Evidence for enhancement of gap junctional coupling between rat island of Calleja granule cells in vitro by the activation of dopamine D3 receptors.

1. Using patch-clamp techniques, we have studied actions of dopamine and related compounds on granule neurones within the islands of Calleja in vitro, in slices if approximately 200 microns thickness or as groups of varying cell number following enzymic digestion. 2. Prior to agonist application, island of Calleja granule cells displayed spontaneous stepwise shifts in whole-cell conductance ranging from 104 to 632 pS. The reversal potentials of these conductance changes ranged widely and matched the distribution of the cells' membrane potentials. Reversal potentials and membrane potentials shifted equally when cells were uniformly depolarized in 24 mM external K+. 3. Bath-applied dopamine elicited, after a delay of 4-9 min, an exaggerated form of the spontaneous behaviour that frequently gave way to a sudden large (up to thirtyfold) conductance change. At concentrations of 100-300 nM, a range of agonists with increasing affinity for the D3 receptor (apomorphine, quinpirole, 7-OH DPAT and PD 128907) triggered the response. The actions were neither mimicked by SKF-38393 nor antagonized by SCH-23390 (a selective D1 agonist and antagonist, respectively). Haloperidol reversibly blocked responses elicited by the D3/D2 agonist quinpirole. The action of effective agonists was maintained when transmitter release was abolished. Given the reported lack of D2 receptors in the islands of Calleja, these findings indicate a direct action of dopamine at the D3 receptor. 4. The dopaminergic effects were not affected by Gd3+ or substantial replacement of external Na+ with TEA, Tris or choline, eliminating stretch-activated channels but suggesting that if transmembrane channels were to be involved in this dopaminergic action they posseses a non-selective permeability to large cations. The reported presence of gap junctions in the islands of Calleja offers the explanation that these effects derive from enhanced activity of such channels or their hemi-constituents. 5. In testing the possible involvement of gap junctional coupling the following experimental observations were made: (i) alkalinization of slices mimicked the effect of D3 agonists; (ii) in cell groups, recording from pairs provided evidence of intercellular coupling, and mechanical separation of recorded neurones from neighbouring cells during the agonist-evoked response caused shutdown of the additional conductance; (iii) when applied to slices, the gap junctional blocker, 18 alpha-glycyrrhetinic acid, whilst not preventing the full-blown dopamine response, significantly reduced both the variance of recorded granule cell input conductance and the cells' apparent capacitance. 6. Taken together the results indicate a D3 action in granule cells, which is best explained by a dopaminergic promotion of intercellular coupling. The physiological relevance of such a mechanism is discussed.

Membrane properties of the granule cells of the islands of Calleja of the rat studied in vitro.

1. Using patch-clamp techniques, we have studied granule neurones from the islands of Calleja in vitro: as isolated cells or as groups of varying numbers following enzymic digestion, or within untreated slices of approximately 100 microns thickness. 2. Recordings were made with patch pipettes in conventional or nystatin-perforated whole-cell mode. Current-clamp recordings indicated that these granule cells are excitable and at resting potential produce irregular spontaneous activity. In voltage clamp the transient inward current underlying these action potentials could be evoked. This current had a threshold for activation of about -50 mV and was sensitive to TTX. In some cells a TTX-resistant transient inward current was observed with a threshold for activation of about -70 mV. 3. Island of Calleja granule cells also exhibited outward currents. A rapidly activating transient current was observed that was resistant to TEA and sensitive to 4-AP, and therefore resembled IA. The current was half-maximally activated at -6 mV and steady-state inactivation was half-complete at -65 mV. 4. More sustained outward currents were also observed. Although some cells appeared to express a Ca(2+)-activated K+ current, the most common finding was a rapidly activating, slowly inactivating, voltage-dependent K+ current that was sensitive to TEA and Ba2+. This current resembled M-current more than delayed rectifier but displayed a number of idiosyncratic kinetic properties. Chief amongst these was the accumulation of an inactivating process when the current was repeatedly evoked from potentials near the cells' resting value by voltage steps that by themselves produced no observable inactivation during the voltage command; this behaviour was similar to the 'C-terminal' inactivation exhibited by lymphocytes and certain expressed K+ channel clones (Kv1.3). 5. These results indicate that the granule cells of the islands of Calleja are excitable and contain a number of additional regulatory conductances. The implications of these findings in, and the usefulness of this preparation to, the elucidation of the function(s) of the islands of Calleja are discussed.

