Affinity of the inverse agonist methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to the benzodiazepine binding site of the GABA(A) receptor is abolished by a phenylalanine (F) to ...isoleucine (I) substitution at position 77 of the gamma2 subunit. We tested the effects of DMCM in gene knockin gamma2I77 mice carrying this mutation. Unlike in wild-type mice, DMCM was not able to reverse the GABA-induced reduction of the picrotoxin-sensitive t-butylbicyclophosphoro-35Sthionate (35STBPS) binding to GABA(A) receptor channels in the forebrain sections of gamma2I77 mice. Accordingly, DMCM was not convulsant in the mutant mice even at doses 20-fold higher (60mg/kg, i.p.) than those producing convulsions in wild-type littermate controls (3 mg/kg, i.p.). Neither did DMCM raise the c-Fos levels in gamma2I77 mouse brain. DMCM additionally exhibits a less well described agonistic effect on GABA(A) receptors that is normally masked by its strong inverse agonist effect. DMCM agonistically enhanced the GABA-induced reduction in 35STBPS binding to the cerebellar granule cell layer in control and mutant mice. In vivo DMCM (20-60 mg/kg i.p.) produced modest anxiolytic-like effects in gamma2I77 mice as assessed by elevated plus maze and staircase tests, but no motor impairment was found in the rotarod test. The results suggest only minor agonistic efficacy for the beta-carboline DMCM.
Abstract
Zolpidem is a hypnotic benzodiazepine site agonist with some γ‐aminobutyric acid (GABA)
A
receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of γ2 ...subunit (γ2F77I) point mutant mice. Analysis of forebrain GABA
A
receptor expression with immunocytochemistry, quantitative
3
Hmuscimol and
35
S t‐butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with
3
Hflunitrazepam and
3
Hmuscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous γ2I77/I77 and γ2F77/F77 mice. However, quantitative immunoblot analysis of γ2I77/I77 hippocampi showed some increased levels of γ2, α1, α4 and δ subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 µ
m
) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, γ2F77/F77) mice by ∼ 60%, and peak amplitude by ∼ 20% at 33–34 °C
in vitro
. The actions of zolpidem (100 n
m
or 1 µ
m
) were substantially reduced in γ2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 µ
m
) on carbachol‐induced oscillations in the CA3 area of γ2I77/I77 mice was significantly different compared with controls. Thus, the γ2F77I point mutation virtually abolished the actions of zolpidem on GABA
A
receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the γ2 subunit.
Inhalation anesthetics activate and cannabinoid agonists inhibit TWIK-related acid-sensitive K(+) channels (TASK)-1 two-pore domain leak K(+) channels in vitro. Many neuromodulators, such as ...noradrenaline, might also manifest some of their actions by modifying TASK channel activity. Here, we have characterized the basal behavioral phenotype of TASK-1 knockout mice and tested their sensitivity to the inhalation anesthetics halothane and isoflurane, the alpha(2) adrenoreceptor agonist dexmedetomidine, and the cannabinoid agonist WIN55212-2 mesylate R-(+)-2,3-dihydro-5-methyl-3-(morpholinyl)methylpyrrolo1,2,3,-de-1,4-benzoxazinyl-(1-naphtalenyl)methanone mesylate). TASK-1 knockout mice had a largely normal behavioral phenotype. Male, but not female, knockout mice displayed an enhanced acoustic startle response. The knockout mice showed increased sensitivity to thermal nociception in a hot-plate test but not in a tail-flick test. The analgesic, sedative, and hypothermic effects of WIN55212-2 (2-6 mg/kg s.c.) were reduced in TASK-1 knockout mice. These results implicate TASK-1-containing channels in supraspinal pain pathways, in particular those modulated by endogenous cannabinoids. TASK-1 knockout mice were less sensitive to the anesthetic effects of halothane and isoflurane than wild-type littermates, requiring higher anesthetic concentrations to induce immobility as reflected by loss of the tail-withdrawal reflex. Our results support the idea that the activation of multiple background K(+) channels is crucial for the high potency of inhalation anesthetics. Furthermore, TASK-1 knockout mice were less sensitive to the sedative effects of dexmedetomidine (0.03 mg/kg s.c.), suggesting a role for the TASK-1 channels in the modulation of function of the adrenergic locus coeruleus nuclei and/or other neuronal systems.
