Much inhibitory synaptic transmission in the spinal cord and brain stem is mediated by the glycinergic system which classically generates fast, chloride-mediated, inhibitory postsynaptic responses. Here we describe the co-existence of a tonic glycine receptor-mediated conductance with phasic glycine receptor-mediated IPSCs, in neurones of the LSO, and its regulation by glycine transporters.
Brain stem slices (~175 µm) containing the LSO were prepared by standard means from P8-P13 male Lister-hooded rats killed humanely. Whole-cell voltage-clamp recordings -70 mV, ~35°C) were made from visualised LSO neurones with CsCl-containing pipettes. Data are presented as mean ± S.E.M. Statistical significance was assessed with paired Student’s t tests.
Under control conditions, spontaneous inward-going synaptic currents with a broad range of amplitudes and kinetic profiles were observed. The frequency of events was decreased by 70 ± 9 % following blockade of glutamate receptor-mediated EPSCs with NBQX (10 µM) and D-AP5 (50 µM), and GABAA receptor-mediated IPSCs with SR95531 (10 µM). The remaining population of spontaneous currents arose with a median inter-event interval of 1.18 ± 0.17 s and had a median amplitude of -50 ± 6 pA (n = 11). These currents had a median 10-90 % rise time of 0.6 ± 0.1 ms and decayed exponentially with a median time constant of 2.7 ± 0.3 ms (n = 11). Event frequency was insensitive to TTX (1 µM), but all events were eliminated by the glycine antagonist strychnine (2 µM). Strychnine also decreased both the mean steady-state holding current -128 ± 14 pA control;-113 ± 15 pA, strychnine, P < 0.02) and its variance (24.1 ± 3.9 pA2, control; 12.9 ± 2.1 pA2, strychnine, P < 0.02). An inhibitor of the type 1 glycine transporter, ALX5407, produced large, strychnine-sensitive increases in both holding current -150.8 ± 18.5 pA, control;-309.3 ± 45.9 pA, ALX5407, n = 10, P = 0.001) and variance (51.9 ± 9.4 pA2, control; 254.5 ± 31.5 pA2, ALX5407, n = 10, P < 0.001). An inhibitor of the type 2 glycine transporter, ORG-25543, caused a small increase in holding current that was not statistically significant -167.1 ± 31.2 pA, control;-185.7 ± 35.1 pA, ORG-25543, P = 0.16) but did produce a two-fold increase in variance (33.5 ± 5.3 pA2, control; 64.8 ± 7.8 pA2, ORG-25543, P = 0.002).
These data demonstrate that LSO neurones receive both phasic and tonic glycinergic inputs, the latter predominantly under control of GlyT1. It will be interesting to determine the roles played by these two forms of inhibition in auditory processing and their relative importance in postnatal development.