The persistent effects of predator odor stressor enhance interoceptive sensitivity to alcohol through GABAA receptor adaptations in the prelimbic cortex in male, but not female rats

Abstract

Background: Traumatic stress is associated with high rates of problematic alcohol use, but how the persistent effects of trauma impact sensitivity to alcohol remain unknown. This study examined the persistent effects of traumatic stress exposure on sensitivity to alcohol and underlying neurobiological mechanisms in rats.

Methods: Male (N=98) and female (N=98) Long-Evans rats were exposed to the predator odor TMT, and two weeks later, molecular, neuronal, and behavioral sensitivity to alcohol were assessed. Next, rats were trained to discriminate alcohol from water (male N=70; female N=56), and the impact of TMT on interoceptive sensitivity to alcohol and the alcohol-like effects of systemic GABA_A receptor activation were evaluated. Lastly, functional involvement of GABA_A and NMDA receptors in the prelimbic cortex (PrL) and the anterior insular cortex (aIC) was investigated.

Results: TMT exposure sex-dependently altered PrL Gabra1, and elevated aIC Grin2b and Grin2c in males. TMT increased PrL c-Fos in males, which was attenuated by alcohol administration. Alcohol-induced locomotor and startle response effects were attenuated in the TMT group in both sexes. TMT exposure potentiated interoceptive sensitivity to alcohol in males but not in females, and this effect was driven by GABA_A receptors in the PrL. Greater stress reactivity during TMT exposure was associated with higher interoceptive sensitivity to alcohol, and alcohol exposure history was linked to a heightened stress response to TMT in males.

Conclusions: Traumatic stress increased interoceptive sensitivity to alcohol in males, but not females, through PrL GABA_A receptor adaptations, potentially enhancing the stimulatory, and by extension the rewarding, effects of alcohol.

Keywords: 2,5-dihydro-2,4,5-trimethylthiazoline; PTSD; alcohol use disorder; anterior insular cortex; drug discrimination; individual differences.

Figure 1–. TMT exposure sex-dependently altered Gabra1 expression in PrL and upregulated NMDA receptor subunits in aIC in males.

For NMDA receptor sub-unit gene expression in the PrL (A-D), there was no effect of TMT exposure or sex. For the GABA_A α−1 sub-unit gene expression (E), TMT exposure decreased expression in males but increased expression in females (interaction: F(1, 28)=18.12, p=0.0002). Other GABA_A receptor sub-unit genes (F-I) were not affected by TMT exposure or sex. For NMDA receptor sub-unit gene expression in the aIC, there was no effect of TMT exposure or sex on (A) Grin1 and (B) Grin2a levels. TMT exposure increased levels of (L) Grin2b and (M) Grin2c in males, but not in females (interaction: F(1, 33)=5.10, p=0.03). GABA_A α−1 subunit gene expression (N-R) was not affected by TMT exposure or sex. Male/CTRL n=7–9, Male/TMT n=6–10, Female/CTRL n=7–10, Female/TMT n=8–9. *p≤0.05.

Figure 2–. TMT exposure increased PrL c-Fos expression in males, which was not observed in rats pre-treated with alcohol.

For c-Fos positive cells/mm² in the PrL in males (A), TMT exposure induced an increase but alcohol did not affect c-Fos expression (TMT: F(1, 22)=12.70, p=0.002; TMT × alcohol: F(1, 22)=6.83, p=0.02). In the PrL in females (B), TMT exposure nor alcohol affected c-Fos positive cell number/mm². In the aIC in males (C) and females (D), TMT exposure nor alcohol administration affected c-Fos expression. (E) Regions of quantification for the PrL (square) and the aIC (oval). (F) Representative images of Fig. 2A data – c-Fos in PrL of male rats. Scale bar is 200 μm. Male/CTRL n=6–8, Male/TMT n=6–7, Female/CTRL n=5–6, Female/TMT n=5–8. ***p≤0.001.

Figure 3–. Alcohol produced locomotor and startle response effects in the control group, but not in the TMT-exposed group.

For the open field test in males (A), alcohol administration (alcohol: F(1, 39)=10.63, p=0.002) decreased distance traveled in the control group (t(18)=4.31, p=0.0004), but not in the TMT group. In the open field test in females (B), alcohol administration (alcohol: F(1, 41)=4.64, p=0.04) resulted in increased distance traveled in the control group (t(20)=2.08, p=0.05), but not in the TMT group. For the acoustic startle response in males (C), alcohol administration (alcohol: F(1, 40)=11.65, p=0.002) decreased the average peak startle response in the control group (t(19)=2.79, p=0.01), but not in the TMT group. For the acoustic startle response in females (D), alcohol administration (alcohol: F(1, 37)=5.35, p=0.03) resulted in decreased average peak startle response in controls (t(18)=2.18, p=0.04), but not in the TMT group. Male/CTRL n=9–11, Male/TMT n=11–12, Female/CTRL n=10–12, Female/TMT n=10–11. *p≤0.05.

