Glycine Receptors in Cultured Chick Sympathetic Neurons are Excitatory and Trigger Neurotransmitter Release

1 Total RNA isolated from embryonic chick paravertebral sympathetic ganglia was used in a reverse transcription‐polymerase chain reaction (RT‐PCR) assay with a pair of degenerate oligonucleotide primers deduced from conserved regions of mammalian glycine receptor α‐subunits. Three classes of cDNA were identified which encode portions of the chicken homologues of the mammalian glycine receptor αl, α2 and α3 subunits. 2 The presence of functional glycine receptors was investigated in the whole‐cell configuration of the patch‐clamp technique in neurons dissociated from the ganglia and kept in culture for 7–8 days. In cells voltage clamped to −70 mV, glycine consistently induced inward currents in a concentration‐dependent manner and elicited half‐maximal peak current amplitudes at 43 μ m . 3 The steady‐state current–voltage relation for glycine‐induced currents was linear between ±80 and −60 mV, but showed outward rectification at more hyperpolarized potentials. Reversal potentials of these currents shifted with changes in intracellular chloride concentrations and matched the calculated Nernst potentials for chloride. 4 β‐Alanine and taurine were significantly less potent than glycine in triggering inward currents, with half‐maximal responses at 79 and 86 μ m , respectively. At maximally active concentrations, β‐alanine‐evoked currents were identical in amplitude to those induced by glycine. Taurine‐evoked currents, in contrast, never reached the same amplitude as glycine‐induced currents. 5 The classical glycine receptor antagonist strychnine reversibly reduced glycine‐induced currents, with half‐maximal inhibition occurring at 62 n m . Two more recently characterized glycine receptor antagonists, isonipecotic acid (half‐maximal inhibition at 2 m m ) and 7‐trifluoromethyl‐4‐hydroxyquinoline‐3‐carboxylic acid (half‐maximal inhibition at 67 μ m ), also blocked glycine‐evoked currents in a reversible manner. The chloride channel blocker picrotoxin reduced glycine‐evoked currents, with half‐maximal effects at 348 μ m . Inhibition by the glycine receptor channel blocker cyanotriphenylborate was half‐maximal at 4 μ m . 6 Apart from evoking inward currents, glycine occasionally triggered short (< 100 ms) spike‐like currents which were abolished by hexamethonium and thus reflected synaptic release of endogenous acetylcholine. In addition, glycine caused Ca 2+ ‐dependent and tetrodotoxin‐sensitive tritium overflow from neurons previously labelled with [ 3 H] noradrenaline. This stimulatory action of glycine was reduced in the presence of strychnine and after treatment with the chloride uptake inhibitor furosemide (frusemide). 7 In 65% of neurons loaded with the Ca 2+ indicator fura‐2 acetoxymethyl ester, glycine increased the ratio of the fluorescence signal obtained with excitation wavelengths of 340 and 380 nm, respectively, which indicates a rise in intracellular Ca 2+ concentration. 8 The results show that sympathetic neurons contain transcripts for different glycine receptor α‐subunits and carry functional heteromeric glycine receptors which depolarize the majority of neurons to trigger transmitter release.