Activation of presynaptic glycine receptors facilitates glycine release from presynaptic terminals synapsing onto rat spinal sacral dorsal commissural nucleus neurons

Abstract

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Here we report the novel finding that presynaptic glycine autoreceptors modulate release from terminals synapsing onto rat spinal sacral dorsal commissural nucleus (SDCN) neurons. In mechanically dissociated SDCN neurons, in which functional presynaptic nerve terminals remain adherent to the isolated neurons, exogenously applied glycine (3 microM) increased the frequency of glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) without affecting their amplitudes or decay times. This suggests that glycine acts presynaptically to increase glycine release probability. Picrotoxin, at a concentration that had little direct effect on sIPSC frequency and amplitude (30 microM), significantly attenuated glycine-induced presynaptic sIPSC facilitation. The glycine-induced sIPSC frequency facilitation was completely abolished either in a Ca(2+)-free external solution or in the presence of 100 microM Cd2+, suggesting the involvement of extracellular Ca2+ influx into the nerve terminals. The glycine action was also completely occluded in the presence of 300 nM tetrodotoxin. In recordings from SDCN neurons in spinal cord slices, glycine (10 microM) increased evoked IPSC (eIPSC) amplitude and decreased the extent of paired-pulse facilitation. In response to brief high frequency stimulus trains the eIPSCs displayed a profound frequency-dependent facilitation that was greatly reduced by picrotoxin (30 microM). These results indicate that glycine acts at presynaptic autoreceptors, causing depolarization of the glycinergic nerve terminals, the subsequent activation of voltage-dependent Na+ and Ca2+ channels, and facilitation of glycine release. Furthermore, this presynaptic facilitation was observed under more physiological conditions, suggesting that these glycinergic autoreceptors may contribute to the integration of local inhibitory inputs to SDCN neurons.

Figure 1. Glycine facilitates glycinergic sIPSC frequency by presynaptic action

Aa, typical trace of glycinergic sIPSCs before, during and after the application of 3 μM glycine. Insets represent the traces from each condition on an expanded time scale. b, the time course of sIPSC frequency during the application of glycine at a V_H of −30 mV. The number of events in every 10 s period (•, presence of glycine; ○, absence of glycine) was summed and plotted. Each point is the mean from 12 neurons. B, current response to a 10 mV, 300 ms hyperpolarizing voltage step, with or without glycine. C, averaged sIPSCs in control conditions (n = 22 events) and in the presence of glycine (n = 28 events). The two averages are superimposed so as to demonstrate the lack of change in sIPSC kinetics. D, typical traces of glycinergic sIPSCs observed before, during and after the application of 3 μM glycine in a standard external solution (a) and in the presence of 0.5 μM strychnine (b). Note that strychnine completely eliminated all sIPSCs and application of glycine did not evoke any response.

Figure 2. Concentration dependence of the pre- and postsynaptic actions of glycine

A, typical traces of sIPSCs and sustained postsynaptic currents in the presence of various concentrations of glycine. The current response to 30 μM glycine (bottom trace) has been truncated. B, mean concentration–response relationship between glycine and sIPSC frequency (a) and sIPSC amplitude (b). Each column is the mean and s.e.m. of data from 8 neurons. Note that concentrations of glycine above 3 μM did not cause a further increase in sIPSC frequency. *P < 0.05, **P < 0.01. C, concentration—response relationship for postsynaptic currents induced by exogenous application of glycine. a, typical current responses induced by various concentrations of glycine. b, concentration–response curves in control conditions (•) and with co-application of glycine and 30 μM picrotoxin (○). All data were normalized to the current amplitude in response to 100 μM glycine. The smooth curves indicate the fit of the data to the Hill equation. The EC₅₀ values were 57 μM (•) and 61 μM (○).