Figure 4–. TMT exposure potentiated interoceptive sensitivity to alcohol in male but not female rats.

Prior to TMT exposure, in males (A), there was a main effect of alcohol on discrimination scores (F(1, 59)=262.2, p<0.0001) as would be expected confirming that the interoceptive effects of alcohol were guiding behavior, and no difference between the TMT and control group. There was no effect on locomotor rate for alcohol administration or group (TMT vs. CTRL). Prior to TMT exposure in females, there was a main effect of alcohol on discrimination scores (F(1, 31)=211.8, p<0.0001) and no difference between groups. Likewise for locomotor rate (D), there was a main effect of alcohol (F(1, 31)=6.48, p=0.02), but no group differences. Following a 2-week period after the TMT exposure, (E) male rats exposed to TMT showed potentiated sensitivity to the interoceptive effects of alcohol (TMT: F(1, 59)=11.32, p=0.001; alcohol dose: F(2.38, 140.3)=63.83, p<0.0001; TMT × alcohol dose: F (3, 177) = 3.09, p = 0.03) specifically at the 0.5 (p=0.02) and 1.0 g/kg (p=0.009) alcohol doses. (F) There was no effect of TMT exposure or alcohol dose on locomotor rate. In females after TMT exposure, (G) there was a main effect of alcohol dose (F(3, 93)=27.03, p<0.0001), but no effect of TMT exposure on discrimination scores. For locomotion in females, (H) there was a main effect of alcohol dose (F(3, 93)=11.77, p<0.0001), no effect of TMT exposure, but there was a significant alcohol dose by TMT exposure interaction (F(3, 93)=3.70, p=0.01). Locomotion was decreased at every alcohol dose compared to vehicle (^#p≤0.05) in controls, but not in the TMT group. For pentobarbital substitution in males, (I) there was no effect of TMT exposure on discrimination scores, but (J) the TMT group showed a decreased locomotor rate compared to controls (t(32)=2.45, p=0.02). Pentobarbital did not produce substitution for the alcohol training dose in males. For pentobarbital substitution in females, there was no effect of TMT exposure on (K) discrimination scores or (L) locomotor rate. Pentobarbital produced substitution for the alcohol training dose in females. The dotted lines on panels E, G, I, and K represent the mean (groups combined because this was prior to the TMT exposure) of the alcohol session discrimination scores prior to the TMT exposure taken from panel A and C, receptively, and serve as a comparator for full substitution of the 2 g/kg alcohol training dose. For males, the mean was 6.41 ± 0.42 SEM, and for females the mean was 6.27 ± 0.23 SEM. Discrimination scores reflect the degree of substitution for the 2.0 g/kg alcohol training dose, or the 2.0 g/kg alcohol-like effects. Alcohol dose response (A-H) sample sizes: Male/CTRL n=21, Male/TMT n=40, Female/CTRL n=18, Female/TMT n=15. Pentobarbital substitution (I-L) sample sizes: Male/CTRL n=15, Male/TMT n=19, Female/CTRL n=9, Female/TMT n=9. Only rats that met criteria for discrimination before and after the TMT exposure were included in analyses. Discrimination was defined as an alcohol session discrimination score ≥ 2 the water session score. *p≤0.05. **p≤0.01. ****p≤0.0001. ^#p≤0.05 relative to vehicle in the CTRL group.

Figure 5–. TMT exposure potentiated the alcohol-like stimulus effects of GABA_A receptor agonism in the prelimbic cortex in male rats.

(A) Representative image and injector tip placement from individual rats with accurate placements in the prelimbic cortex (PrL). Site-specific injection of muscimol into the PrL of male rats resulted in (B) greater discrimination scores (t(9.05)=2.10, p=0.03) and (C) greater locomotor rate (t(14.33)=2.34, p=0.02) in the TMT group compared to controls. Site-specific injection of MK-801 into the PrL did not produce group differences for (D) discrimination scores, but did show potentiation for (E) locomotor rate (t(8.06)=5.86, p=0.0002) in the TMT group compared to controls. (F) Representative image and injector tip placement from individual rats with accurate placements in the anterior insular cortex (aIC). Site-specific injection of muscimol into the aIC of male rats resulted in no group difference for (G) discrimination scores or (H) locomotor rate. Discrimination scores reflect the degree of substitution for the 2.0 g/kg alcohol training dose, or the 2.0 g/kg alcohol-like effects. PrL/Muscimol: CTRL n=5, TMT=12. PrL/MK-801: CTRL n=3, TMT n=9. aIC/Muscimol: CTRL n=6, TMT n=6. 5 rats from the PrL and 1 from the aIC were removed from analyses due to incorrect bilateral cannulae placements. *p≤0.05. ***p≤0.001.

