was responsible for the anticonvulsant effects of diazepam in either the PTZ or MES assay and neither the α3 nor α5 subtypes appeared to confer anticonvulsant activity. Moreover, whereas the α1 and α2 subtypes played a modest role with respect to the PTZ assay, they had a negligible role in the MES assay. With respect to subtype-selective compounds, zolpidem and TP003 had much reduced anticonvulsant efficacy relative to diazepam in both the PTZ and MES assays whereas TP13 had high anticonvulsant efficacy in the PTZ but not the MES assay. Taken together, these data not only indicate a role for α2-containing GABAA receptors in mediating PTZ and MES anticonvulsant activity but also suggest that efficacy at more than one subtype is required and that these subtypes act synergistically. [ABSTRACT FROM AUTHOR]

In the mammalian central nervous system, transporter-mediated reuptake may be critical for terminating the neurotransmitter action ofd-serine at the strychnine insensitive glycine site of the NMDA receptor. The Na+ independent amino acid transporter alanine–serine–cysteine transporter 1 (Asc-1) has been proposed to account for synaptosomald-serine uptake by virtue of its high affinity ford-serine and widespread neuronal expression throughout the brain. Here, we sought to validate the contribution of Asc-1 tod-serine uptake in mouse brain synaptosomes using Asc-1 gene knockout (KO) mice. Total [3H]d-serine uptake in forebrain and cerebellar synaptosomes from Asc-1 knockout mice was reduced to 34 ± 5% and 22 ± 3% of that observed in wildtype (WT) mice, respectively. When the Na+ dependent transport components were removed by omission of Na+ ions in the assay buffer,d-serine uptake in knockout mice was reduced to 8 ± 1% and 3 ± 1% of that measured in wildtype mice in forebrain and cerebellum, respectively, suggesting Asc-1 plays a major role in the Na+ independent transport ofd-serine. Potency determination ofd-serine uptake showed that Asc-1 mediated rapid high affinity Na+ independent uptake with an IC50 of 19 ± 1 µm. The remaining uptake was mediated predominantly via a low affinity Na+ dependent transporter with an IC50 of 670 ± 300 µm that we propose is the glial alanine–serine–cysteine transporter 2 (ASCT2) transporter. The results presented reveal that Asc-1 is the only high affinityd-serine transporter in the mouse CNS and is the predominant mechanism ford-serine reuptake. [ABSTRACT FROM AUTHOR]

The GABAA receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that α2- rather than α3-containing GABAA receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an α3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2′-difluoro-5′-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an α3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders α2-containing receptors BZ insensitive (α2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of α3-containing GABAA receptors is sufficient to produce the anxiolytic effects of BZs and that α2 potentiation may not be necessary. [ABSTRACT FROM AUTHOR]

Background TAK-831 is a potent inhibitor of the DAAO enzyme being developed for the cognitive deficits and negative symptoms of schizophrenia. Inhibition of DAAO results in an increase in D-serine, a co-agonist of the NMDA receptor and a full agonist of the δ2 glutamate receptor. Augmentation of NMDA and δ2 glutamate receptor function may be beneficial in schizophrenia. To determine this, rats given TAK-831 were tested in the Novel Object Recognition (NOR) test, a basic cognition test, the Attentional Set Shifting Task (ASST), an animal assay to assess executive function in rodents, and Social Interaction (SI), an animal model of negative symptoms. To determine the impact that a target localized in the cerebellum has on learning, the compound was also profiled in eyeblink conditioning (EBC), a model of cerebellar-based associative learning. Methods Rats were given an acute and chronic dose of TAK-831 and cerebrospinal fluid (CSF); cerebellar tissue and plasma were harvested after 6 hours. For NOR, mice were dosed acutely and chronically (14d) with TAK-831 and assessed in their ability to differentiate between novel and previously seen objects after a 4-hour inter-trial interval (ITI). Rats treated with a subchronic phencyclidine (PCP) regimen (2 mg/kg, 7 days bid) showed a deficit in the extra-dimensional shift (EDS) in the ASST. Acute and chronic (14d) doses of TAK-831 were assessed for reversal of the PCP-induced deficit. TAK-831 was dosed acutely and chronically (0.3, 1, 3 mg/kg, po) and assessed for its ability to reverse a naturally occurring SI deficit seen in Balb/c mice. The same dose range was used when the SI deficit was induced by maternal treatment with Poly(I:C). In EBC, a periorbital electrical stimulus was paired with a corneal air puff resulting in classical conditioning. TAK-831 (0.1 and 1 mg/kg, po, 10d chronic dosing) was assessed for its ability to improve acquisition of the conditioned response in the presence and absence of scopolamine. Results TAK-831 produced a dose-dependent increase in D-serine levels across CSF, cerebellum, and in plasma. After a 4-hour ITI, vehicle-treated mice were unable to differentiate between a novel and familiar object in NOR. Animals were able to identify the novel object when treated with TAK-831 at 0.3, 1, 3 and 10 mg/kg acute doses and at 0.003, 0.01, 0.03, 0.1, 1.0 and 3 mg/kg chronic doses. TAK-831 was able to reverse the PCP-induced deficit in rats in the EDS in the ASST at 3, 10, and 30 mg/kg acute doses and at 0.1, 1.0 and 10 mg/kg chronic doses. TAK-831 was efficacious in the Balb/c SI model at 1 and 3 mg/kg acute doses and at 0.01, 0.03, 0.1, 1.0 and 10 mg/kg chronic doses; it was efficacious at 3 mg/kg acute dosing in the Poly(I:C) SI model. TAK-831 caused an improvement in both the acquisition of the conditioned response and in reversing the scopolamine-induced deficit at 0.1 and 1 mg/kg in the EBC paradigm. Discussion TAK-831 shows utility at a range of doses in animal models associated with negative symptoms and cognitive impairment in schizophrenia. Its efficacy in EBC shows the impact on cerebellar-based learning; this region of the brain is now considered to also be involved in schizophrenia. TAK-831 shows a different range of efficacies pre-clinically, depending on whether the compound is dosed acutely or chronically, indicating a potential plasticity of effect in the brain of preclinical species. [ABSTRACT FROM AUTHOR]

The GABA(A) receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that alpha2-rather than alpha3-containing GABA(A) receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an alpha3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2'-difluoro-5'-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an alpha3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders alpha2-containing receptors BZ insensitive (alpha2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of alpha3-containing GABA(A) receptors is sufficient to produce the anxiolytic effects of BZs and that alpha2 potentiation may not be necessary.

The i.v. agent etomidate exerts its anaesthetic actions through potentiation of gamma-aminobutyric acid-A receptors containing beta2 and beta3 subunits. It was recently shown that the beta2 subunit contributes to the sedative properties of etomidate, whereas the beta3 subunit is responsible for its anaesthetic properties. However, these studies evaluated anaesthetic effects in point mutation mice in which the effect of etomidate was decreased, but not abolished, at the beta2 subunit. Here we have used beta2 knockout mice to completely remove any contribution of the beta2 subunit to the effects of etomidate. Etomidate was equally anaesthetic in wildtype and knockout mice, thus further confirming that efficacy at the beta3 subunit only is sufficient to induce general anaesthesia.