Modulation of gamma-aminobutyric acid-mediated inhibitory synaptic currents in dissociated cortical cell cultures.

Abstract

Inhibitory y-aminobutyric acid-mediated synaptic currents were studied in dissociated primary cultures of neonatal rat cortex with the whole-cell patch-clamp technique. Immunocytochemical staining of the cultures showed the presence of a large number of glutamic acid decarboxylasecontaining neurons, and electrical stimulation of randomly selected neurons produced in many cases chloride-mediated and bicuculline-sensitive inhibitory synaptic currents in postsynaptic cells. The amplitude and decay time of the inhibitory synaptic currents were increased by flunitrazepam and decreased by the /3-carboline derivative methyl 6,7-dimethoxy-4-ethyl-)3-carboline-3-carboxylate, two high-affimity ligands for the allosteric regulatory sites of r-aminobutyric acid receptors. The imidazobenzodiazepine Ro 15-1788, another high-affinity ligand of the y-aminobutyric acid receptor regulatory sites that has negligible intrinsic activity, blocked the action of flunitrazepam and /3-carboline. However, Ro 15-1788 also increased the decay rate of the inhibitory synaptic currents. This might suggest that an endogenous ligand for the benzodiazepine-f8-carboline binding site is operative in Yaminobutyric acid-mediated synaptic transmission.The development of the patch-clamp technique (1) has greatly facilitated the study of receptor function in terms of specific ion currents. The intracellular perfusion characteristic of this technique (2) allows manipulation of the ionic environment that is useful for distinguishing and characterizing different ion currents.Among the functional synaptic connections that have been described in primary neuronal cultures from fetal rat cortex (3) are those mediated by y-aminobutyric acid (GABA), an inhibitory neurotransmitter that is synthesized, released, and taken up by these cells (4, 5). Voltage-clamp analysis of GABA-mediated inhibitory postsynaptic currents (IPSCs) has been reported on various synapses. The best characterized is the neuromuscular junction of the crayfish and locust (6-10). IPSCs were also recorded in hippocampal slices (11) and in primary cultures of hippocampal neurons in which diazepam, a ligand for the allosteric modulatory site of GABA receptors, potentiated the IPSCs (12).Another anxiolytic benzodiazepine, flunitrazepam, and an anxiogenic /3-carboline derivative, methyl 6,7-dimethoxy-4-ethyl-,8-carboline-3-carboxylate (DMCM), potentiate and reduce, respectively, the increase in chloride conductance induced by direct application of GABA on the cell bodies of spinal cord neurons (13). We studied the action of these drugs and of the imidazobenzodiazepine MATERIALS AND METHODS Primary Culture. Primary neuronal cultures were obtained from 1-day-old rat cerebral cortices by the dissociation procedure described (15) for neonatal rat cerebellar cultures. Cells were plated (2 x 106 cells per dish) on 35-mm Nunc dishes coated with poly (L-lysine) (10 ,Ag/ml; Mr, 70-150 X 103) and were cultured in basal Eagle's medium/10% (vol/vol) fetal bovine serum (both from GIBCO)/25 mM KC1/2 mM ...

Endogenous Positive Allosteric Modulation of GABAA Receptors by Diazepam binding inhibitor

“…Similarly, FLZ has been observed to reduce evoked inhibitory postsynaptic potential (IPSP) amplitude in hippocampal CA1 pyramidal neurons (King et al, 1985) and eIPSC duration in dissociated cortical neurons (Vicini et al, 1986). Some reports have indicated agonist effects of FLZ (Skerritt and Macdonald, 1983; De Deyn and Macdonald, 1987; Weiss et al, 2002), including at receptors carrying α3 subunits (Ramerstorfer et al, 2010).…”

Synthesis and Anticonvulsant Activity of N‐(2‐Hydroxy‐ethyl)amide Derivatives

“…Bicuculline (BIC) is a known competitive antagonist of the inhibitory amino acid GABA at the central GABA A receptors [14]. BIC produces convulsions through its antagonism of GABA A -receptor-mediated inhibition [15].…”

Subregion-Specific Impacts of Genetic Loss of Diazepam Binding Inhibitor on Synaptic Inhibition in the Murine Hippocampus

“…In the hippocampus, the amplitude of inhibitory postsynaptic potentials in rat CA1 pyramidal cells was reduced after FLZ application (King et al, 1985), suggesting that the blockade of the GABA A R BZ-binding site impeded the potentiation of inhibition by an endogenous ligand. Additionally, FLZ reduced IPSC decay time in the thalamic reticular nucleus (Christian et al, 2013; Christian and Huguenard, 2013) and in cultured cortical neurons (Vicini et al, 1986). Similarly, FLZ reduced the amplitude of electrically evoked inhibitory postsynaptic currents (eIPSCs) in CA1 pyramidal cells following the induction of long-term potentiation (Xu and Sastry, 2005).…”