Chronic dizocilpine (MK-801) reversibly delays GABA(A) receptor maturation in cerebellar granule neurons in vitro

We investigated the effect of chronically blocking NMDA receptor stimulation to examine changes in GABA(A) receptor expression and pharmacology in cerebellar granule cells at different stages of maturation. We have previously shown that NMDA-selective glutamate receptor stimulation alters GABA(A) receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunits. When NMDA receptor stimulation is blocked with MK-801 during the first week in vitro, a decrease in the alpha1, gamma2S, and gamma2L receptor subunit mRNAs occurred. When present only during the second week, changes were limited to the alpha1 and gamma2L mRNAs. Finally, if MK-801 was present during the first week and removed during the second week, these changes reversed. Whole-cell voltage-clamp recordings showed that treatment with MK-801 during either the first or second week increased the EC50 of the receptors for GABA and attenuated the potentiation mediated by flunitrazepam. Last, these properties were reversed if MK-801 was removed after the first week in vitro. Our results suggest that MK-801 reversibly inhibits GABA(A) receptor maturation by modulating receptor subunit expression and that the altered pharmacological responses appear to be dominated by changes in the levels of allosteric modulation mediated by the gamma2 receptor subunit.     

Differential distribution of (GABA)A receptor subunits on bulbospinal serotonergic and nonserotonergic neurons of the ventromedial medulla of the rat

Spinally projecting neurons of the ventromedial medulla (VMM) compose an important efferent pathway for the modulation of nociception. These neurons receive a substantial gamma-aminobutyric acid (GABA)-ergic input, but the GABA receptor that mediates this input is unknown. This study examined the distribution of GABA(A) receptor alpha1 and alpha3 subunits in serotonergic and nonserotonergic neurons of the VMM that project to the dorsal horn in the rat. A pledget of Gelfoam soaked in Fluoro-Gold was placed at the thoracolumbar junction of the spinal cord to label spinally projecting neurons. Alternate sections of the medulla were then incubated with a mixture of antisera to either serotonin and the alpha1 subunit, or to serotonin and the alpha3 subunit of the GABA(A) receptor. Nearly 30% of spinally projecting neurons in the VMM were immunoreactive for the alpha1 subunit. A similar percentage of spinally projecting neurons in the VMM were immunoreactive for the alpha3 subunit, although diffuse cellular labeling combined with intense staining of processes in the neuropil precluded a rigorous semi-quantitative estimation of this population. No alpha1-subunit-immunoreactive neurons colocalized serotonin. In contrast, serotonergic neurons were immunoreactive for the alpha3 subunit. However, these double-labeled neurons were a modest percentage of the serotonergic population. A small percentage of spinally projecting serotonergic neurons was immunoreactive for the alpha3 subunit. These results suggest that significant numbers of spinally projecting serotonergic and nonserotonergic neurons of the VMM possess GABA(A) receptors that differ in their respective subunit compositions and that both classes of neurons may mediate the antinociception produced by the microinjection of GABA(A) receptor antagonists in the VMM.     

Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures

The GABA(A) receptor is a ligand gated chloride channel consisting of five membrane spanning proteins for which 13 different genes have been identified in the mammalian brain. The present review summarizes recent work from our laboratory on the characterization of the immunocytochemical distribution of these GABA(A) receptor subunits in the rat brain and changes in immunoreactivity and mRNA expression after kainic acid-induced status epilepticus. A heterogeneous distribution of immunoreactive GABA(A) receptor subunits was observed. The most abundant ones were: alpha1, alpha2, alpha4, alpha5, beta2, beta3, gamma2, and delta. Alpha1, beta2, and gamma2 were about equally distributed in all subfields of the hippocampus; alpha4- and delta-subunits were preferentially found in the dentate molecular layer and in CA1; alpha2 was localized to the dentate molecular layer and CA3; alpha5 was found in the dendritic areas of CA1 to CA3; and beta1 was preferentially seen in CA2. Alpha1, beta2, gamma2 and delta were highly concentrated in interneurons. Kainic acid-induced seizures caused acute and chronic changes in the expression of mRNAs and immunoreactive proteins. Acute changes included decreases in alpha2, alpha5, beta1, beta3, gamma2 and delta mRNA levels (by about 25-50%), accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages in granule cells (after 6-12 h). Chronic changes, characterized by losses in mRNA and immunoreactive proteins in CA1 and CA3, are undoubtedly due to seizure-related cell damage. However, compensatory expression of alpha2 and beta3 subunits, especially in CA3b/c, was observed. Furthermore, increases in mRNAs and immunoreactive proteins were seen for alpha1, alpha2 alpha4, beta1, beta2, beta3 and gamma2 in granule cells and in the molecular layer of the dentate gyrus at 7-30 days after kainic acid injection. The changes in the expression of GABA(A) receptor subunits, observed in practically all hippocampal subfields, may reflect altered GABA-ergic transmission during development of the epileptic syndrome. Increased expression of GABA(A) receptor subunits in the dendritic field of granule cells and CA3 suggest that GABA-ergic inhibition may be augmented at these levels. However, the lasting preservation of alpha1-, beta2-, and gamma2-subunits in interneurons could provide a basis for augmented inhibition of GABA-ergic interneurons, leading to net disinhibition.     

Tolerance to diazepam and changes in GABA(A) receptor subunit expression in rat neocortical areas

Long-term treatment with diazepam, a full allosteric modulator of the GABA(A) receptor, results in tolerance to its anticonvulsant effects, whereas an equipotent treatment with the partial allosteric modulator imidazenil does not produce tolerance. Use of subunit-specific antibodies linked to gold particles allowed an immunocytochemical estimation of the expression density of the alpha1, alpha2, alpha3, alpha5, gamma(2L&S) and beta(2/3) subunits of the GABA(A) receptor in the frontoparietal motor and frontoparietal somatosensory cortices of rats that received long-term treatment with vehicle, diazepam (three times daily for 14 days, doses increasing from 17.6 to 70.4 micromol/kg), or imidazenil (three times daily for 14 days, doses increasing from 2.5 to 10.0 micromol/kg). In this study, tolerance to diazepam was associated with a selective decrease (37%) in the expression of the alpha1 subunit in layers III-IV of the frontoparietal motor cortex, and a concomitant increase in the expression of the alpha5 (150%), gamma(2L&S) and beta(2/3) subunits (48%); an increase in alpha5 subunits was measured in all cortical layers. In the frontoparietal somatosensory cortex, diazepam-tolerant rats had a 221% increase in the expression of alpha5 subunits in all cortical layers, as well as a 35% increase in the expression of alpha3 subunits restricted to layers V-VI. Western blot analysis substantiated that these diazepam-induced changes reflected the expression of full subunit molecules. Rats that received equipotent treatment with imidazenil did not become tolerant to its anticonvulsant properties, and did not show significant changes in the expression of any of the GABA(A) receptor subunits studied, with the exception of a small decrease in alpha2 subunits in cortical layers V-VI of the frontoparietal somatosensory cortex. The results of this study suggest that tolerance to benzodiazepines may be associated with select changes in subunit abundance, leading to the expression of different GABA(A) receptor subtypes in specific brain areas. These changes might be mediated by a unique homeostatic mechanism regulating the expression of GABA(A) receptor subtypes that maintain specific functional features of GABAergic function in cortical cell layers.     

Differential regulation of GABA(A) receptor gene expression by ethanol in the rat hippocampus versus cerebral cortex

Previous research has shown that chronic ethanol consumption dramatically alters GABA(A) receptor alpha1 and alpha4 subunit gene expression in the cerebral cortex and GABA(A) receptor alpha1 and alpha6 subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABA(A) receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABA(A) receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABA(A) receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABA(A) receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABA(A) receptor alpha4 subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABA(A) receptor alpha1, alpha2, alpha3, beta(2/3), or gamma2 was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABA(A) receptor alpha4 subunits and significantly decreased the relative expression of cerebral cortical GABA(A) receptor alpha1 subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABA(A) receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.     

