Article Figures & Data
Figures
- Figure 1.
Taurine decreases the excitability of VB neurons through GABAA receptors. A, Representative current-clamp traces demonstrate AP firing evoked by a 0.1 nA current step (duration, 500 ms) in a VB neuron. Membrane resistance (Rm) was measured by injecting hyperpolarizing current (−0.02 nA). After taurine (50 μm) perfusion, AP firing decreased. The inhibitory effects of taurine were completely reversible on washing out taurine. B, Pooled data show that taurine reduces the excitability of VB neurons (**p < 0.01; n = 5). C, Bar graph illustrates the normalized Rm from the same (as in B) VB neurons (**p < 0.01). Under control conditions, Rm was 267 ± 11 MΩ, whereas in the presence of taurine and after the washout of taurine, Rm values were 242 ± 15 and 267 ± 8 MΩ, respectively. D, Representative current-clamp trace demonstrating AP firing evoked by a 0.1 nA current step (duration, 500 ms) in another VB neuron; after taurine (50 μm) perfusion, AP firing decreased. Subsequent gabazine application increased the number of evoked APs. Strychnine (1 μm) was present throughout the recordings to prevent the activation of glycine receptors. E, Pooled data show that taurine reduces the excitability of VB neurons (**p < 0.01 vs control; n = 6). Gabazine was able to block the inhibition produced by taurine (**p < 0.01 vs control; n = 6). F, Bar graph illustrates parallel changes (as in E) in membrane resistance (Rm) produced by taurine and gabazine (**p < 0.01 vs control; n = 6). Under control conditions, Rm was 234 ± 19 MΩ, whereas in the presence of taurine and taurine plus gabazine, Rm was 213 ± 17 and 253 ± 20 MΩ, respectively. Error bars represent SE.
- Figure 2.
Low concentrations of taurine preferentially activate GABAA receptors. A, Exemplar traces of taurine-induced currents in a VB neuron voltage clamped at 0 mV (here and in subsequent figures). Application of 50 μm taurine evoked a modest (∼50 pA) outward current in a VB neuron, whereas in the presence of the GABAA receptor antagonist gabazine, 50 μm taurine produced very little current shift in a different VB neuron. In another VB neuron, 50 μm taurine produced a modest (∼50 pA) current shift in the presence of the glycine receptor antagonist strychnine. The bars above the traces indicate the period of drug application at the indicated concentration in this and subsequent figures. B, Exemplar current trace demonstrating that application of 500 μm taurine evoked large outward currents in VB relay neurons (the scale bars here are different from those in A). In the presence of GABAA or glycine receptors antagonist, 500 μm taurine still elicited current shifts in each instance, but had no effect on the holding current when both GABAA and glycine receptors are blocked. All traces are from different VB neurons. C, Concentration–response curves of taurine-activated currents in the absence or presence of GABAA and glycine receptors antagonist applied individually. Taurine-activated currents mediated by GABAA receptors are larger than currents mediated by glycine receptors. Error bars represent SE. D, Concentration–response curves in C were enlarged to illustrate the differences in current amplitude evoked by taurine of low concentrations (10–100 μm). Taurine-activated currents mediated by GABAA receptors are significantly larger than currents mediated by glycine receptors. When glycine receptors were blocked by strychnine, taurine evoked concentration-dependent outward currents in thalamic relay neurons. Control group: 10 μm, 11.8 ± 4.3 pA, n = 6; 50 μm, 66.8 ± 12.7 pA, n = 6; 100 μm, 133.8 ± 21.5 pA, n = 5; strychnine group: 10 μm, 12.4 ± 1.8 pA, n = 12; 50 μm, 43.6 ± 5.3 pA, n = 16; 100 μm, 103.4 ± 13.0 pA, n = 8; gabazine group: 10 μm, 0.4 ± 0.8 pA, n = 8; 50 μm, 11.1 ± 3.0 pA, n = 8; 100 μm, 29.4 ± 5.0 pA, n = 5. Error bars represent SE.
- Figure 3.
The effects of taurine on synaptic versus extrasynaptic GABAA receptors. A, A typical recording of sIPSCs in a VB neuron in the presence of 50 μm taurine. Strychnine (1 μm) was present throughout so as to exclude the contribution of glycine receptors (as well as recordings in Figs. 4 and 6). Averaged sIPSC traces (>100 events per ensemble trace) before (black) and after (gray) taurine application; superimposed traces illustrate the similarity in amplitude and decay time. B, The normalized amplitude (0.97 ± 0.06), decay time constant (1.03 ± 0.03), and frequency (1.03 ± 0.09) of sIPSCs after 50 μm taurine perfusion. These parameters are not significantly different from those obtained under control conditions (p > 0.05; n = 11). C, A representative current trace demonstrating that in the presence of 50 μm taurine, 500 nm midazolam did not shift the baseline current. D, In a different neuron than in C, ZnCl2 (20 μm) partially blocked the taurine-induced outward current as evidenced by the small (∼20 pA) shift in the current baseline. E, In a different neuron than in C and D, gabazine (10 μm) effectively blocked the taurine-induced outward current as well as all sIPSCs. The shift in the current baseline is ∼60 pA. F, Averaged current shifts produced by gabazine (n = 7), Zn2+ (n = 6), and midazolam (n = 7) after taurine application. Error bars represent SE.
