Alpha subunit isoform influences GABA(A) receptor modulation by propofol.

We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.

Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the gamma2 subunit of the gamma-aminobutyrate type A receptor.

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.

Excitation of rat substantia nigra pars reticulata neurons by 5-hydroxytryptamine in vitro: evidence for a direct action mediated by 5-hydroxytryptamine2C receptors.

Single-unit extracellular and whole-cell patch clamp recording were used to study the actions of exogenously applied 5-hydroxytryptamine on substantia nigra pars reticulata neurons in parasaggital slices of rat midbrain. Seventy-six per cent of substantia nigra pars reticulata cells (254/334) recorded extracellularly were excited by 5-hydroxytryptamine (EC50 = 9.56 microM); in the remainder, inhibitions (13.5%), biphasic responses (4.2%) or lack of response (6.3%) were observed. Using whole-cell patch recording, 5-hydroxytryptamine (10 microM) caused either an inward current (9/9 cells) or a depolarization (3/3 cells) at membrane potentials in the range -50 to -90 mV, which was resistant to tetrodotoxin (4/4 cells), indicating that the predominant, excitatory action of 5-hydroxytryptamine was due to a direct action on substantia nigra pars reticulata neurons. The 5-hydroxytryptamine excitation (recorded extracellularly) was reduced to 24 +/- 6% of control values by methysergide (0.1 microM) and to 17 +/- 5% of control by ketanserin (10 microM), but was unaffected by the 5-hydroxytryptamine antagonists spiperone (0.1 microM), yohimbine (0.1 microM), pindolol (1 microM), GR113808A (1 microM) or ICS 205930 (10 microM). In addition, the 5-hydroxytryptamine excitation was mimicked by the 5-hydroxytryptamine2C receptor--preferring agonist alpha-methyl 5-hydroxytryptamine (10 microM), but the agonists CP93, 129 (0.1-1 microM) and (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (0.1-1 microM) were without effect. Taken together, this pharmacology indicated involvement of the 5-hydroxytryptamine2C receptor in the 5-hydroxytryptamine excitation, while other candidate receptors known to be present in rat substantia nigra pars reticulata (5-hydroxytryptamine1B, 5-hydroxytryptamine2A and 5-hydroxytryptamine4) could be excluded from consideration. While in accord with current information on the location of 5-hydroxytryptamine receptor subtypes in substantia nigra pars reticulata, and the consequence of activation of neuronal 5-hydroxytryptamine2C receptors, these results contrast with data from in vivo experiments which suggest that the net effect of 5-hydroxytryptamine is to inhibit substantia nigra pars reticulata neurons. The reason for this apparent discrepancy may lie in detailed consideration of the microcircuitry of the substantia nigra pars reticulata. This may lead to a re-evaluation of the influence of 5-hydroxytryptamine on this basal ganglia output relay nucleus, and its role in motor control and the gating of generalized seizure activity.

Neurosteroids act on the GABA(A) receptor at sites on the N-terminal side of the middle of TM2.

Two sets of chimeras between alphaxalone-sensitive GABA(A) receptor alpha2 or beta1 subunits and the alphaxalone-insensitive glycine receptor alpha1 subunit were constructed to determine the structural domains important for the modulatory actions of neuroactive steroids. These data suggest that the site of action for neurosteroids on GABA(A) receptors is not the same as that for volatile anesthetics and ethanol, but is on the N-terminal side of the middle of TM2.

Rat substantia nigra pars reticulata neurones are tonically inhibited via GABAA, but not GABAB, receptors in vitro.

Extracellular single unit recordings were made from substantia nigra pars reticulata (SNr) neurones in slices of rat brain. Cells fired spontaneous action potentials at 11.4 +/- 0.8 Hz. The GABAA receptor agonist isoguvacine (1-10 microM) reduced firing rate in a concentration-dependent manner [50% of maximal inhibition (IC50) with 3.2 microM], as did the GABAB agonist baclofen (0.3-10 microM; IC50 1.4 microM). The GABAA antagonist bicuculline (30 microM) not only blocked the action of isoguvacine, but also increased the basal firing rate to 187.5 +/- 12.6% of control. The GABAB antagonist CGP 55845A (0.1 microM), while blocking the inhibitory action of baclofen, was without effect on spontaneous firing rate, as was strychnine (10 microM), the antagonist of glycine and taurine, and also Met-enkephalin (10 microM). Tiagabine (50 microM), the blocker of GABA uptake, caused an inhibition of firing which could be reversed with bicuculline (30 microM) but not CGP 55845A (1 microM). We conclude that the firing rate of SNr neurones is under tonic inhibition by GABA in vitro, which can be relieved by antagonists of GABAA, but not GABAB receptors, and enhanced by blockade of GABA reuptake. The source of this GABA tone is likely to be from recurrent axon collaterals of SNr neurones themselves.