GABA(A) receptors in the primate basal ganglia: an autoradiographic and a light and electron microscopic immunohistochemical study of the alpha1 and beta2,3 subunits in the baboon brain

The distribution of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the alpha1 and beta2,3 subunits of the GABA(A) receptor by light and electron microscopy. In the caudate-putamen, the alpha1 subunit was distributed in high densities in the matrix compartment, and the beta2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the alpha1 and beta2,3 subunits. Four types of alpha1 subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, alpha1 and beta2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, alpha1 and beta2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABA(A) receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus.     

The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current

Propofol (2,6-diisopropylphenol), an intravenous general anesthetic in active clinical use today, potentiates the action of gamma-aminobutyric acid (GABA) at the type-A receptor and also directly induces current in the absence of GABA. We expressed different combinations of murine GABA(A) receptor alpha1, beta3 and gamma2 subunits in Xenopus oocytes to investigate the subunit dependence of propofol potentiation of pentobarbital-induced current. Pentobarbital induces current in all beta3-subunit-containing receptors, whereas current gating by GABA requires the presence of both alpha1 and beta3 subunits. Therefore, pentobarbital rather than GABA was used to induce current in order to separate the subunit dependence of current gating from the subunit dependence of potentiating action of propofol. alpha1beta3gamma2, alpha1beta3, beta3gamma2, or beta3 subunit combinations all responded to pentobarbital in a dose-dependent manner. True potentiation was defined as the current magnitude to simultaneous application of pentobarbital and propofol exceeding the additive responses to individual drug applications. A dose-dependent propofol potentiation of pentobarbital-induced current was observed in oocytes injected with alpha1beta3 or alpha1beta3gamma2 but not in beta3gamma2 or beta3 subunits, suggesting that the alpha1 subunit was necessary for this modulatory action of propofol. Further examination of the propofol potentiation in chimeras between the alpha1 and beta3 subunits showed that the extracellular amino-terminal half of the alpha1 subunit was sufficient to support propofol potentiation. The different requirements of the receptor structure for the agonistic (gating) and the potentiating actions suggest that these two actions of propofol are distinct processes mediated through its action at distinct sites.     

SB-205384: a GABA(A) receptor modulator with novel mechanism of action that shows subunit selectivity

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.     

Action of ethanol and zolpidem on gamma-aminobutyric acid responses from cerebellar Purkinje neurons: relationship to beta-adrenergic receptor input

The observation that cerebellar Purkinje cells contain type-I benzodiazepine-sensitive GABA(A) receptors is consistent with findings in the present work that the majority of Purkinje neurons are sensitive to enhancement of GABA by the type-1 benzodiazepine agonist, zolpidem. Previous work has demonstrated a relation between zolpidem and ethanol enhancement of GABA responses in several brain regions, but had not tested Purkinje neurons. Therefore, given that a majority of Purkinje neurons were found to be sensitive to zolpidem, ethanol would have been expected to enhance GABA responses from this cell type. However, in agreement with earlier electrophysiological studies, ethanol enhanced GABA inhibitory responses from only a small proportion of these cerebellar Purkinje neurons. Rather than enhancement of GABA, local application of ethanol either inhibited or did not affect responses to GABA from a majority of cerebellar-Purkinje neurons. Nonetheless, as previously reported, a portion of the Purkinje neurons initially insensitive to ethanol enhancement of GABA became sensitive to this action of ethanol with co-application of the beta-adrenergic agonist, isoproterenol. Thus, these results collectively implicate a beta-adrenergic input dependency for ethanol enhancement of GABA from some, but not all, cerebellar Purkinje neurons sensitive to zolpidem. Because a beta-adrenergic input did not allow ethanol enhancement of GABA from all Purkinje neurons, future studies should explore the possibility that other auxiliary neural inputs to zolpidem-sensitive cerebellar Purkinje neurons may be required for ethanol enhancement of GABA responsiveness when a beta-adrenergic input does not have this action. Likewise, knowing that the action of zolpidem can predict ethanol enhancement of GABA in other brain regions, the present findings suggest that a future determination be made concerning whether zolpidem-sensitive neurons in these other regions of brain require a beta-adrenergic or an alternative neural input for ethanol enhancement of GABA responses.     

