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Understanding the genetic variation in GABAA receptor subunit genes (GABRs), GABRA1-6, GABRB1-3, GABRG1-3 and GABRD, in individuals affected by epilepsy may improve the diagnosis and treatment of epilepsy syndromes through identification of disease-associated variants. However, the lack of functional analysis and validation of many novel and previously reported familial and de novo mutations have made it challenging to address meaningful gene associations with epilepsy syndromes. GABAA receptors belong to the Cys-loop receptor family. Even though GABAA receptor mutant residues are widespread among different GABRs, their frequent occurrence in important structural domains that share common functional features suggests associations between structure and function.

Understanding the genetic variation in GABA

We studied the effects of ATP-gamma-S on the T, N and L calcium current components of nodose ganglion neurons using the whole cell variation of the patch clamp technique. ATP-gamma-S can serve as a phosphate donor in kinase-mediated reactions, the donated phosphate group being resistant to the action of phosphatases. We therefore compared the effect of ATP-gamma-S to that of the catalytic subunit of the cyclic AMP-dependent protein kinase (AK-C), included in the recording pipette with 5 mM ATP. AK-C (50 micrograms/ml) had no effect on the T current, and caused a approximately 30% increase in currents containing the N and L components during a 20-min recording, as compared to a approximately 45% decrease in control currents. In contrast, in the presence of 2.5 mM ATP-gamma-S, T currents declined approximately 30%, and currents containing the N and L components declined to a greater extent than control currents, about 65%. In addition, the time to peak current was increased from approximately 14 ms to approximately 40 ms. This effect of ATP-gamma-S on calcium currents was similar to that of certain neurotransmitters or GTP-gamma-S, an activator of G proteins, except that the effects of ATP-gamma-S were delayed 5-7 min relative to GTP-gamma-S. The effects of both ATP-gamma-S and GTP-gamma-S were reduced or abolished in neurons treated with pertussis toxin. We conclude that AK-C regulates neuronal calcium currents, presumably by phosphorylation of channels or associated proteins, and that the ATP-gamma-S-induced reduction of calcium currents cannot be due to its serving as a phosphate donor for endogenous AK.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P (SP) produced membrane depolarization and decreased membrane conductance of mouse spinal cord neurons in primary dissociated cell culture. SP-responses were abolished by intracellular tetraethylammonium suggesting that SP decreased potassium conductance. Reversal of SP-responses was not observed with membrane hyperpolarization suggesting that SP reduced a voltage-dependent potassium conductance that was activated by membrane depolarization.

Synaptic interactions between spinal cord neurons grown in dissociated cell culture were studied electrophysiologically, and presynaptic cells were subsequently injected by intracellular iontophoresis with horseradish peroxidase (HRP). Following histochemical processing, injected cells were filled with dense reaction product which facilitated the light and electron microscopic identification of the individual physiologically typed neurons. This technique applied to neurons in monolayer culture allowed the visualization of complex intercellular relationships in essentially two dimensions. The number and distribution of morphologically defined synaptic contacts was determined for correlation with individual evoked postsynaptic potentials. HRP-filling of inhibitory and excitatory neurons revealed differences with respect to cellular geometry, axonal projection, and the number, location and ultrastructure of synaptic contacts.

Murine spinal cord neurons grown in dissociated cell culture were used to study the effects of barbiturate (phenobarbital, mephobarbital) and benzodiazepine (diazepam, chlordiazepoxide( anticonvulsants on amino acid responses. Both types of anticonvulsant augmented GABA-mediated postsynaptic inhibition without augmenting beta-alanine or glycine-mediated postsynaptic inhibition. Barbiturates, but not benzodiazepines, antagonized glutamate-mediated postsynaptic excitation. Augmentation of GABA-mediated inhibition by the anticonvulsants should contribute to their anticonvulsant action; antagonism of glutamate-mediated excitation by barbiturates should also contribute to their anticonvulsant action and could be at least in part responsible for their sedative actions.

GABA (4-aminobutyric acid) and its bicyclic analog THIP (4,5,6,7-tetrahydroisoxazolo-[4,5-c]-pyridin-3-ol) produced membrane hyperpolarization and increased chloride ion conductance of mouse spinal cord neurons in cell culture. Above 1 nM diazepam enhanced the actions of both GABA and THIP with similar potency and efficacy. Diazepam has been shown to enhance the binding of [3H]GABA to rat brain membranes over similar concentration ranges, with the EC50 values for enhancement of [3H]GABA binding and increase in membrane conductance being similar. In contrast, binding of [3H]THIP has been shown to be unaltered by diazepam under a variety of conditions. The possible reasons for such a discrepancy between these electrophysiological and neurochemical results with THIP are discussed.