A deficit of functional GABA(A) receptors in neurons of beta 3 subunit knockout mice.

Mice whose gamma-aminobutyric acid type A (GABA(A)) beta3 subunit gene is inactivated ('beta3 knockout mice') have been previously shown to have epilepsy, hypersensitive behavior, cleft palate, and a high incidence of neonatal mortality. In this study, we analyze whole-cell responses to GABA in neurons from beta3+/+, beta3+/- and beta3-/- mice. We demonstrate markedly decreased responses to GABA in both hippocampal and dorsal root ganglion neurons isolated from beta3-/- mice without major differences in the GABA concentration-response curves. We also utilize the subunit selective pharmacology of Zn2+ and the anticonvulsant drug loreclezole to help infer the presence of beta2 and gamma subunits in the GABA(A) receptors remaining in neurons from beta3-/- mice.

Differentiated dopaminergic MN9D cells only partially recapitulate the electrophysiological properties of midbrain dopaminergic neurons.

The cell line MN9D, a fusion of embryonic ventral mesencephalic and neuroblastoma cells, is extensively used as a model of dopamine (DA) neurons because it expresses tyrosine hydroxylase and synthesizes and releases DA. These cells are also used to test mechanisms and potential therapeutics relevant to the loss of DA neurons in Parkinson's disease. To date, little work has been done to determine whether MN9D cells electrophysiologically resemble mature DA neurons. We examined sodium, calcium and potassium currents in undifferentiated and differentiated MN9D cells, and compared these to those found in acutely dissociated mouse substantia nigra pars compacta DA neurons. It was observed that undifferentiated MN9D cells bore no resemblance to DA neurons. Upon differentiation with butyric acid with or without a prior treatment with glial cell line-derived neurotrophic factor, differentiated MN9D cells produce an electrophysiological profile that more closely resembles substantia nigra pars compacta DA neurons even though the A-type potassium current remains noticeably absent. These observations demonstrate that undifferentiated MN9D cells are not reasonable models of DA neurons. Although differentiated MN9D cells are closer to the mature DA neuronal phenotype, they do not fully mimic DA neurons and are likely to be of questionable value as a model because of their substantive differences, including the lack of the characteristic A-type potassium current. The future use of one or a combination of growth or other factors to differentiate MN9D cells may yield a more useful model system for Parkinson's disease studies in vitro.

Two sulphonated dye compounds which compete for inositol 1,4, 5-trisphosphate binding to rat liver microsomes: effects on 5'-phosphatase activity.

The ability of heparin to interact with the Ins 1,4,5-P3 receptor is dependent on its chain length and degree of sulphation. Here we report results obtained with two sulphonated dye compounds of known structures and molecular weights below 1000, cibacron blue and Patent blue. Both compounds compete for Ins 1,4,5-P3 binding to rat liver microsomes and also inhibit Ins 1,4,5-P3 5'-phosphatase activity in the same preparation. Comparison with the effects of heparin show these to be two separate actions of the compounds.

Propofol and other intravenous anesthetics have sites of action on the gamma-aminobutyric acid type A receptor distinct from that for isoflurane.

Both volatile and intravenous general anesthetics allosterically enhance gamma-aminobutyric acid (GABA)-evoked chloride currents at the GABA type A (GABAA) receptor. Recent work has revealed that two specific amino acid residues within transmembrane domain (TM)2 and TM3 are necessary for positive modulation of GABAA and glycine receptors by the volatile anesthetic enflurane. We now report that mutation of these residues within either GABAA alpha2 (S270 or A291) or beta1 (S265 or M286) subunits resulted in receptors that retain normal or near-normal gating by GABA but are insensitive to clinically relevant concentrations of another inhaled anesthetic, isoflurane. To determine whether receptor modulation by intravenous general anesthetics also was affected by these point mutations, we examined the effects of propofol, etomidate, the barbiturate methohexital, and the steroid alphaxalone on wild-type and mutant GABAA receptors expressed in human embryonic kidney 293 cells. In most cases, these mutations had little or no effect on the actions of these intravenous anesthetics. However, a point mutation in the beta1 subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct activation of the receptor by high concentrations of propofol. These data indicate that the receptor structural requirements for positive modulation by volatile and intravenous general anesthetics may be quite distinct.

Mice devoid of gamma-aminobutyrate type A receptor beta3 subunit have epilepsy, cleft palate, and hypersensitive behavior.

gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The beta3 subunit is an essential component of the GABA(A)-R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a beta3 gene inactivated by gene targeting. GABA(A)-R density is approximately halved in brain of beta3-deficient mice, and GABA(A)-R function is severely impaired. Most beta3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. beta3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the beta3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. beta3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and/or pleiotropic developmental defects.