Glycine receptor α3 and α2 subunits mediate tonic and exogenous agonist-induced currents in forebrain

Abstract

Neuronal inhibition can occur via synaptic mechanisms or through tonic activation of extrasynaptic receptors. In spinal cord, glycine mediates synaptic inhibition through the activation of heteromeric glycine receptors (GlyRs) composed primarily of α1 and β subunits. Inhibitory GlyRs are also found throughout the brain, where GlyR α2 and α3 subunit expression exceeds that of α1, particularly in forebrain structures, and coassembly of these α subunits with the β subunit appears to occur to a lesser extent than in spinal cord. Here, we analyzed GlyR currents in several regions of the adolescent mouse forebrain (striatum, prefrontal cortex, hippocampus, amygdala, and bed nucleus of the stria terminalis). Our results show ubiquitous expression of GlyRs that mediate large-amplitude currents in response to exogenously applied glycine in these forebrain structures. Additionally, tonic inward currents were also detected, but only in the striatum, hippocampus, and prefrontal cortex (PFC). These tonic currents were sensitive to both strychnine and picrotoxin, indicating that they are mediated by extrasynaptic homomeric GlyRs. Recordings from mice deficient in the GlyR α3 subunit (Glra3^-/-) revealed a lack of tonic GlyR currents in the striatum and the PFC. In Glra2^-/Y animals, GlyR tonic currents were preserved; however, the amplitudes of current responses to exogenous glycine were significantly reduced. We conclude that functional α2 and α3 GlyRs are present in various regions of the forebrain and that α3 GlyRs specifically participate in tonic inhibition in the striatum and PFC. Our findings suggest roles for glycine in regulating neuronal excitability in the forebrain.

Keywords: Cys-loop receptor; alpha subunits; glycine receptor; strychnine; tonic inhibition.

Fig. 1.

Exogenous glycine-activated GlyR currents in the forebrain. (A and B) Whole-cell currents elicited by exogenous glycine applications (30 μM–10 mM) were recorded from MSNs in vitro in the NAc (A), and glycine concentration-response curves were generated (B), confirming the presence of measurable glycine-activated currents. (C) To demonstrate that the currents measured in A and B were due to glycine action at strychnine-sensitive GlyRs and not other neurotransmitter receptors, whole-cell currents elicited by glycine were first measured in the absence and then in the presence of a concurrent application of the GlyR-specific antagonist strychnine (100 nM). The amplitude of current produced by applications of 300 μM glycine was reduced in the presence of strychnine. (D) Currents elicited by 1 mM glycine were measured in several brain regions. Glycine-activated currents were present in the dorsolateral striatum (DS), NAc, hippocampus (CA1), layer II/III of the medial PFC, basolateral amygdala (BLA), central nucleus of the amygdala (CeA), and the BNST. Data represent means ± SEM (n = 8–26 neurons from 7–11 mice).

Fig. S1.

Exogenous glycine-activated GlyR currents are not mediated by NMDA receptors or GABA_ARs. Whole-cell currents elicited by exogenous glycine applications (300 μM or 1 mM) were recorded from MSNs in the NAc in the presence of the NMDA receptor antagonist AP5 (50 μM) or the GABA_AR antagonist gabazine (20 μM). The amplitude of the glycine-activated current was not significantly altered by the application of either antagonist [F(2, 33) = 0.7327, P = 0.49], and there was no significant interaction between glycine concentration and antagonist [F(2, 60) = 0.25, P = 0.78]. Data are presented as mean ± SEM (n = 4–9 neurons from two to four mice).

Fig. S4.

The glycine transporter inhibitor sarcosine enhances the strychnine-induced current. To confirm that strychnine-induced shifts in the holding current were the result of GlyR conductance, the glycine transporter inhibitor sarcosine (500 μM) was washed onto the slice before 1 μM strychnine, and shifts in the holding current were measured in the NAc. (A and B) Sample traces are shown for recordings from the NAc (A) and the BNST (B). (C) Sarcosine alone produced significant shifts in the holding current in both the NAc [t(9) = 6.16, P = 0.0002] and the BNST [t(4) = 4.29, P = 0.0127]. (D) Sarcosine application significantly increased the amplitude of the strychnine-induced current in both the NAc [t(47) = 3.98, P = 0.0002] and the BNST [t(23) = 9.68, P < 0.0001]. Data are presented as mean ± SEM (n = 5–43 neurons from 2 to 18 mice; *P < 0.05 compared with the strychnine-induced current shift for aCSF alone).