The experimentally proven convulsant alpha-keto-delta-guanidinovaleric acid (alpha-K-delta-GVA) was applied to mouse spinal cord neurons in primary dissociated cell culture to assess its effects on postsynaptic gamma-aminobutyric acid (GABA)- and glycine (GLY)-responses. Intracellular microelectrode recording techniques were used. alpha-K-delta-GVA reversibly inhibited both GABA- and GLY-responses in a concentration-dependent manner. The effect of alpha-K-delta-GVA on GABA-responses was not antagonized by co-application of the benzodiazepine receptor antagonist CGS 9896. The results suggest that alpha-K-delta-GVA inhibited responses to the inhibitory neurotransmitters GABA and GLY by blocking the chloride channel. This action might underlie the convulsant effect of this compound in rabbit. The possible pathophysiological importance of alpha-K-delta-GVA in hyperargininemic patients is discussed.

The effects of a series of caprolactam derivatives with central depressant, convulsant or muscle relaxant activity were investigated upon gamma-aminobutyric acid (GABA) receptor-ionophore binding to rat brain membranes using [3H]GABA, [3H]GABA, [3H]muscimol and [35S]-tert.-butylbicyclophophorothionate ([35S]TBPS) as ligands, and GABA responses in mouse spinal cord neurones in dissociated cell culture. Some caprolactams produced a picrotoxin-like chloride-dependent partial inhibition of muscimol binding and were potent inhibitors of TBPS binding. One compound that was further investigated (4,4,6,6-tetramethylhexahydro-2H-azepin-2-one), inhibited GABA responses and increased the frequency of paroxysmal depolarizations in cultured neurones. Other caprolactams enhanced muscimol binding and were relatively weak inhibitors of TBPS binding, and one (3,3-diallyl-6,6-dimethylhexahydro-2H-azepin-2,4-dione) was shown to enhance GABA responses and produced quiescence of activity in cultured neurones. There was a direct correlation between caprolactam effects on muscimol binding in the presence of chloride ions and their effects on TBPS binding suggesting a similar site of action for the caprolactams influencing the binding of these two ligands. For the two classes of caprolactams, with respect to inhibition or enhancement of muscimol binding, there appeared to be a relationship between in vitro effects and their convulsant or depressant activity in mice. Caprolactams may be useful low molecular weight probes for the study of GABA receptor-ionophore complexes.

Calcium-dependent action potentials were recorded from mouse spinal cord neurons in primary dissociated cell culture following addition of the potassium channel blockers tetraethylammonium ion and 3-aminopyridine. The pharmacologically active barbiturates, pentobarbital and phenobarbital, but not the pharmacologically inactive barbiturate, barbituric acid, produced reversible, dose-dependent reduction of action potential duration at sedative-hypnotic and anesthetic concentrations. Pentobarbital reduced action potential duration at concentrations from 25 to 600 microM (50% reduction at 170 microM) while phenobarbital reduced action potential duration at concentrations from 100 to 5000 microM (50% reduction at 900 microM). The barbiturate concentrations which reduced calcium-dependent action potential duration in this study correlate with reduction of neurotransmitter release from other neuronal preparations and with reduction of calcium uptake by synaptosomes. The results suggest that barbiturates may produce anesthesia in part by reduction of presynaptic calcium entry and consequent reduction of neurotransmitter release in addition to postsynaptic increase of membrane chloride ion conductance. Barbiturate anticonvulsant actions are probably due to postsynaptic augmentation of GABA-mediated inhibition and depression of excitatory synaptic transmission. The major difference between anticonvulsant (phenobarbital) and anesthetic (pentobarbital) barbiturates was the dose-dependency of these actions. Phenobarbital produced postsynaptic modulation of neurotransmitter responses at low concentrations and decreased calcium-dependent action potential duration and increased chloride ion conductance at high concentrations. In contrast, pentobarbital produced all actions at low concentrations. Thus for phenobarbital there would be a large therapeutic index for anticonvulsant activity compared to anesthetic activity but for pentobarbital there would be a small therapeutic index.

Following blockade of membrane potassium conductance with tetraethylammonium ions or 3-aminopyridine, long-duration action potentials were recorded from mouse spinal cord neurons in primary dissociated cell culture. The action potentials were calcium-dependent since they: (1) were not blocked by the sodium-channel blocker tetrodotoxin, (2) could be recorded in sodium-free, calcium-containing medium (3) could not be evoked in sodium-containing, calcium-free medium, (4) were blocked by calcium channel blockers manganese and cobalt and (5) had overshoot amplitudes that varied linearly with the log of the extracellular calcium concentration (slope of 27.5 mV/decade change in calcium concentration).