Figure 6–. Heightened stress reactivity to TMT exposure is associated with higher interoceptive sensitivity to alcohol, but not with the alcohol-like effects of pentobarbital.

(A) The ratio of time spent digging over time spent immobile during the 15-min TMT exposure was calculated as the D/I ratio. (B) Rats with a D/I ratio < 1 were assigned to the TMT-1 subgroup, and rats with a D/I ratio > 1 were assigned to the TMT-2 subgroup. (C) The post-TMT alcohol dose response curve was reanalyzed using the TMT subgroup. There was a main effect of group (F(2, 58)=10.20, p=0.0002), alcohol dose (F(2.43, 140.9)=74.30, p<0.0001), and group × alcohol dose interaction (F(6, 174)=2.90, p=0.01). (C) The TMT-2 group showed higher discrimination scores compared to the control group at the 1.0 g/kg alcohol dose. (D) There was no effect of alcohol dose or group for locomotor rate. (E) At the 1.0 g/kg alcohol dose, the TMT-2 group showed higher discrimination scores compared to both TMT-1 and CTRL groups (group: F(2, 58)=8.05, p=0.0008), but there were no group differences in locomotor rate (F). There were no group differences for pentobarbital (G) discrimination scores or (F) locomotor rate. Discrimination scores reflect the degree of substitution for the 2.0 g/kg alcohol training dose, or the 2.0 g/kg alcohol-like effects. Alcohol dose response sample size: CTRL n=21, TMT-1 n=27, TMT-2 n=13. Pentobarbital sample size: CTRL n=15, TMT-1 n=12, TMT-2 n= 7. *p≤0.05, **p≤0.01, ****p≤0.0001 for TMT-2 compared to controls. ^#p≤0.05 for TMT-2 compared to TMT-1.

Figure 7–. TMT exposure increases digging and immobility behavior in male and female rats: comparing stress-reactivity in alcohol-experienced and alcohol-naïve cohorts.

During the 15-min TMT exposure, rats engaged in both digging (pushing bedding with the forepaws) and immobility (captures freezing behavior) behaviors. (A) In males rats, the TMT group spent more time digging bedding (F(1, 164)=166.0, p<0.0001) during early time bins compared to the control group in both the alcohol-experienced and alcohol-naïve rats. In the TMT group, alcohol-experienced rats engaged in more digging behavior (F(1, 164)=15.80, p=0.0001) than the alcohol-naïve rats at time bin 9. (B) In female rats, the TMT group spent more time digging bedding (F(1, 150)=130.5, p<0.0001) during early time bins compared to the control group in both the alcohol-experienced and alcohol-naïve rats. There were no differences between the alcohol-experienced and alcohol-naïve groups. (C) In male rats, the TMT group spent more time immobile (F(1, 160)=51.50, p<0.0001) than the control group in the alcohol-naïve group, but not in the alcohol-experienced group. In the TMT groups, the alcohol-experienced rats spent less time immobile (F(1, 160)=10.31, p=0.0016) at time bins 12 and 15 compared to the alcohol-naïve rats. (D) In female rats, the TMT groups spent more time immobile (F(1, 150)=69.14, p<0.0001) compared to the control groups for both alcohol-experienced and alcohol-naïve rats. There was a main effect of alcohol-experience (F(1, 150)=13.75, p=0.0003), but post-hoc tests showed no differences between the alcohol-experienced and the alcohol naïve rats. CTRL/Male/Alcohol-Experienced n=27, TMT/Male/Alcohol-Experienced n=43, CTRL/Male/Alcohol-naïve n=50, TMT/Male/Alcohol-naïve n=48, CTRL/Female/Alcohol-Experienced n=28, TMT/Female/Alcohol-Experienced n=28, CTRL/Female/Alcohol-naïve n=49, TMT/Female/Alcohol-naïve n=49. *p ≤ 0.05 for TMT vs. control groups. ^#p ≤ 0.05 for alcohol-experienced vs. alcohol-naïve groups.