Chronic GABA treatment downregulates the GABAA receptor alpha 2 and alpha 3 subunit mRNAS as well as polypeptide expression in primary cultured cerebral cortical neurons

Chronic GABA exposure of mammalian primary cultured cortical neurons results in a downregulation of the GABA-benzodiazepine receptor complex. In the present study, the mRNA levels, as well as polypeptide expression, for the GABAA receptor alpha 2 and alpha 3 subunits in cultured embryonic mouse cerebral cortical neurons (7 day old) were examined using northern analysis and immunoblotting techniques following chronic GABA treatment. The alpha 1 subunit mRNA or polypeptide could not be detected in these neurons. The steady state levels of mRNA for the GABAA receptor alpha 2 and alpha 3 subunits showed a decrease in comparison with untreated neurons. There was no change in the level of the beta actin or poly(A)+ RNA under the same experimental conditions. This agonist-induced reduction in the GABAA receptor alpha 2 and alpha 3 subunit mRNA was blocked by the concomitant exposure of neurons to R 5135, an antagonist of GABAA receptor. The polypeptide expression for the GABAA receptor alpha 2 and alpha 3 subunits in chronically GABA-treated neurons also showed a decline and this change was also blocked by the concomitant exposure of cells to GABA and R 5135. These results indicate that the chronic exposure of the GABAA receptor complex to agonist downregulates the expression of the alpha subunits of the receptor complex, which may be related to an observed decreases in the number of binding sites and GABA-induced 36Cl-influx in the cortical neurons.     

Properties and sex-specific differences of GABAA receptors in neurons expressing gamma1 subunit mRNA in the preoptic area of the rat

Gamma-aminobutyric acid type A (GABAA) receptors expressed within the medial preoptic area (mPOA) are known to play a critical role in regulating sexual and neuroendocrine functions. In the rat brain, high levels of expression of the gamma1 subunit mRNA of the GABAA receptor are restricted to a limited number of regions that mediate sexual behaviors, including the mPOA. The biophysical and pharmacological profiles of native gamma1-containing receptors in neurons are unknown. Here, we have characterized the properties of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) and currents elicited by fast perfusion of GABA to isolated mPOA neurons of juvenile male and female rats. No significant sex-specific differences were evident in the mean peak amplitude, distribution of event amplitudes, kinetics of current decay, or the frequency of sIPSCs. The profile of modulation of sIPSCs by diazepam, beta-CCM and zolpidem, allosteric modulators that act at the benzodiazepine (BZ) site of the GABAA receptor, support the assertion that mPOA neurons of both sexes express functional gamma1-containing receptors. The ability of zolpidem to modulate both sIPSC amplitude and currents elicited by rapid perfusion of GABA to mPOA neurons differed significantly between the sexes. Zolpidem reversibly induced negative modulation of currents in mPOA neurons isolated from male rats, but had no effect in mPOA neurons from female rats. Concentration-response analysis of responses in neurons acutely isolated from male rats indicated an IC50 of 58 nM with maximal decreases of approximately 50% of control peak current amplitude. In situ hybridization analysis demonstrated that levels of the gamma1 subunit mRNA are significantly higher in mPOA neurons from male than female rats. No significant sex-specific differences were detected in the levels of alpha1, alpha2, or alpha5 mRNAs. These results suggest that native gamma1-containing receptors are expressed in primary neurons of the mPOA and that sex-specific differences in the expression of this subunit may contribute to sexual dimorphism in GABAA receptor modulation by compounds acting at the BZ site.     