The anesthetic-sedative barbiturate pentobarbital (PB) and the anticonvulsant barbiturate phenobarbital (PhB) were applied to mammalian spinal cord neurons in primary dissociated cell culture to assess their effects on: (1) postsynaptic GABA-responses; (2) paroxysmal activity produced by the convulsant bicuculline; (3) resting membrane properties; and (4) spontaneous neuronal activity. The results demonstrated that: (1) anticonvulsant actions occurred at barbiturate concentrations which augmented GABA-responses; (2) anesthetic actions occurred at barbiturate concentrations which produced direct increases in chloride conductance; (3) both anticonvulsant and anesthetic actions occurred at clinically relevant concentrations; and (4) concentrations of PhB, but not PB, which produced GABA-augmentation and direct conductance changes were widely separated. These findings support the hypotheses that augmentation of GABA-mediated inhibition and possibly reduction of glutamate (GLU)-mediated excitation form the basis at least in part for barbiturate anticonvulsant action and that addition of direct increases in chloride conductance to augmentation of GABA-mediated inhibition and reduction of GLU-mediated excitation may partially underlie anesthetic-sedative barbiturate action.

The convulsant compounds bicuculline (BICUC) and penicillin (PCN) are antagonists of GABA-mediated synaptic inhibition. In addition, we have shown that BICUC and PCN produced membrane depolarization of mouse spinal cord neurons in primary dissociated cell culture by blocking a potassium conductance, a non-synaptic direct effect. Both compounds also prolonged calcium-dependent action potentials of mouse dorsal root ganglion and spinal cord neurons in cell culture. Thus, BICUC and PCN had both synaptic and non-synaptic actions. The possibility that both synaptic and non-synaptic actions of BICUC and PCN are involved in their convulsant mechanism of action is discussed.

The actions of diazepam and itsp-chloro-derivative Ro 5-4864 were compared on mouse spinal cord and dorsal root ganglion neurons in cell culture. Diazepam enhanced but Ro 5-4864 reduced iontophoretic GABA responses in a concentration-dependent manner. Both diazepam and Ro 5-4864 limited sustained, high frequency repetitive firing of spinal cord neurons but diazepam was more potent. Ro 5-4864 was, however, more potent than diazepam in inhibiting spontaneous neuronal activity of spinal cord neurons and reducing the duration of calcium-dependent action potentials of dorsal root ganglion neurons. The differeing actions of diazepam and Ro 5-4864 may account for the contrasting pharmalogical spectra of the two benzodiazepines.

The neutral amino acid gamma-aminobutyric acid (GABA) produced membrane hyperpolarization and increased membrane chloride ion conductance of spinal cord (SC) and cortical (CTX) neurons in cell culture. GABA dose-response curves were obtained for SC neurons by pressure applying known concentrations of GABA from micropipettes with large tips (miniperfusion pipettes). GABA response threshold was about 2 micrometers and large responses were elicited at GABA concentrations greater than 10 micrometers. Bicuculline (BICUC) (0.1-10 micrometers) reversibly antagonized GABA responses on both SC and CTX neurons with a half maximal inhibitory concentration of about 1 micrometer. BICUC antagonism of GABA responses was competitive (Lineweaver-Burke analysis). These results are compared with data on GABA and BICUC displacement of [3H]GABA binding to membranes of SC and CTX neurons in cell culture. It is suggested that high affinity GABA receptors are likely to be relevant for postsynaptic GABA responses while low affinity GABA receptors may be presynaptic.

We investigated opioid peptide actions on somatic calcium-dependent action potentials of dorsal root ganglion (DRG) neurons grown in primary dissociated cell culture. We report that leucine-enkephalin decreased the duration and amplitude of DRG somatic calcium-dependent action potentials. The opioid peptide action was dose-dependent over 20 nM to 5 microM and was antagonized by naloxone, consistent with mediation by opiate receptors. Thus, DRG neuron membranes have opiate receptors which act to decrease calcium influx. It is more likely, therefore, that opiate receptors on the somata of DRG neurons in culture are functionally similar to opiate receptors on primary afferent terminals.

Opiate alkaloid and opioid peptide actions on spontaneous neuronal activity and postsynaptic amino acid responsiveness were assessed using intracellular recording techniques applied to murine spinal cord neurons in primary dissociated cell culture. Application of opiates was by superfusion and amino acids by iontophoresis. Glycine and GABA but not glutamate responses were antagonized by the opiate alkaloids. Since opiate effects on glycine and GABA responses were not naloxone-reversible, only weakly stereospecific, and not produced by the opioid peptide [D-Ala2]-Met-enkephalinamide, it is unlikely that these effects were mediated by opiate receptors. Opiate depression of glycine inhibition was correlated with the induction of paroxysmal depolarizations in cultured spinal cord neurons, suggesting that antagonism of inhibitory amino acid transmission may underlie the convulsant actions of high concentrations of the opiate alkaloids.