Figure 3. Effects of picrotoxin on glycine-induced sIPSC frequency facilitation

A, typical traces of glycinergic sIPSCs before, during and after the application of 3 μM glycine in standard external solution (a) and in the presence of 30 μM picrotoxin (b). The 2 traces were obtained from the same neuron. Ba, effect of 30 μM picrotoxin on glycinergic sIPSC frequency and amplitude. b, effect of picrotoxin on the facilitation of sIPSCs by glycine. In both a and b, data have been normalized relative to the original control sIPSC frequency and amplitude. Each column represents the mean and s.e.m. of data from 6 neurons. **P < 0.01.

Figure 4. Glycine-induced sIPSC frequency facilitation is mediated by Ca²⁺ influx through VDCCs

A, typical traces of glycinergic sIPSCs before, during and after the application of 3 μM glycine in standard external solution (a), in Ca²⁺-free external solution (b) and in external solution containing 100 μM Cd²⁺ (c). All traces were obtained from the same neuron. B, cumulative probability plots for sIPSC inter-event interval (a; P = 0.67, Ca²⁺ free vs. Ca²⁺ free + glycine) and sIPSC amplitude (b; P = 0.40) obtained from the trace shown in Ab (102 events for the Ca²⁺-free condition and 21 events for the Ca²⁺-free + glycine condition). The insets show the effects of the various manipulations on the mean sIPSC frequency and amplitude. Each column represents mean and s.e.m. data from 7 neurons, with the values all being normalized to the initial control values. **P < 0.01. C, cumulative probability plots for sIPSC inter-event interval (a; P = 0.23, Cd²⁺vs. Cd²⁺ + glycine) and sIPSC amplitude (b; P = 0.45) obtained from the trace shown in Ac (225 events for the Cd²⁺ condition and 29 events for the Cd²⁺ + glycine condition). As in B, insets show the mean and s.e.m., normalized to the initial control values (n = 6). ** P < 0.01.

Figure 5. Glycine-induced sIPSC frequency facilitation requires TTX-sensitive Na⁺ channels

A, typical traces of glycinergic sIPSCs before, during and after the application of 3 μM glycine in standard external solution (a) and in the presence of 300 nM TTX (b). The 2 traces were obtained from the same neuron. B, cumulative probability plots for sIPSC inter-event interval (a; P = 0.29, TTX vs. TTX + glycine) and current amplitude (b; P = 0.76) for the data shown in Ab (183 events for the TTX condition and 23 events for the TTX + glycine condition). The insets show mean and s.e.m. for sIPSC frequency and amplitude from 11 neurons, with data normalized to the original control conditions. **P < 0.01.

Figure 6. Effect of glycine on IPSCs evoked by paired pulses

A, typical traces of glycinergic eIPSCs obtained in response to paired-pulse stimulation (inter-stimulus interval = 50 ms), in control conditions (left trace) and again in the presence of 10 μM glycine (middle trace). Each trace represents the average of 20 trials. The right trace shows the superimposition of these two recordings. Ba, bar graph showing the averaged glycine-induced increase in the first eIPSC (eIPSC₁) amplitude (n = 5). Error bars represent s.e.m.b, effects of glycine on the paired-pulse facilitation ratio (eIPSC₂/eIPSC₁). Connections between open circles represent the glycine-induced change in the paired-pulse facilitation ratio in each individual experiment, whereas filled circles represent the mean ±s.e.m. from 5 neurons. *P < 0.05.

Figure 7. Picrotoxin reduces frequency-dependent facilitation of glycinergic eIPSCs

A, typical averaged traces of eIPSCs in the absence and presence of 30 μM picrotoxin during a brief stimulation train presented at a frequency of 100 Hz (left), 33.3 Hz (middle) and 10 Hz (right). All traces represent the average of 20–40 trials, which were then superimposed in each experimental condition. B, plots of the normalized mean peak amplitudes of each eIPSC during brief stimulus trains presented at 100 Hz (left, n = 7 neurons), 33.3 Hz (middle, 5 neurons) and 10 Hz (right, 4 neurons). Each point represents the mean and s.e.m. ○, data recorded in control conditions; •, data recorded in the presence of picrotoxin. *P < 0.05,**P < 0.01.