Affinity of the inverse agonist methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) to the benzodiazepine binding site of the GABA
A receptor is abolished by a phenylalanine (F) to ...isoleucine (I) substitution at position 77 of the γ2 subunit. We tested the effects of DMCM in gene knockin γ2I77 mice carrying this mutation. Unlike in wild-type mice, DMCM was not able to reverse the GABA-induced reduction of the picrotoxin-sensitive
t-butylbicyclophosphoro-
35Sthionate (
35STBPS) binding to GABA
A receptor channels in the forebrain sections of γ2I77 mice. Accordingly, DMCM was not convulsant in the mutant mice even at doses 20-fold higher (60
mg/kg, i.p.) than those producing convulsions in wild-type littermate controls (3
mg/kg, i.p.). Neither did DMCM raise the c-Fos levels in γ2I77 mouse brain. DMCM additionally exhibits a less well described agonistic effect on GABA
A receptors that is normally masked by its strong inverse agonist effect. DMCM agonistically enhanced the GABA-induced reduction in
35STBPS binding to the cerebellar granule cell layer in control and mutant mice. In vivo DMCM (20–60
mg/kg i.p.) produced modest anxiolytic-like effects in γ2I77 mice as assessed by elevated plus maze and staircase tests, but no motor impairment was found in the rotarod test. The results suggest only minor agonistic efficacy for the β-carboline DMCM.
TASK two-pore-domain leak K(+) channels occur throughout the brain. However, TASK-1 and TASK-3 knockout (KO) mice have few neurological impairments and only mildly reduced sensitivities to ...inhalational anesthetics, contrasting with the anticipated functions and importance of these channels. TASK-1/-3 channel expression can compensate for the absence of GABA(A) receptors in GABA(A) alpha6 KO mice. To investigate the converse, we analyzed the behavior of TASK-1 and -3 KO mice after administering drugs with preferential efficacies at GABA(A) receptor subtypes: benzodiazepines (diazepam and flurazepam, active at alpha1betagamma2, alpha2betagamma2, alpha3betagamma2, and alpha5betagamma2 subtypes), zolpidem (alpha1betagamma2 subtype), propofol (beta2-3-containing receptors), gaboxadol (alpha4betadelta and alpha6betadelta subtypes), pregnanolone, and pentobarbital (many subtypes). TASK-1 KO mice showed increased motor impairment in rotarod and beam-walking tests after diazepam and flurazepam administration but not after zolpidem. They also showed prolonged loss of righting reflex induced by propofol and pentobarbital. Autoradiography indicated no change in GABA(A) receptor ligand binding levels. These altered behavioral responses to GABAergic drugs suggest functional up-regulation of alpha2beta2/3gamma2 and alpha3beta2/3gamma2 receptor subtypes in TASK-1 KO mice. In addition, female, but not male, TASK-1 KO mice were more sensitive to gaboxadol, suggesting an increased influence of alpha4betadelta or alpha6betadelta subtypes. The benzodiazepine sensitivity of TASK-3 KO mice was marginally increased. Our results underline that TASK-1 channels perform such key functions in the brain that compensation is needed for their absence. Furthermore, because inhalation anesthetics act partially through GABA(A) receptors, the up-regulation of GABA(A) receptor function in TASK-1 KO mice might mask TASK-1 channel's significance as a target for inhalation anesthetics.
Zolpidem is a hypnotic benzodiazepine site agonist with some γ‐aminobutyric acid (GABA)A receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of γ2 subunit ...(γ2F77I) point mutant mice. Analysis of forebrain GABAA receptor expression with immunocytochemistry, quantitative 3Hmuscimol and 35S t‐butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with 3Hflunitrazepam and 3Hmuscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous γ2I77/I77 and γ2F77/F77 mice. However, quantitative immunoblot analysis of γ2I77/I77 hippocampi showed some increased levels of γ2, α1, α4 and δ subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 µm) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, γ2F77/F77) mice by ∼ 60%, and peak amplitude by ∼ 20% at 33–34 °C in vitro. The actions of zolpidem (100 nm or 1 µm) were substantially reduced in γ2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 µm) on carbachol‐induced oscillations in the CA3 area of γ2I77/I77 mice was significantly different compared with controls. Thus, the γ2F77I point mutation virtually abolished the actions of zolpidem on GABAA receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the γ2 subunit.
Zolpidem is a hypnotic benzodiazepine site agonist with some gamma-aminobutyric acid (GABA)(A) receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of gamma2 ...subunit (gamma2F77I) point mutant mice. Analysis of forebrain GABA(A) receptor expression with immunocytochemistry, quantitative (3)Hmuscimol and (35)S t-butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with (3)Hflunitrazepam and (3)Hmuscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous gamma2I77/I77 and gamma2F77/F77 mice. However, quantitative immunoblot analysis of gamma2I77/I77 hippocampi showed some increased levels of gamma2, alpha1, alpha4 and delta subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 microm) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, gamma2F77/F77) mice by approximately 60%, and peak amplitude by approximately 20% at 33-34 degrees C in vitro. The actions of zolpidem (100 nm or 1 microm) were substantially reduced in gamma2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 microm) on carbachol-induced oscillations in the CA3 area of gamma2I77/I77 mice was significantly different compared with controls. Thus, the gamma2F77I point mutation virtually abolished the actions of zolpidem on GABA(A) receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the gamma2 subunit.
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