Sodium-independent [3H]GABA and [3H]muscimol binding was determined in adult rat cerebral cortical and spinal cord membranes and in membranes from fetal mouse cortical and spinal cord neurons in primary dissociated cell culture. In adult rat cerebral cortical membranes, [3H]GABA bound to two sites (Kd=8nM,Bmax=0.62pmol/mg protein; Kd=390nM,Bmax=3.9pmol/mg protein) whereas the GABA agonist, [3H]muscimol, bound only to a high affinity site (Kd=5.6nM,Bmax=1.9pmol/mg protein). In adult rat spinal cord, only a low affinity site was seen with [3H]GABA (Kd=340nM,Bmax=9.8pmol/mg protein) and only a high affinity site was seen with [3H]muscimol (Kd=5.6nM,Bmax=0.25pmol/mg protein). The inability to measure a high affinity [3H]GABA binding site in spinal cord probably reflects the high ratio of low to high affinity sites in spinal cord (39:1). In membranes from mouse neurons in cel; culture, [3H]GABA bound to two sites on cortical neurons (Kd=9nM,Bmax=0.24pmol/mg protein; Kd=510nM,Bmax=1.3pmol/mg protein) and spinal cord neurons (Kd=13nM,Bmax=0.12pmol/mg protein; Kd=640nM,Bmax=3.2pmol/mg protein). Again, the ratio of low to high affinity sites in cultured mouse spinal cord neurons was high (27:1). The effects of the potent GABA antagonist, (+)bicuculline, on both low and high affinity [3H]GABA binding was determined. Bicuculline appeared to inhibit binding to both sites competitively but theKi for inhibiting the high affinity site was 5 μM and for inhibiting the low affinity site was 115 μM. Bicuculline inhibited [3H]muscimol binding in both brain and spinal cord competitively withKis of 4μM and 10 μM respectively. Bicuculline inhibition of [3H]muscimol binding in cultured neuronal membranes was similar to that in adult rat membranes. The binding of the potent GABA agonist, muscimol, only to the high affinity site in both adult rat and cultured mouse neuronal membranes suggests that the high affinity site is the physiologically relevant postsynaptic GABA receptor. The fact that bicuculline inhibits the high affinity site (but not the low affinity site) in concentrations similar to those needed to block GABA-responses in physiological experiments28 supports this hypothesis.

Cerebellar macroneurons, including Purkinje cells, survive and differentiate in long-term monolayer cultures, which are prepared by a partial dissociation procedure we refer to as a microexplant technique. Intracellular recording demonstrated that these neurons were functional, showing spontaneous spiking activity and electrical excitability, and both spontaneous and evoked synaptic activity. In order to further characterize cell types, light and electron microscopic studies were performed after intracellular iontophoresis of horseradish peroxidase. Purkinje neurons were identified by their form of dendritic arborization and numerous dendritic spines. Cortical granule cells and macroneurons derived from the deep nuclei could also be demonstrated.

Cerebellar neurons derived from 17- to 19-day-old fetal rats have been grown in a monolayer in microexplant cell culture, and intracellular recording coupled with iontophoresis of amino acid neurotransmitters has been employed to characterize their amino acid chemosensitivity. Although these cultures contain at least 3 different neuronal cell types, intracellular recordings were obtained from large neurons (diameter greater than 15 microns) with 1-5 dendritic shafts and fine dendritic arborizations and which could, on morphological grounds, be identified as Purkinje cells. All neurons with resting membrane potentials greater than 25 mV and with action potentials evoked by intracellular stimulation, responded to iontophoretically applied glutamate and GABA. There was essentially no chemosensitivity to glycine, beta-alanine or taurine. Aspartate application evoked only small responses at high iontophoretic currents. GABA reversibly increased membrane conductance and produced hyperpolarization at resting membrane potential with reversal potentials between -50 and -40 mV (5-10 mV more negative than resting membrane potential). Glutamate reversibly increased membrane conductance and produced depolarizing responses with extrapolated reversal potentials between 0 and -10 mV. Aspartate augmented glutamate responses at low iontophoretic currents which did not directly alter membrane potential or conductance. Thus Purkinje cells grown in the absence of parallel fiber and climbing fiber input develop autonomous neuropharmacologic specificity similar to that of Purkinje cells in vivo.