Effect of zolpidem on miniature IPSCs and occupancy of postsynaptic GABAA receptors in central synapses

GABAA-mediated miniature IPSCs (mIPSCs) were recorded from layer V pyramidal neurons of the visual cortex using whole-cell patch-clamp recording in rat brain slices. At room temperature, the benzodiazepine site agonist zolpidem enhanced both the amplitude (to 138 +/- 26% of control value at 10 microM) and the duration (163 +/- 14%) of mIPSCs. The enhancement of mIPSC amplitude was not caused by an increase of the single-channel conductance of the postsynaptic receptors, as determined by peak-scaled non-stationary fluctuation analysis of mIPSCs. The effect of zolpidem on fast, synaptic-like (1 msec duration) applications of GABA to outside-out patches was also investigated. The EC50 for fast GABA applications was 310 microM. In patches, zolpidem enhanced the amplitude of currents elicited by subsaturating GABA applications (100-300 microM) but not by saturating applications (10 mM). The increase of mIPSC amplitude by zolpidem provides evidence that the GABAA receptors are not saturated during miniature synaptic transmission in the recorded cells. By comparing the facilitation induced by 1 microM zolpidem on outside-out patches and mIPSCs, we estimated the concentration of GABA seen by the postsynaptic GABAA receptors to be approximately 300 microM after single vesicle release. We have estimated a similar degree of receptor occupancy at room and physiological temperature. However, at 35 degreesC, zolpidem did not enhance the amplitude of mIPSCs or of subsaturating GABA applications on patches, implying that, in these neurons, zolpidem cannot be used to probe the degree of receptor occupancy at physiological temperature.     

Effects of temperature and volatile anesthetics on GABA(A) receptors

BACKGROUND: Potentiation of the activity of the gamma-aminobutyric acid type A (GABA(A)) receptor channel by volatile anesthetic agents is usually studied in vitro at room temperature. Systematic variation of temperature can be used to assess the relevance of this receptor to general anesthesia and to characterize the modulation of its behavior by volatile agents at normal body temperature. METHODS: Potentiation of the GABA(A) receptor by halothane, sevoflurane, isoflurane, and methoxyflurane was studied at six temperatures in the range 10-37 degrees C using the whole-cell patch-clamp technique and mouse fibroblast cells stably transfected with defined GABA(A) receptor subunits. RESULTS: Control GABA concentration-response plots showed small and physically reasonable changes in the GABA concentration required for a half-maximal effect, the Hill coefficient, and maximal response over the range 10-30 degrees C. Potentiations of GABA (1 microM) responses by aqueous minimum alveolar concentrations of the volatile anesthetic agents decreased with increasing temperature from 10-37 degrees C in an agent-specific manner (methoxyflurane > isoflurane > sevoflurane > halothane) but tended to equalize at normal body temperature (37 degrees C). These findings are in line with published results on the temperature dependence of anesthetic potencies in animals. CONCLUSIONS: These results are consistent with direct binding of volatile anesthetic agents to the GABA(A) receptor channel playing an important role in general anesthesia. The finding that the degree of anesthetic potentiation was agent-specific at low temperatures but not at 37 degrees C emphasizes the importance of doing in vitro experiments at normal body temperature.     

Functional properties of recombinant GABA(A) receptors composed of single or multiple beta subunit subtypes

GABA(A) receptor (GABAR) isoforms in the central nervous system are composed of combinations of alpha(1-6), beta(1-4), gamma(1-4), delta(1) and epsilon(1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of beta subunit subtypes in the functional properties of GABARS and to determine whether multiple beta subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat beta1 and/or beta3, alpha5 and gamma2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The alpha5beta1gamma2L isoform was less sensitive to GABA than the alpha5beta3gamma2L isoform. alpha5beta1gamma2L isoform currents were also insensitive to the allosteric modulator loreclezole, while alpha5beta3gamma2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both beta1 and beta3 subtypes along with alpha5 and gamma2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different beta1 subunit subtypes with properties different from receptors that contain only a single beta1 subtype and that the beta1 subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.     

