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.
Abstract
GABA
A
receptors mediate fast phasic inhibitory postsynaptic potentials and participate in slower tonic extrasynaptic inhibition. Thy1α6 mice with ectopic forebrain expression of GABA
A
...receptor α6 subunits exhibit increased extrasynaptic GABA
A
receptor‐mediated background conductance and reduced synaptic GABA
A
receptor currents in hippocampal CA1 neurons W. Wisden
et al
. (2002)
Neuropharmacology
43, 530–549. Here we demonstrate that isolated CA1 neurons of these mice showed furosemide‐sensitivity of GABA‐evoked currents, confirming the functional expression of α6 subunit. In addition, receptor autoradiography of the CA1 region of Thy1α6 brain sections revealed pharmacological features that are unique for α6βγ2 and α6β receptors. The existence of atypical α6β receptors was confirmed after completely eliminating GABA
A
receptors containing γ1, γ2, γ3 or δ subunits using serial immunoaffinity chromatography on subunit‐specific GABA
A
receptor antibodies. Behaviourally, the Thy1α6 mice showed normal features with slightly enhanced startle reflex and struggle‐escape behaviours. However, they were more sensitive to GABA
A
antagonists DMCM (shorter latency to writhing clonus) and picrotoxinin (shorter latency to generalized convulsions). Tiagabine, an antiepileptic GABA‐uptake inhibitor that increases brain GABA levels, delayed picrotoxinin‐induced convulsions at a low dose of 3.2 mg/kg in Thy1α6 mice, but not in control mice; however, the overall effect of higher tiagabine doses on the convulsion latency remained smaller in the Thy1α6 mice. Altered balance between extrasynaptic and synaptic receptors thus affects seizure sensitivity to GABAergic convulsants. Importantly, the increased extrasynaptic inhibition, even when facilitated in the presence of tiagabine, was not able fully to counteract enhanced seizure induction by GABA
A
antagonists.
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) sub(A) receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of ...gamma 2 subunit ( gamma 2F77I) point mutant mice. Analysis of forebrain GABA sub(A) receptor expression with immunocytochemistry, quantitative super(3)Hmuscimol and super(35)S t-butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with super(3)Hflunitrazepam and super(3)Hmuscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous gamma 2I77-I77 and gamma 2F77-F77 mice. However, quantitative immunoblot analysis of gamma 2I77-I77 hippocampi showed some increased levels of gamma 2, alpha 1, alpha 4 and delta subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 mu m) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL-6J, gamma 2F77-F77) mice by similar to 60%, and peak amplitude by similar to 20% at 33-34 degree C in vitro. The actions of zolpidem (100 nm or 1 mu m) were substantially reduced in gamma 2I77-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 mu m) on carbachol-induced oscillations in the CA3 area of gamma 2I77-I77 mice was significantly different compared with controls. Thus, the gamma 2F77I point mutation virtually abolished the actions of zolpidem on GABA sub(A) receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the gamma 2 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.
Agonists of the allosteric benzodiazepine site of GABA
A receptors bind at the interface of the α and γ subunits. Here, we tested the in vivo contribution of the γ2 subunit to the actions of ...zolpidem, an α1 subunit selective benzodiazepine agonist, by generating mice with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the γ2 subunit. The γ2F77I mutation has no major effect on the expression of GABA
A receptor subunits in the cerebellum. The potency of zolpidem, but not that of flurazepam, for the inhibition of
3Hflunitrazepam binding to cerebellar membranes is greatly reduced in γ2I77/I77 mice. Zolpidem (1 μM) increased both the amplitude and decay of miniature inhibitory postsynaptic currents (mIPSCs) in Purkinje cells of control C57BL/6 (34% and 92%, respectively) and γ2F77/F77 (20% and 84%) mice, but not in those of γ2F77I mice. Zolpidem tartrate had no effect on exploratory activity (staircase test) or motor performance (rotarod test) in γ2I77/I77 mice at doses up to 30 mg/kg (i.p.) that strongly sedated or impaired the control mice. Flurazepam was equally effective in enhancing mIPSCs and disrupting performance in the rotarod test in control and γ2I77/I77 mice. These results show that the effect of zolpidem, but not flurazepam, is selectively eliminated in the brain by the γ2F77I point mutation.
Agonists of the allosteric benzodiazepine site of GABAA receptors bind at the interface of the alpha and gamma subunits. Here, we tested the in vivo contribution of the gamma2 subunit to the actions ...of zolpidem, an alpha1 subunit selective benzodiazepine agonist, by generating mice with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma2 subunit. The gamma2F77I mutation has no major effect on the expression of GABAA receptor subunits in the cerebellum. The potency of zolpidem, but not that of flurazepam, for the inhibition of 3Hflunitrazepam binding to cerebellar membranes is greatly reduced in gamma2I77/I77 mice. Zolpidem (1 microM) increased both the amplitude and decay of miniature inhibitory postsynaptic currents (mIPSCs) in Purkinje cells of control C57BL/6 (34% and 92%, respectively) and gamma2F77/F77 (20% and 84%) mice, but not in those of gamma2F77I mice. Zolpidem tartrate had no effect on exploratory activity (staircase test) or motor performance (rotarod test) in gamma2I77/I77 mice at doses up to 30 mg/kg (i.p.) that strongly sedated or impaired the control mice. Flurazepam was equally effective in enhancing mIPSCs and disrupting performance in the rotarod test in control and gamma2I77/I77 mice. These results show that the effect of zolpidem, but not flurazepam, is selectively eliminated in the brain by the gamma2F77I point mutation.
Agonists of the allosteric benzodiazepine site of GABA sub(A) receptors bind at the interface of the alpha and gamma subunits. Here, we tested the in vivo contribution of the gamma 2 subunit to the ...actions of zolpidem, an alpha 1 subunit selective benzodiazepine agonist, by generating mice with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma 2 subunit. The gamma 2F77I mutation has no major effect on the expression of GABA sub(A) receptor subunits in the cerebellum. The potency of zolpidem, but not that of flurazepam, for the inhibition of super(3)Hflunitrazepam binding to cerebellar membranes is greatly reduced in gamma 2I77/I77 mice. Zolpidem (1 mu M) increased both the amplitude and decay of miniature inhibitory postsynaptic currents (mIPSCs) in Purkinje cells of control C57BL/6 (34% and 92%, respectively) and gamma 2F77/F77 (20% and 84%) mice, but not in those of gamma 2F77I mice. Zolpidem tartrate had no effect on exploratory activity (staircase test) or motor performance (rotarod test) in gamma 2I77/I77 mice at doses up to 30 mg/kg (i.p.) that strongly sedated or impaired the control mice. Flurazepam was equally effective in enhancing mIPSCs and disrupting performance in the rotarod test in control and gamma 2I77/I77 mice. These results show that the effect of zolpidem, but not flurazepam, is selectively eliminated in the brain by the gamma 2F77I point mutation.
