Effects of monoamines on the intrinsic excitability of lateral orbitofrontal cortex neurons in alcohol-dependent and non-dependent female mice

Abstract

Changes in brain reward and control systems of frontal cortical areas including the orbitofrontal cortex (OFC) are associated with alcohol use disorders (AUD). The OFC is extensively innervated by monoamines, and drugs that target monoamine receptors have been used to treat a number of neuropsychiatric diseases, including AUDs. Recent findings from this laboratory demonstrate that D2, α2-adrenergic and 5HT_1A receptors all decrease the intrinsic excitability of lateral OFC (lOFC) neurons in naïve male mice and that this effect is lost in mice exposed to repeated cycles of chronic intermittent ethanol (CIE) vapor. As biological sex differences may influence an individual's response to alcohol and contribute to the propensity to engage in addictive behaviors, we examined whether monoamines have similar effects on lOFC neurons in control and CIE exposed female mice. Dopamine, norepinephrine and serotonin all decreased spiking of lOFC neurons in naïve females via activation of G_iα-coupled D2, α2-adrenergic and 5HT_1A receptors, respectively. Firing was also inhibited by the direct GIRK channel activator ML297, while blocking these channels with barium eliminated the inhibitory actions of monoamines. Following CIE treatment, evoked spiking of lOFC neurons from female mice was significantly enhanced and monoamines and ML297 no longer inhibited firing. Unlike in male mice, the enhanced firing of neurons from CIE exposed female mice was not associated with changes in the after-hyperpolarization and the small-conductance potassium channel blocker apamin had no effect on current-evoked tail currents from either control or CIE exposed female mice. These results suggest that while CIE exposure alters monoamine regulation of OFC neuron firing similarly in males and female mice, there are sex-dependent differences in processes that regulate the intrinsic excitability of these neurons.

Keywords: Chronic intermittent ethanol exposure; Female mice; G protein-coupled inwardly rectifying potassium channel; Intrinsic excitability; Lateral orbitofrontal cortex; Monoamine.

Figure 1

Dopamine decreases current-induced spiking of lOFC neurons from female mice via D2 receptors. (A) Representative traces show decreased action potential (AP) spiking in the presence of 50 μM DA as compared with control. The graph shows the effect of dopamine on AP spiking (mean ± SEM) induced by a series of current injections (40–220 pA) in lOFC neurons. In comparison with control, DA produced a significant decrease in basal firing rates of lOFC neurons (two-way repeated measures ANOVA: main effect of DA, F_(1,11) = 6.134, *p=0.03; N=12). (B) The D1 receptor blocker SCH23390 (10 μM) itself had no effect on DA-induced inhibition (q=0.79, p>0.05). A significant reduction of spiking was observed when a combination of DA and SCH23390 was applied (two-way ANOVA, F_(2,12) = 9.38, q=3.293, *p=0.0035; N=7). (C) Sulpiride (10 μM) by itself did not alter AP spiking (q=0.0908, p>0.05) but prevented the inhibition of firing by DA (two-way ANOVA, F_(2,26) = 0.1226, q=0.467, p>0.05; N=14). (D) The D2 receptor agonist quinpirole (5 μM) significantly decreased AP spiking as compared with control (two-way ANOVA, F_(1,10) = 17.95, **p=0.0017; N=10).

Figure 2

Acute application of norepinephrine or serotonin decreases current-induced spiking of lOFC neurons from female mice via α2-adrenergic or 5-HT_1A receptors, respectively. Representative traces show decreased action potential (AP) spiking in the presence of 50 μM NE or 5-HT as compared with control. (A) Effect of NE on AP spiking (mean ± SEM) induced by a series of current injections (40–220 pA) in lOFC neurons. NE (50 μM) produced a significant decrease in current-evoked AP spiking (two-way ANOVA, F_(1,7) = 6.223, *p=0.04; N=8). (B) The α2-adrenergic receptor blocker yohimbine (100 nM) by itself did not alter cell firing (two-way ANOVA, F_(2,12) = 0.37, q=0.5927, p>0.05; N=7), but prevented NE-induced inhibition of spiking (no difference in spiking between yohimbine alone and yohimbine + NE; q=0.2486, p>0.05). (C) The α1-adrenergic receptor blocker naftopidil (10 μM) alone did not alter spike firing (two-way ANOVA, F_(2,20) = 5.12, q=1.332, p>0.05; N=11) or prevent NE inhibition of spike firing (naftopidil alone versus naftopidil + NE; q=3.186, **p<0.01). (D) Effect of 5-HT on AP spiking (mean ± SEM) induced by a series of current injections (40–220 pA). 5-HT (50 μM) produced a significant decrease in current-evoked spiking (two-way repeated measures ANOVA, F_(1,7) = 7.698, *p=0.0275; N=8). (E) The 5-HT_1A receptor antagonist WAY100635 (100 nM) alone had no effect on AP firing of lOFC neurons (control versus WAY100635, q=2.368, p>0.05) but attenuated spike inhibition by 50 μM 5-HT (no difference in number of spikes between WAY100635 alone WAY100365 + 5-HT; two-way ANOVA, F_(2,12) = 9.688, q=2.03, p>0.05; N=7). (F) The 5-HT2 receptor agonist α-methyl-5HT (50 μM) did not affect AP spiking (two-way ANOVA, F_(1,7) = 1.004, p=0.3496; N=8).

