SYNAPSE 11:259-261 (1992)
Short Communications
Heterogeneity of Responses to Agents That Act at the Benzodiazepine Site on GABA, Receptors of Dorsal Root Ganglion Neurons Freshly Isolated From Adult Rats GEOFFREY WHITE Neurogen Corporation, Branford, Connecticut 06405
KEY WORDS
Inhibition, DRG, Patch-clamp, Ion channel
GABA exerts a n influence on neuronal excitability by activating a C1- current through the GABAAreceptor1 ionophore complex. This complex is a heteromultimeric protein (Boormann et al., 1987; Olsen and Tobin, 1990; Vicini, 1991), whose various subunits are differentially expressed within the brain and possibly within neurons themselves (Juiz et al., 1989; Olsen and Tobin, 1990; Vicini, 1991; Zimprich et al., 1991). Such observations raise the possibility that a topographical pattern of pharmacological and physiological properties of GABA, receptorlionophore complexes exists within the nervous system (Olsen and Tobin, 1990; Vicini, 1991). Functional heterogeneity is well documented in oocytes and transfected cultured mammalian tumor cells with respect to sensitivity to GABA and agents that act at the benzodiazepine receptor (Puia et al., 1991; Sigel et al., 1990). Functional heterogeneity in sensitivity to GABA has recently been demonstrated for neurons isolated from adult rats (White, 1992). No electrophysiological studies have systematically identified heterogeneity in responses to benzodiazepines €or intrinsically expressed GABAAreceptor complexes in neurons from adult mammals. This report details heterogeneity in such responses and suggests that individual neurons can express more than one benzodiazepine receptor subtype. Male Sprague-Dawley rats (200-500 g) were anesthetized with chloral hydrate (400 mg/kg) and decapitated. Dorsal root ganglia (DRG) were removed and placed in oxygenated DMEM, 0.4 mglml type I11 trypsin, 0.3 mg/ml type VII collagenase, and 0.1 mg/ml type IV DNase 11. This solution was incubated at 35°C for 45-60 minutes. Individual neurons were dissociated from the ganglia by vigorous shaking. Neurons were devoid of processes as described previously (White et al., 1990). Electrophysiological responses were obtained from 40 neurons using the whole-cell patch-clamp technique 0 1992 WILEY-LISS, INC.
with a n Axopatch 1D amplifier (Axon Instruments) and pClamp software (Axon Instruments) as described previously (White, 1990). Briefly, subsequent to gaining access to the cell interior, membrane potential was voltage-clamped to -60 mV. Series resistance (3-6 m n ) was compensated by 90%.Current amplitude was
Short Communications
Heterogeneity of Responses to Agents That Act at the Benzodiazepine Site on GABA, Receptors of Dorsal Root Ganglion Neurons Freshly Isolated From Adult Rats GEOFFREY WHITE Neurogen Corporation, Branford, Connecticut 06405
KEY WORDS
Inhibition, DRG, Patch-clamp, Ion channel
GABA exerts a n influence on neuronal excitability by activating a C1- current through the GABAAreceptor1 ionophore complex. This complex is a heteromultimeric protein (Boormann et al., 1987; Olsen and Tobin, 1990; Vicini, 1991), whose various subunits are differentially expressed within the brain and possibly within neurons themselves (Juiz et al., 1989; Olsen and Tobin, 1990; Vicini, 1991; Zimprich et al., 1991). Such observations raise the possibility that a topographical pattern of pharmacological and physiological properties of GABA, receptorlionophore complexes exists within the nervous system (Olsen and Tobin, 1990; Vicini, 1991). Functional heterogeneity is well documented in oocytes and transfected cultured mammalian tumor cells with respect to sensitivity to GABA and agents that act at the benzodiazepine receptor (Puia et al., 1991; Sigel et al., 1990). Functional heterogeneity in sensitivity to GABA has recently been demonstrated for neurons isolated from adult rats (White, 1992). No electrophysiological studies have systematically identified heterogeneity in responses to benzodiazepines €or intrinsically expressed GABAAreceptor complexes in neurons from adult mammals. This report details heterogeneity in such responses and suggests that individual neurons can express more than one benzodiazepine receptor subtype. Male Sprague-Dawley rats (200-500 g) were anesthetized with chloral hydrate (400 mg/kg) and decapitated. Dorsal root ganglia (DRG) were removed and placed in oxygenated DMEM, 0.4 mglml type I11 trypsin, 0.3 mg/ml type VII collagenase, and 0.1 mg/ml type IV DNase 11. This solution was incubated at 35°C for 45-60 minutes. Individual neurons were dissociated from the ganglia by vigorous shaking. Neurons were devoid of processes as described previously (White et al., 1990). Electrophysiological responses were obtained from 40 neurons using the whole-cell patch-clamp technique 0 1992 WILEY-LISS, INC.
with a n Axopatch 1D amplifier (Axon Instruments) and pClamp software (Axon Instruments) as described previously (White, 1990). Briefly, subsequent to gaining access to the cell interior, membrane potential was voltage-clamped to -60 mV. Series resistance (3-6 m n ) was compensated by 90%.Current amplitude was
