thapsigargin and gabazine

The present study was performed to investigate the effects of depleting intracellular Ca(2+) stores on bicuculline- or gabazine-induced epileptiform excitability. Studies were performed on monolayer rat hippocampal neuronal networks utilising a system that allowed simultaneous multiple extracellular single-unit recordings of neuronal activity. Hippocampal neuronal networks were prepared from enzymatically dissociated hippocampi from 18-day-old fetal Wistar rats. The cells were cultured in Neurobasal medium with B27 serum-free supplements directly onto the surface of planar multiple microelectrode arrays with a central recording array of 64 (4 x 16) indium-tin thin-film recording electrodes. All cells recorded at 21 days-in-vitro exhibited spontaneous discharge activity with firing rates between 0.3-30.7 Hz. gamma-aminobutyric acid (GABA) produced a concentration-dependent decrease in firing (EC(50)=9.1 microM) which could be blocked by pre-application of bicuculline methobromide (10 microM). Addition of the GABA(A)-receptor antagonists gabazine (10 microM) or bicuculline (10 microM) resulted in the rapid generation of synchronised bursting within all the cells recorded. Bicuculline exhibited heterogeneity of action on firing rate, whereas gabazine always increased firing. Pre-incubation with thapsigargin, which depletes intracellular calcium stores, resulted in a decrease in the amount of neuronal excitation produced by bicuculline, but not by gabazine, suggesting that bicuculline-induced neuronal excitation requires release of Ca(2+) from intracellular stores.

Topics: Animals; Bicuculline; Calcium; Cells, Cultured; Convulsants; Electrophysiology; Enzyme Inhibitors; Epilepsy; GABA Antagonists; Hippocampus; Immunohistochemistry; Microelectrodes; Nerve Net; Neurons; Pyridazines; Rats; Rats, Wistar; Thapsigargin

Evoked field potentials were recorded in the CA3 region of rat hippocampal slices to detect whether intracellular Ca2+ stores are involved in the epileptiform effects of the two prototypic GABA(A) antagonists, bicuculline methiodide (BMI) and gabazine (SR-95531; GBZ). Field population spikes gradually increased and became repetitive (epileptiform bursting) in the presence of either BMI (5 microM), or GBZ (5 microM). Thapsigargin (2 microM), a depletor of intracellular Ca2+ stores, reduced the epileptiform effect of BMI, but had no significant effect on the GBZ-induced hyperexcitability. These data suggest that Ca2+ release from intracellular stores participates in the epileptiform response of hippocampal CA3 neurons to BMI, but not in the response to GBZ.

Topics: Action Potentials; Animals; Bicuculline; Calcium; Convulsants; Electrophysiology; Enzyme Inhibitors; GABA Antagonists; Hippocampus; Male; Organ Culture Techniques; Pyridazines; Rats; Rats, Sprague-Dawley; Thapsigargin

We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters > 15 microns and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 microM gamma-aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 microM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 microM), an inhibitor of intracellular calcium pumps, and ryanodine (20 microM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 microM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3'5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 microM) and cyclosporin A (20 microM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 micrograms/ml) and calcineurin (300 micrograms/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases.

Topics: Alkaline Phosphatase; Animals; Bicuculline; Caffeine; Calcineurin; Calcium; Calcium Channels; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; GABA Antagonists; gamma-Aminobutyric Acid; Imidazoles; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Membrane Potentials; Phosphodiesterase Inhibitors; Phosphoric Monoester Hydrolases; Pyridazines; Receptors, Cytoplasmic and Nuclear; Receptors, GABA-A; Retinal Ganglion Cells; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin; Turtles