Presence of ethanol-sensitive and ethanol-insensitive glycine receptors in the ventral tegmental area and prefrontal cortex in mice

Abstract

Background and purpose: Glycine receptors composed of α1 and β subunits are primarily found in the spinal cord and brainstem and are potentiated by ethanol (10-100 mM). However, much less is known about the presence, composition and ethanol sensitivity of these receptors in higher CNS regions. Here, we examined two regions of the brain reward system, the ventral tegmental area (VTA) and the prefrontal cortex (PFC), to determine their glycine receptor subunit composition and sensitivity to ethanol.

Experimental approach: We used Western blot, immunohistochemistry and electrophysiological techniques in three different models: wild-type C57BL/6, glycine receptor subunit α1 knock-in and glycine receptor subunit α2 knockout mice.

Key results: Similar levels of α and β receptor subunits were detected in both brain regions, and electrophysiological recordings demonstrated the presence of glycine-activated currents in both areas. Sensitivity of glycine receptors to glycine was lower in the PFC compared with VTA. Picrotoxin only partly blocked the glycine-activated current in the PFC and VTA, indicating that both regions express heteromeric αβ receptors. Glycine receptors in VTA neurons, but not in PFC neurons, were potentiated by ethanol.

Conclusion and implications: Glycine receptors in VTA neurons from WT and α2 KO mice were potentiated by ethanol, but not in neurons from the α1 KI mice, supporting the conclusion that α1 glycine receptors are predominantly expressed in the VTA. By contrast, glycine receptors in PFC neurons were not potentiated in any of the mouse models studied, suggesting the presence of α2/α3/α4, rather than α1 glycine receptor subunits.

Keywords: PFC; VTA; ethanol; glycine receptors; reward system; subunit composition.

FIGURE 1

Presence of glycine receptors in the mesolimbic circuit. (a) Western blot shows the presence of the α subunits (MW 48 kDa) of glycine receptors (α GlyR) in the VTA and PFC. (b) Graph represents the normalized signal of α GlyRs to Gβ. Data shown are means ± SEM; n = 5 (from duplicates). ^* P < 0.05, significantly different as indicated; unpaired Student's t‐test. (c, d) Low magnification image of VTA and PFC (25×), respectively, in coronal brain slices from a GlyT2‐GFP mouse with TH (red) and anti‐GFP (green) signals in the VTA and MAP2 (red) and anti‐GFP (green) signals in the PFC. GlyT2‐positive projections appear in both regions. (e, f) High magnification image of the VTA and PFC (63×), respectively, in brain slices of a GlyT2‐GFP mouse showing GlyT2‐GFP (green) and GlyR α (magenta) signals. Confocal images of the VTA and PFC were replicated in three different mice

FIGURE 2

Presence of glycinergic synaptic activity in VTA and PFC in WT mice. (a, b) Representative traces of 1 min of synaptic activity in the VTA and PFC, respectively. The first trace shows the total mPSC, the second shows the isolated mIPSC mediated by glycine receptors and the third trace shows the blockade of the glycinergic mIPSC by strychnine (STN). (c) Average glycinergic mIPSC in the VTA and PFC. (d, e) Percentage of neurons recorded with and without glycinergic activity in the VTA and PFC, respectively. Graph shows the average frequency (f), amplitude (g) and the average decay constant (10–90%) (h) of the glycinergic currents in the VTA and PFC. Graph shows the average cumulative probability for interevent interval (i), amplitude (j) and decay constant (k). Data presented as means ± SEM; n = 8 neurons for VTA and n = 15 neurons for PFC. ^* P < 0.05, significantly different from VTA; unpaired Student's t test for (f)–(h), and Kolmogorov–Smirnov test for (i)–(k) where significance was achieved for all three parameters

FIGURE 3

Glycine‐activated currents in dissociated neurons from VTA and PFC in WT mice. (a) Representative traces of currents activated with 1 to 1000 μM glycine in neurons from the VTA. (b) Glycine concentration–response curve normalized to the maximum response. (c) Representative traces of currents activated with 10 to 1000 μM glycine in neurons from the PFC. (d) Glycine concentration–response curve normalized to the maximum response. Data presented are means ± SEM. n = 15 from three mice for VTA and n = 11 from six mice for PFC. The curves were significantly different (P < 0.05); Student's t test