A novel recognition site for somatostatin-14 on the GABA(A) receptor complex

Functional interactions between gamma-aminobutyric acid (GABA) and somatostatin are suggested by the presence of synaptic contacts between GABA and somatostatin neurons, colocalisation of GABA and somatostatin and reciprocal modulation of somatostatin and GABA release. Nevertheless, a direct interaction of somatostatin with the GABA(A) receptor complex has not yet been investigated. A quantitative autoradiographic technique was used to determine the ability of somatostatin to interact with the [35S]t-butylbicyclophosphothionate [35S]TBPS binding sites of the GABA(A) receptor complex: somatostatin inhibited [35S]TBPS binding with IC50 values in the micromolar range in all brain regions studied. These results demonstrate for the first time a direct interaction between somatostatin and the GABA(A) receptor complex.     

Alcohol modulation of single GABA(A) receptor-channel kinetics

Alcohol modulation of single-channel kinetics of GABA(A) receptor currents was studied with rat dorsal root ganglion neurons using the excised outside-out patch clamp technique. GABA (1 microM) alone or GABA (1 microM) plus ethanol (30-300 mM) or n-Octanol (30-300 microM) were applied by pressure ejection to evoke single-channel currents. The main single-channel conductance was not changed by either ethanol or n-Octanol at 25 pS. Both alcohols exerted the same effects on the single-channel kinetics, although n-Octanol was more potent than ethanol. The frequency of openings, the mean open time, the percentage of open time, the frequency of bursts, and the mean burst duration were all increased, but the mean closed time was decreased. These changes in channel kinetics account for the increase in whole-cell current amplitude caused by ethanol and n-Octanol.     

Developmental changes of inhibitory synaptic currents in cerebellar granule neurons: role of GABA(A) receptor alpha 6 subunit

Eye opening and increased motor activity after the second postnatal week in rats imply an extensive development of motor control and coordination. We show a parallel development change in spontaneous IPSC (sIPSC) kinetics in cerebellar granule neurons. sIPSCs were studied by whole-cell recordings in cerebellar slices, prepared from 7-30 postnatal day old rats. Early in development, sIPSCs had slow decay kinetics whereas in older rats faster decaying sIPSCs were found in larger proportion. Currents elicited by 1 mM GABA pulses (GABACs) in nucleated patches excised from cerebellar granule neurons revealed that GABACs kinetics better approximate sIPSC decay in young but not in more developed rats. The expression of alpha 6 subunit of GABAA receptors, unique in cerebellar granule neurons, has been shown to increase during development. Therefore, we took advantage of the recently reported selective inhibition of GABAA receptors by furosemide to characterize the relative contribution of alpha 6 subunits to native receptors in inhibitory synapses of cerebellar granule neurons. Although furosemide inhibition of sIPSCs amplitude was highly variable among distinct granule cells, it increased during development. At the same time, furosemide failed to inhibit sIPSCs recorded from Purkinje neurons. From the comparison of furosemide inhibition and kinetics of sIPSCs with GABACs recorded from mammalian HEK293 cells transfected with combinations of alpha 1 and alpha 6 GABAA receptor subunits together with beta 2 gamma 2 subunits, we propose that an increased alpha 6 subunit contribution in the molecular assembly of postsynaptic receptors in cerebellar glomeruli is responsible for the developmental changes observed.     