The influence of the gamma 2L subunit on the modulation of responses to GABAA receptor activation.

1. Whole-cell patch clamp recordings were made from L-cells transfected with 2 combinations of subunits of the GABAA receptor. Log concentration-response curves were constructed to gamma-aminobutyric acid (GABA) on alpha 1,beta 1,gamma 2L containing cells and compared to those from alpha 1,beta 1 containing cells. The effects of flunitrazepam, pentobarbitone and alphaxalone on the concentration-response relationships were also examined. 2. From the log concentration-response curves, GABA had a mean (+/- s.e. mean) pEC50 = 5.2 +/- 0.09 and slope factor = 1.7 +/- 0.02 on alpha 1,beta 1,gamma 2L cells which were significantly different from the values obtained from alpha 1,beta 1 cells where the pEC50 = 5.6 +/- 0.02 and the slope = 1.5 +/- 0.02. 3. Flunitrazepam produced a parallel leftward shift of GABA concentration-response curves on alpha 1,beta 1,gamma 2L cells. The EC50 for flunitrazepam = 6.3 +/- 2.7 nM. No increase in the maxima of the GABA concentration-response curves was found in the presence of flunitrazepam. Flunitrazepam did not potentiate responses from alpha 1,beta 1 cells. 4. The log concentration-response curves from both populations of cells were shifted to the left by equal amounts by pentobarbitone. A significant increase in the maximal response to GABA was also produced by pentobarbitone. This occurred at lower concentrations of pentobarbitone on alpha 1,beta 1 cells. 5. Alphaxalone produced leftward shifts of GABA log concentration-response curves of similar magnitudes in both populations of cells. Significant increases in the maxima were found at 100 nM in alpha 1, beta 1 cells but not up to 1 microM in alpha 1,beta 1,gamma 2L cells.6. These results provide further evidence of the modulatory role of the gamma 2L subunit of the GABAA receptor containing alpha 1 and beta 2 subunits. As well as influencing the apparent affinity of GABA and conferring benzodiazepine modulation, it also appeared to regulate the increase in maximal response produced in the presence of barbiturates and steroids. This latter effect may imply that barbiturates and steroids increase the channel open-state probability in the presence of GABA and that this effect is diminished by the presence of the gamma 2L subunit.

The pharmacology of recombinant GABAA receptors containing bovine alpha 1, beta 1, gamma 2L sub-units stably transfected into mouse fibroblast L-cells.

1. Responses to gamma-aminobutyric acid (GABA) were evoked in mouse fibroblast L-cells stably transfected with bovine, alpha 1, beta 1, gamma 2L sub-units of the GABAA receptor. Expression was stimulated via a steroid-inducible promoter system. 2. In near symmetrical intracellular and extracellular chloride concentrations, GABA evoked inward currents at negative holding potentials that reversed at +5 mV and displayed slight outward rectification. Concentration-response curves were fitted well by the logistic equation. GABA had a pEC50 = 5.1 +/- 0.1 and the curves had a slope of 1.9 +/- 0.1. 3. Responses to GABA were antagonized by bicuculline, picrotoxin and penicillin. The action of bicuculline was competitive (pA2 = 6.4) whilst the block by picrotoxin was uncompetitive and strongly agonist-dependent. 4. Benzodiazepine receptor agonists potentiated responses to 3 microM GABA. The rank order of potency was FG 8205 > flunitrazepam > zolpidem > C1218872. FG 8205 and C1218872 produced markedly lower maximal potentiations with efficacies 0.4 and 0.6 x that of flunitrazepam, respectively. The potencies of zolpidem and C1218872 observed are in agreement with the BZ1 type pharmacology of this sub-unit combination. The potentiation of GABA by flunitrazepam was antagonized by flumazenil with a Ki of 3.8 nM. 5. GABA responses were potentiated in the presence of pentobarbitone and alphaxalone. The response was also noticeably broadened by these compounds due to a decrease in the response decay rate. Concentrations of pentobarbitone of 100 microM and above evoked an inward current in the absence of GABA. Alphaxalone up to 10 microM did not evoke a direct response. 6. This expression system produced functional receptors that behaved in a fashion analogous to those found endogenously in other preparations. Thus, this system appears to provide a useful and versatile preparation for the analysis of sub-unit regulation of GABAA receptor pharmacology.

Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites.

The differential sensitivity of type A gamma-aminobutyric acid (GABAA) receptors to benzodiazepine ligands seen in the mammalian nervous system is thought to be generated by the existence of a number of different receptor subtypes, assembled from a range of closely related subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression of cloned cDNAs encoding the subunits of interest. Two transient expression systems have so far been employed in the study of the ligand-binding characteristics and chloride channel properties of such GABAA receptors--Xenopus oocytes and transfected mammalian cells. Here we report on the use of a steroid-inducible promoter expression system for the production of a permanently transfected clonal cell line expressing the alpha 1 beta 1 gamma 2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gel-exclusion chromatography, we have shown that the alpha 1, beta 1, and gamma 2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same benzodiazepine pharmacology as native alpha 1-containing GABAA receptors and function as GABA-gated chloride channels. Such cell lines expressing individual GABAA receptor subtypes will prove important tools in the study of the structure, function, and pharmacology of GABAA receptors and in the development of subtype-specific drugs.

The effect of omega-conotoxin GVIA on synaptic transmission within the nucleus accumbens and hippocampus of the rat in vitro.

1. The actions of two calcium channel antagonists, the N-channel blocker omega-conotoxin GVIA (omega-CgTx) and the L-channel antagonist nisoldipine, on synaptic transmission were investigated in the hippocampus and nucleus accumbens of the rat in vitro. 2. omega-CgTx (100 nM for 10 min) produced a marked and irreversible reduction of focally evoked population spikes and intracellularly recorded excitatory postsynaptic potentials (e.p.s.ps) in the nucleus accumbens, which could not be overcome by increasing the stimulus strength. 3. Nisoldipine (10 microM for 10 min) had no effect on population spikes in the nucleus accumbens or the CA1 of the hippocampus. 4. In the hippocampus, population spikes were not irreversibly reduced by omega-CgTx (100 nM for 10 min) but rather, multiple population spikes were produced along with spontaneous synchronous discharges. This indicated that inhibitory synaptic transmission was being preferentially reduced. 5. Intracellular recordings demonstrated that omega-CgTx powerfully reduced inhibitory synaptic transmission in an irreversible manner and that excitatory transmission was also reduced but to a lesser extent. Unlike excitatory transmission in the nucleus accumbens and inhibitory transmission in the hippocampus, increasing the stimulus strength overcame the reduction of hippocampal excitatory transmission. 6. It is concluded that omega-CgTx-sensitive calcium channels are involved in the calcium entry that precedes the synaptic transmission in all these synapses. The apparent lower sensitivity of the hippocampal excitatory fibres to omega-CgTx may indicate that calcium entry that promotes transmitter release at central synapses may be mediated by pharmacologically distinct calcium channels.

Synaptic potentials mediated by excitatory amino acid receptors in the nucleus accumbens of the rat, in vitro.