FIGURE 4

Inhibition of glycine receptors in VTA and PFC neurons by picrotoxin (PTX) in WT mice. (a, b) The blocking action of PTX (20 μM) on glycine currents mediated in neurons from the VTA and PFC, respectively. The traces were evoked with 30 μM glycine for VTA and 50 μM for PFC. (c) Graph shows the percentage of inhibition of the current from control after application of PTX for VTA and PFC. Data presented are means ± SEM. n = 22 for VTA and n = 10 for PFC. % inhibition = 100 − (a × 100∕b)

FIGURE 5

Effects of intracellular GTP‐γ‐S on glycine receptors in VTA and PFC in WT mice. (a, b) Currents in activated glycine receptors were assayed using 15 and 30 μM glycine every 3 min for VTA and PFC neurons, respectively, with intracellular dialysis of GTP‐γ‐S. Traces show only the first and last evoked currents (at 1 and 15 min). (c) Time course of GTP‐γ‐S mediated potentiation of glycine receptors in VTA and PFC. Data represented as mean ± SEM; n = 11 neurons for VTA and n = 10 neurons for PFC. ^* P < 0.05, VTA significantly different from PFC; two‐way ANOVA and Bonferroni post hoc test. ^# P < 0.05, significantly different from the control in the VTA curve; one‐way ANOVA

FIGURE 6

Effects of ethanol on glycine receptors of VTA and PFC from WT and α1 KI mice. (a, c) Representative traces of glycine‐activated currents and the effects of 1 to 100 mM ethanol in neurons of the VTA from WT and α1 KI mice, respectively. (b, d) Representative traces of glycine‐activated currents and the effects of 10 to 100 mM ethanol in neurons of the PFC from WT and α1 KI mice, respectively. The currents were activated with 7 and 30 μM glycine for VTA and PFC, respectively. (e) Graph shows ethanol potentiation (1–100 mM) of glycine receptors of VTA from WT and α1 KI mice. One outlier was found and excluded from this graph. (f) Graph shows ethanol potentiation (10–100 mM) of glycine receptors in PFC from WT and α1 KI mice. Data presented are means ± SEM; n = 10 for 1 mM, n = 10 for 5 mM, n = 16 for 10 mM, n = 15 for 50 mM and n = 14 for 100 mM in VTA of WT mice; n = 7 for 1 mM, n = 7 for 5 mM, n = 8 for 10 mM, n = 8 for 50 mM and n = 8 for 100 mM in VTA of α1 KI mice. n = 6 for 10 mM, n = 6 for 50 mM and n = 6 for 100 mM in PFC of WT mice; n = 14 for 10 mM, n = 14 for 50 mM and n = 14 for 100 mM in PFC α1 KI mice. ^* P < 0.05, significant effects of the different ethanol concentrations between the different potentiation curves from different brain regions; two‐way ANOVA and Bonferroni post hoc test. ^# P < 0.05, significant effects of the different ethanol concentrations within the same potentiation curve of the same brain region; one‐way ANOVA

FIGURE 7

Effects of ethanol on glycine receptors of VTA and PFC from α2 KO mice. (a) Representative traces of currents from VTA neurons evoked with 15 μM of glycine and the effect of 1–100 mM of ethanol. (b) Representative traces of PFC neurons evoked with 40 μM of glycine and the effect of 10–100 mM of ethanol. (c) Graph shows the effect of several concentrations of ethanol on the amplitude of glycine‐activated currents for VTA and PFC of α2 KO mice. Data presented are means ± SEM; n = 9 for 1 mM, n = 9 for 5 mM, n = 21 for 10 mM, n = 20 for 50 mM and n = 18 for 100 mM of VTA. n = 3 for 10 mM, n = 3 for 50 mM and n = 3 for 100 mM of PFC. ^# P < 0.05, significant effects of the different ethanol concentrations in VTA; one‐way ANOVA