Amino acid residue 200 on the alpha1 subunit of GABA(A) receptors affects the interaction with selected benzodiazepine binding site ligands

Mutant alph1 subunits of the GABA(A) receptor were coexpressed in combination with the wild-type beta2 and gamma2 subunits in human embryonic kidney (HEK) 293 cells. The binding properties of various benzodiazepine site ligands were determined by displacement of ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]-[1,4]benzodia zepine-3-carboxylate ([3H]Ro 15-1788). The mutation G200E led to a decrease in zolpidem and 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine (CL 218872) affinity amounting to 16- and 8-fold. Receptors containing a conservative T206V substitution showed a 41- and 38-fold increase in methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and CL 218872 affinity combined with a decrease in diazepam and zolpidem affinity, amounting to 7- and 10-fold. Two mutations, Q203A and Q203S showed almost no effects on the binding of benzodiazepine site ligands, indicating that this residue is not involved in the binding of benzodiazepines and related compounds.     

Pharmacological modulation of GABA(A) receptor-mediated postsynaptic potentials in the CA1 region of the rat hippocampus

It is unclear whether GABA(A) receptor-mediated hyperpolarizing and depolarizing synaptic potentials (IPSP(A)s and DPSP(A)s, respectively) are evoked by (a) the same populations of GABAergic interneurones and (b) exhibit similar regulation by allosteric modulators of GABA(A) receptor function. We have attempted to address these questions by investigating the effects of (a) known agonists for presynaptic receptors on GABAergic terminals, and (b) a range of GABA(A) receptor ligands, on each response. The GABA uptake inhibitor NNC 05-711 (10 microM) enhanced whereas bicuculline (10 microM) inhibited both IPSP(A)s and DPSP(A)s. (-)-Baclofen (5 microM), [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO; 0.5 microM), and carbachol (10 microM) caused substantial depressions (up to 99%) of DPSP(A)s that were reversed by CGP 55845A (1 microM), naloxone (10 microM) and atropine (5 microM), respectively. In contrast, 2-chloroadenosine (CADO; 10 microM) only slightly depressed DPSP(A)s. Quantitatively, the effect of each agonist was similar to that reported for IPSP(A)s. The neurosteroid ORG 21465 (1 - 10 microM), the anaesthetic propofol (50-500 microM), the barbiturate pentobarbitone (100-300 microM) and zinc (50 microM) all enhanced DPSP(A)s and IPSP(A)s. The benzodiazepine (BZ) agonist flunitrazepam (10-50 microM) and inverse agonist DMCM (1 microM) caused a respective enhancement and inhibition of both IPSP(A)s and DPSP(A)s. The BZomega1 site agonist zolpidem (10-30 microM) produced similar effects to flunitrazepam. The anticonvulsant loreclezole (1-100 microM) did not affect either response. These data demonstrate that similar populations of inhibitory interneurones can generate both IPSP(A)s and DPSP(A)s by activating GABA(A) receptors that are subject to similar allosteric modulation.     

Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin

Modification of synaptic strength in the mammalian central nervous system (CNS) occurs at both pre- and postsynaptic sites. However, because postsynaptic receptors are likely to be saturated by released transmitter, an increase in the number of active postsynaptic receptors may be a more efficient way of strengthening synaptic efficacy. But there has been no evidence for a rapid recruitment of neurotransmitter receptors to the postsynaptic membrane in the CNS. Here we report that insulin causes the type A gamma-aminobutyric acid (GABA[A]) receptor, the principal receptor that mediates synaptic inhibition in the CNS, to translocate rapidly from the intracellular compartment to the plasma membrane in transfected HEK 293 cells, and that this relocation requires the beta2 subunit of the GABA(A) receptor. In CNS neurons, insulin increases the expression of GABA(A) receptors on the postsynaptic and dendritic membranes. We found that insulin increases the number of functional postsynaptic GABA(A) receptors, thereby increasing the amplitude of the GABA(A)-receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) without altering their time course. These results provide evidence for a rapid recruitment of functional receptors to the postsynaptic plasma membrane, suggesting a fundamental mechanism for the generation of synaptic plasticity.