- Figure 4.
Endogenous taurine directly evokes GABAergic currents in VB neurons. A, An exemplar current trace (top) demonstrating that SNAP-5114 (50 μm), a GAT-2 and GAT-3 inhibitor, failed to induce a current shift in a VB neuron. An appreciable shift in the baseline current was observed after coapplication of SNAP-5114 and a second reuptake inhibitor, NO-711 (10 μm; bottom trace). B, Exemplar current trace showing that GES (10 μm), a taurine uptake inhibitor, evoked current shift (∼20 pA) in a different VB neuron than in A. GES (50 μm) induced a greater outward current (∼100 pA) in a different VB neuron (note the different vertical scale bar here from that for 10 μm GES trace). C, Bar graph demonstrates that GES induced significant outward currents in VB neurons (10 μm: 21.5 ± 3.9 pA, n = 8; 50 μm: 105.5 ± 18.9 pA, n = 5; 100 μm: 215.3 ± 31.2 pA, n = 7), whereas SNAP-5114 induced little outward current (50 μm: 0.3 ± 2.3 pA, n = 8). Coapplication of NO-711 (10 μm) and SNAP-4114 (50 μm) also produced a significant outward current (193.4 ± 37.9; n = 4). D, Sample current traces demonstrating the effect of taurine (50 μm) on the outward current in the presence of SNAP-4114 (50 μm; top trace) or SNAP-4114 plus NO-711 (10 μm; bottom trace). E, Bar graph summarizing the effect of SNAP-5114 (50 μm), alone and in combination with NO-711 (10 μm), on taurine (50 μm)-evoked currents. Control taurine currents were from Figure 2. In the presence of SNAP-5114 alone, taurine produced a 46.6 ± 7.1 pA current shift (n = 5), whereas taurine alone produced a 67 ± 13 pA current shift (n = 6; same data as in Fig. 2); taurine again produced a somewhat smaller outward current (26.8 ± 6.6 pA; n = 4) in the presence of SNAP-5114 and NO-711. Error bars represent SE.
- Figure 5.
Expression of taurine-evoked currents is age dependent. A, Taurine (50 μm) failed to evoke a current shift in a VB neuron from a P9 mouse. Taurine (100 μm) produced small outward current (∼20 pA) in a VB neuron from a P8 mouse. B, Bar graph demonstrates that taurine (10, 50, and 100 μm) induced much smaller currents in VB neurons from young mice (10 μm, 0.1 ± 0.7 pA, n = 4; 50 μm, 6.5 ± 4.7 pA, n = 9; 100 μm, 16.2 ± 11.1 pA, n = 8; ***p < 0.001 vs >P22 group at all three concentrations). Data for age >P22 group are from Figure 2C. Error bars represent SE.
- Figure 6.
Comparison of taurine-evoked Cl− currents recorded in HEK293 cells expressing α1β2γ2s and α4β2δ GABAA receptors. A, Typical concentration-dependent taurine-activated and maximum GABA-activated currents recorded from α4β2δ GABAA receptors expressed in HEK293 cells. B, Typical concentration-dependent taurine-activated and maximum GABA-activated currents recorded from α1β2δ2s GABAA receptors. C, Averaged relative concentration–response curves (Itaurine/IGABA-max) for α1β2γ2s (n = 8) and α4β2δ (n = 12) GABAA receptors. The averaged concentration–response data in the figure were fitted using a sum of least-squares method to a Hill equation of the form: I = Imax × [agonist]nH/([agonist]nH + EC50nH), where I is the peak current, Imax is the maximum whole-cell current amplitude, [agonist] is the agonist concentration, EC50 is the agonist concentration eliciting a half-maximal current response, and nH is the Hill coefficient. Error bars represent SE.
- Figure 7.
Taurine-mediated currents are absent in VB neurons from mice lacking the GABAA receptor α4 subunit. A, Taurine (50 μm) evoked a marked current (∼50 pA) in a VB neuron from a wild-type mouse. In contrast, taurine produced no current shift in a VB neuron from a Gabra4−/− mouse. B, Bar graph demonstrates that taurine (10 and 50 μm) induced current shifts in wild-type, but not α4 knock-out, VB neurons (***p < 0.001; n = 6–8). Error bars represent SE. WT, Wild type; KO, knock-out.
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