Fig. 2.

Endogenous tonically activated GlyR currents in brain. (A) To identify tonic GlyR currents in MSNs in the nucleus accumbens, the effects of 1 μM strychnine on baseline holding currents were measured. (B) To determine whether tonic GlyR currents are due to activation of α homomers, the effects of 100 μM picrotoxin (in the presence of gabazine) on baseline holding currents were determined. (C) Strychnine induced significant shifts in the holding current of MSNs [t(42) = 6.872, P < 0.0001], indicating the presence of a tonic GlyR conductance. Similarly, picrotoxin applications induced significant shifts in holding currents [t(5) = 5.52, P = 0.003]. The amplitudes of the current shifts induced by strychnine and picrotoxin were not significantly different from each other [t(47) = 0.39, P = 0.70)]; n = 6–43 neurons from 3–18 mice. (D) To identify tonic GlyR currents in each of the brain regions in which exogenous glycine-activated currents were detected, the effects of 1 μM strychnine on baseline holding currents were measured. Significant strychnine-induced shifts in holding current were detected in the dorsolateral striatum (DS) [t(17) = 4.59, P = 0.0003], NAc [t(42) = 6.87, P < 0.0001], hippocampus (CA1) [t(6) = 5.12, P = 0.0022], and PFC (layer II/III) [t(11) = 4.97, P = 0.0004], indicating the presence of tonic GlyR currents in these regions. In contrast, there was no effect of strychnine on the holding current of neurons recorded from the basolateral amygdala (BLA) [t(9) = 1.00, P = 0.34], central nucleus of the amygdala (CeA) [t(6) = 1.82, P = 0.12], or BNST [t(10) = 2.15, P = 0.06], indicating the absence of tonic GlyR currents in these regions. Data are presented as means ± SEM (n = 7–43 neurons from 5–18 mice); *P < 0.05.

Fig. S2.

Strychnine does not affect the amplitude or frequency of spontaneous postsynaptic currents. To determine the possible role of strychnine in inhibiting postsynaptic currents, spontaneous postsynaptic currents were measured in the NAc before and after the administration of strychnine (100 nM and 1 µM). Gabazine (10 µM) and NBQX (10 µM) were also used to block GABA_AR- and AMPA receptor-mediated postsynaptic currents. There was a significant effect of NBQX on the amplitude [F(6, 62) = 74.13, P < 0.0001] (A) and frequency [F(6, 62) = 6.193, P < 0.0001] (B) of postsynaptic currents. However, neither concentration of strychnine had a significant effect on the amplitude or frequency of spontaneous postsynaptic currents (all P > 0.05). Data are presented as mean ± SEM (n = 7–14 neurons from two to six mice; *P < 0.05 compared with aCSF; ^#P < 0.05 compared with 100 nM strychnine; ^†P < 0.05 compared with 1 µM strychnine).

Fig. S3.

Strychnine-induced shifts in the holding current are mainly mediated by GlyRs. To determine whether strychnine-induced shifts in the holding current were the result of α7 nAChR antagonism, the effects of the α7-specific nAChR inhibitor methyllycaconitine (MLA) (10 nM or 100 nM) on the holding current were measured in the NAc. Both 100 nM methyllycaconitine [t(11) = 2.551, P = 0.027] and 1 μM strychnine [t(42) = 6.872, P < 0.0001] produced significant shifts in the holding current. There was a significant effect of drug on shifts in the holding current [F(2, 60) = 6.87, P = 0.0021], and post hoc analysis with Bonferroni correction revealed that 1 μM strychnine produced a much larger shift in the holding current than either concentration of methyllycaconitine (both P < 0.05). Data are presented as means ± SEM (n = 8–43 neurons from 3–18 mice; *P < 0.05 between drug groups, ^#P < 0.05 compared with baseline holding current).

Fig. S5.

Strychnine block of tonic GlyR currents alters the excitability of neurons. Current-clamp experiments were performed before and after the application of 1 µM strychnine to determine the effects of GlyR inhibition on the excitability of neurons in the NAc. (A) Strychnine application hyperpolarized the RMP of neurons in the NAc [t(14) = 4.494, P = 0.0005]. (B and C) Strychnine also increased the rheobase of neurons in this area [t(12) = 2.378, P = 0.0349] (B), although there was no significant effect on the input resistance of these neurons [t(14) = 0.1542, P = 0.88] (C). Data are presented as mean ± SEM (n = 13–15 neurons from five mice; *P < 0.05; n.s., not significant).