Focal stimulation of the dorsal regions of the nucleus accumbens of the rat, in vitro, evoked field potentials consisting of two negative waves. The first wave probably reflected electrical activation of the presynaptic fibres. This wave was resistant to excitatory amino acid antagonists and sulpiride. The second negative wave arose from a slower positive going component. These probably evince a population spike and field EPSP, respectively. Intracellular studies also indicated the occurrence of EPSP's and action potentials. The presumed field EPSP and population spike were reduced by kynurenic acid (IC50 approximately 300 microM) but not by AP5 nor sulpiride. When the Mg2+ was removed, the field EPSP was prolonged in a manner fully reversible by the addition of 30 microM D(-)AP5. These results indicate that focal stimulation within the nucleus accumbens may activate excitatory amino acid-ergic fibres that make synaptic contact with an indigenous population of neurones. The postsynaptic receptors were probably of a non-NMDA subtype. However NMDA receptor-mediated responses were evident upon removal of the Mg2+, suggesting that these receptors could also contribute to neuronal excitation under the appropriate physiological conditions.

The effect of NMDA receptor glycine site antagonists on hypoxia-induced neurodegeneration of rat cortical cell cultures.

The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration. Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801. Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium. MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC50 of 15 nM against increases in LDH levels. HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC50s of 175 and 18 microM, respectively. Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e. complete protection. This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage. Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death. These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.

The pharmacology of quisqualate and AMPA in the cerebral cortex of the rat in vitro.

The depolarising population response to the excitatory amino acids, quisqualate and AMPA, in slices of cerebral cortex of the rat have been compared. Their respective dose-response curves had a similar maximum but the slope of the curve for AMPA was consistently steeper than that for quisqualate. The dose-response curves for AMPA had a mean log EC50 of -5.18 +/- 0.05, which was significantly different from -4.62 +/- 0.07 the mean log EC50 of the dose-response curves for quisqualate. Responses to both agonists were antagonised by kynurenic acid, barbiturates and gamma-DGT to a similar extent. The antagonism by kynurenate appeared to be competitive whilst the barbiturates were evidently noncompetitive antagonists. These results are in agreement with claims that quisqualate and AMPA act at a similar recognition site. The differences in the slopes of the dose-response curves for quisqualate and AMPA may be explained by the differences in the cellular uptake of the two agonists and/or by differences in efficacy.

Spontaneous paroxysmal activity induced by zero magnesium and bicuculline: suppression by NMDA antagonists and GABA mimetics.

Slices of rat cerebral cortex developed spontaneous paroxysmal discharges when superfused with Krebs medium containing zero Mg2+ or 50 microM bicuculline. In both situations, the N-methyl-D-aspartate (NMDA) antagonists APV, 100 microM, and ketamine, 100 microM substantially reduced the frequency of the paroxysmal events, the reduction being greater in zero Mg2+. gamma-Aminobutyric acid (GABA) 1 mM, the GABA-A agonist muscimol 2 microM and the GABA-B receptor agonist baclofen 10 microM, each reduced the frequency of events in zero Mg2+ while muscimol and GABA also reduced the amplitude of the events. GABA and baclofen were similarly effective against bicuculline-induced events but the muscimol concentration required was 5-10-fold higher. These results suggest that, under our vitro conditions, neocortical cells are normally restrained from paroxysmal discharges by Mg2+. Inhibition by GABA through GABA-A receptors and inhibition by GABA through GABA-B receptors, may also contribute to this restraint.

Effect of aminooxyacetic acid on gamma-aminobutyric acid release from frog retina.

The Ca2+-dependence of the release of [3H]gamma-aminobutyric acid (GABA) from the pre-loaded, superfused frog retina was investigated in the presence and absence of the GABA-transaminase inhibitor, aminooxyacetic acid (AOAA). In the latter case, an ion-exchange column chromatographic technique was used to separate [3H]GABA from tritiated metabolites released with it into the superfusate. In the presence of aminooxyacetic acid, a 2 min 30 mM K+ pulse released similar amounts of [3H]GABA in normal and Ca2+-free Ringer solutions. However, when aminooxyacetic acid was absent, the K+-evoked release of [3H]GABA was substantially reduced under Ca2+-free conditions. It is concluded that the presence of aminooxyacetic acid can reduce the Ca2+-dependence of K+-evoked release of GABA from the frog retina. This is in accordance with its effect on cerebral cortex of the rat, reported previously.