Figure 3

Low concentrations of monoamines decrease current-induced spiking of lOFC neurons from female mice when monoamine transporters are suppressed. Graphs show effect of each monoamine on AP spiking (mean ± SEM) of lOFC neurons induced by a series of current injections (40–220 pA) in the absence (A–C) or presence (D–F) of nomifensine. Without nomifensine, low concentrations (1 and 100 nM) of DA (A; two-way ANOVA: main effect of DA, F_(2,20) = 2.107, p=0.1478), NE (B; two-way ANOVA: main effect of NE, F_(2,10) = 1.181, p=0.3464) or 5HT (C; two-way ANOVA: main effect of 5HT, F_(2,10) = 1.151, p=0.3549) had no effect on AP spiking of lOFC neurons. In the presence of 10 μM nomifensine, DA (D; 1 nM, two-way ANOVA, F_(3,27) = 14.78, q=3.971, **p<0.001; 100 nM (q=5.152, ***p<0.0001)), NE (E; 1 nM, two-way ANOVA, F_(3,27) = 10.5, q=2.843, *p<0.05; 100 nM q=5.271, ***p<0.0001) and 5HT (F; 1 nM, two-way ANOVA, F_(3,21) = 11.98, q=3.597, **p<0.01; 100 nM q=4.447, ***p<0.001) inhibited spike firing. Nomifensine itself did not affect spiking (q=0.1431, p>0.05).

Figure 4

Effects of GIRK channel modulators on current-induced spiking of lOFC neurons from naïve female mice. Graphs show the effect of each drug on AP spiking (mean ± SEM) induced by a series of current injections (40–220 pA) in lOFC neurons. (A) The GIRK activator ML297 (10 μM) produced a significant decrease in current-evoked spiking of lOFC neurons (two-way repeated measures ANOVA, F_(1,15) = 35.99, ***p<0.0001; N=16). (B) The GIRK channel inhibitor barium (100 μM) caused a small but significant increase in spiking (two-way repeated measures ANOVA, F_(1,20) = 33.07, ***p<0.0001; N=19). In the presence of barium, the monoamines (50 μM) no longer inhibited spike firing (C; DA, two-way repeated measures ANOVA, F_(2,10) = 8.951, q=0.1142, p>0.05; N=6; D; NE, two-way repeated measures ANOVA, F_(2,10) = 0.0063, q=1.135, p>0.05; N=6; E; 5HT, two-way repeated measures ANOVA, F_(2,12) = 7.471, q=0.8876, p>0.05; N=7).

Figure 5

CIE exposure of female mice enhances the intrinsic excitability of lOFC neurons without altering the amplitudes of after-hyperpolarization and outward tail currents. (A) Representative traces show increased action potential (AP) spiking in mice withdrawn for three days (3D-WD) and seven days (7D-WD) following CIE treatment as compared with air control. Graph shows number of spikes (mean ± SEM) of lOFC neurons from air and CIE treated female mice plotted against a series of current injections (40–220 pA). In comparison with air control (N=72), basal firing rates of lOFC neurons were significantly enhanced in 3D-WD (N=45) and 7D-WD (N=15) CIE groups (two-way ANOVA: main effect of CIE F_(2,128) = 16.83, ***p<0.0001; post-hoc comparison: air vs 3D-WD CIE, q=5.773, p<0.0001 and air vs 7D-WD CIE, q=2.252, p<0.05). (B) CIE had no significant effect on AHP amplitude as compared to air controls (one-way ANOVA, F_(2,211) = 0.7815, p=0.4591; post-hoc comparison: q=0.2241 for air vs 3D-WD CIE and q=1.243 for air vs 7D-WD CIE). (C–E) Representative traces show total tail current amplitudes in the absence (black) and presence (gray) of the SK channel blocker apamin (100 nM) in air control, 3D-WD and 7D-WD CIE groups, respectively. Graphs show comparison of the peak outward tail current amplitudes (mean ± SEM) in response to depolarizing voltage steps (400 ms, from −20 to +30 mV with 10 mV between steps) in the absence and presence of apamin. The SK channel blocker apamin did not significantly alter the tail current amplitude of lOFC neurons in air controls (C; two-way ANOVA; F_(1,16) = 3.264, p=0.0897; N=17), 3D-WD CIE (D; two-way ANOVA; F_(1,17) = 3.634, p=0.0737; N=19) and 7D-WD CIE (E; two-way ANOVA; F_(1,14) = 3.196, p=0.0955; N=15) groups. (F) Graph shows number of spikes (mean ± SEM) of lOFC neurons from control female mice in the absence and presence of apamin. Apamin did not affect lOFC firing as compared to baseline recordings without apamin (two-way ANOVA; F_(1,12) = 0.467, p=0.5074; N=13).

Figure 6

CIE exposure blunts the inhibitory effects of monoamines. Summary graphs show number of spikes at the highest current injection (220 pA) expressed as percent change from control baseline (without drug) in response to each treatment condition. (A) DA significantly inhibited AP spiking of lOFC neurons in air-treated mice (two-tailed paired t-test, t(13) = 3.877, **p=0.0019) but not those in the 3D-WD group (two-tail paired t-test, t(7) = 1.17, p=0.2786). (B) NE significantly reduced evoked AP spiking in air control mice (two-tailed paired t-test, t(11) = 3.772, **p=0.0031), but not those in the 3D-WD group (two-tailed paired t-test, t(8) = 1.614, p=0.1453). (C) 5HT caused a significant reduction in spike firing in air control mice (two-tailed paired t-test, t(11) = 2.466, *p=0.0313), but not those in the 3D-WD group (two-tailed paired t-test, t(7) = 0.811, p=0.4441). (D) The GIRK channel activator ML297 (10 μM) significantly reduced firing in lOFC neurons from air-exposed mice (two-tailed paired t-test, t(16) = 6.648, ***p<0.0001) but not those in the 3D-WD group (two-tailed paired t-test, t(13) = 0.8796, p=0.395).