Fig. 3.

Tonic GlyR currents in forebrain are mediated by α3 subunit-containing GlyRs. The effects of strychnine on baseline holding currents were measured and compared in Glra2^−/Y and Glra3^−/− mice and their WT littermates. Whole-cell recordings were performed in the striatum and PFC, regions in which significant tonic GlyR currents were identified in C57/BL6J mice. (A) Sample tracings demonstrating the effects of 1 μM strychnine on the holding currents of MSNs in the NAc of Glra3^+/+ (Left) and Glra3^−/− (Right) mice. (B and C) Strychnine-induced shifts in the holding currents of MSNs in the NAc (n = 4–10 cells from three to seven mice) (B) and dorsal striatum (n = 6–9 cells from four to six mice) (C) of Glra3^+/+ mice, indicating the presence of a tonic GlyR current [NAc: t(14) = 2.93, P = 0.011; dorsal striatum: t(11) = 3.58, P = 0.004]; however, no significant shift was observed in Glra3^−/− mice. Strychnine-induced shifts in holding current were detected in MSNs in the NAc (B) and dorsal striatum (C) of both Glra2^+/Y and Glra2^−/Y mice. The amplitude of the tonic GlyR current was not significantly different between the two genotypes in either region of the striatum [NAc: t(10) = 0.12, P = 0.91; dorsal striatum: t(16) = 0.24, P = 0.81]. (D) In the medial prefrontal cortex, applications of 1 μM strychnine produced shifts in the holding currents of layer II/III pyramidal neurons in Glra3^+/+ mice; however, as in the striatum, this effect of strychnine was absent in neurons from Glra3^−/− mice [t(15) = 2.40, P = 0.029]. GlyR tonic currents were detected in neurons recorded from both Glra2^+/Y and Glra2^−/Y mice, and the amplitude of the strychnine-induced currents was not significantly different between the genotypes [t(15) = 0.97, P = 0.35]. In contrast to layer II/III, there were no significant effects of strychnine on the baseline holding currents of pyramidal neurons in layer V/VI in any of the mouse genotypes tested, suggesting the lack of an appreciable tonic GlyR current in this cortical layer. Data represent means ± SEM (n = 6–9 neurons from three to seven mice; *P < 0.05 compared with WT littermates).

Fig. 4.

Large-amplitude glycine-activated currents are mediated by α2 subunit-containing GlyRs in brain. (A) Examples of whole-cell GlyR currents elicited by exogenous applications of glycine recorded from MSNs in the NAc of Glra2^−/Y and Glra3^−/− and WT mice. (B) Glycine (300 μM) produced currents of significantly reduced amplitude in MSNs in the NAc of Glra2^−/Y compared with Glra2^+/Y mice [t(17) = 3.03, P = 0.007]. In contrast, the amplitude of current in MSNs elicited by 300 μM glycine was not significantly different between Glra3^+/+ and Glra3^−/− mice [t(19) = 0.29, P = 0.77]; n = 8–11 cells from three to four mice. (C) Although no tonic GlyR currents were detected in the BNST, GlyR currents were measured in response to exogenous applications of glycine. As in the striatum, the amplitude of current in BNST neurons in response to 300 μM glycine was significantly reduced in Glra2^−/Y compared with Glra2^+/Y mice [t(21) = 2.67, P = 0.014] but was not significantly different between neurons recorded from Glra3^+/+ and Glra3^−/− mice [t(7) = 0.95, P = 0.37]; *P < 0.05; n = 4–12 cells from two to four mice. (D) Similarly, in the PFC, the amplitude of glycine-activated currents in Glra2^−/Y mice was significantly reduced compared with Glra2^+/Y mice in pyramidal neurons in both layer II/III [t(30) = 2.48, P = 0.019] and layer V/VI [t(20) = 4.38, P = 0.0003]; however, unlike in striatum and BNST, the amplitude of glycine-activated currents was also significantly reduced in Glra3^−/− compared with Glra3^+/+ mice in both layer II/III [t(28) = 3.42, P = 0.0019] and layer V/VI [t(26) = 2.41, P = 0.024]; *P < 0.05; n = 9–17 cells from three to five mice. Data are presented as means ± SEM.