3.1.1. Ethanol Self-Administration
In adulthood, adolescent CORT-exposed and control rats were trained to self-administer ethanol or sucrose. Factorial ANOVA analysis of overall numbers of reinforcers earned, active lever presses, and magazine entries across self-administration sessions as a function of adolescent treatment (H
2O vs. CORT) and reinforcer type (sucrose vs. EtOH), indicated no treatment × reinforcer interactions (all
p > 0.05). However, there were significant effects of time and time × reinforcer interactions for reinforcers earned (F
(19, 8) = 58.22,
p < 0.001; F
(19, 8) = 43.99,
p < 0.001), indicating that sucrose self-administration (
Figure 1B) was acquired more quickly for both treatment groups. Inspection of the ethanol self-administration data indicated that once animals had acquired stable self-administration (e.g., the final 3 days of testing), there was a separation between the CORT-exposed and control groups in the number of ethanol reinforcers earned (
Figure 1C). Analysis of the number of reinforcers earned on the last three days of self-administration revealed a significant reinforcer type by adolescent exposure interaction (F
(1, 26) = 8.10,
p = 0.009), and subsequent analysis by separate two-way ANOVAs for each reinforcer type indicated that there was only a significant effect of adolescent CORT exposure on reinforcers earned in rats responding for ethanol (F
(1, 13) = 17.14,
p < 0.001), but not for sucrose (F
(1, 13) = 1.829,
p > 0.05). Therefore, adolescent CORT exposure resulted in a significant increase in the number of ethanol reinforcers earned once self-administration was acquired, but did not affect self-administration of sucrose (
Figure 1B,C). Similarly, analysis of ethanol intake (g/kg) during the same time frame revealed a significant effect of adolescent condition (F
(1, 26) = 6.94,
p = 0.02), but no interaction with session day (F
(2, 26) = 0.32,
p > 0.05), indicating that the adolescent CORT-exposed animals self-administered significantly more ethanol as a g/kg dose during all three of the last self-administration sessions (
Figure 1C, inset).
3.1.3. Phosphoproteomic Analysis
Two weeks after the reinstatement test, rats were euthanized and their brains were analyzed for changes in the levels of phosphorylated proteins in the amygdala. A discovery-based mass spectrometry approach was used to identify potentially novel biological signaling differences in the brains of rats that self-administered sucrose vs ethanol or that were exposed to CORT in adolescence, and their interaction. A total of 156 unique proteins were identified on which sites of phosphorylation could be resolved. Within these proteins, 478 unique phosphorylation patterns were identified, and of these, 270 phosphopeptides were significantly regulated in at least one of the experimental conditions (
Suppl. Table S1, phosphopeptides above yellow row are significant). Next, volcano plots were created to compare the magnitude of change in phosphopeptide abundance based on the main effect of adolescent CORT exposure (
Figure 2A) versus the main effect of ethanol self-administration (
Figure 2B) relative to the −log10 of the
p-value from the ANOVA to identify highly significant differences (
y-axis) of large effect size (
x-axis). This analysis revealed that adolescent CORT exposure produced 16 changes in protein phosphorylation (red dots in
Figure 2A) that were both significantly different from H
2O exposure (points above gray line =
p < 0.05 after correcting for multiple comparisons) and that were of large effect size (either increases or decreases with an effect size greater than a four-fold change from H
2O exposed control = log2(ratio) >2 or <−2). On the other hand, there was only one significant difference of large effect size identified, based on the reinforcer that was previously self-administered (
Figure 2B), suggesting that adolescent CORT exposure had a larger long-term effect on the amygdala phosphoproteome than the prior ethanol self-administration experience. Indeed, the protein seemingly regulated by ethanol self-administration, microtubule-associated protein 2 (MAP2), was also regulated by CORT exposure, and both effects were likely driven by a few large values in the CORT–sucrose group.
We went on to test for potential interaction effects between adolescent CORT exposure and ethanol self-administration. After correcting for multiple comparisons, significant interactions (
p < 0.1) were identified for 10 phosphopeptides (
Suppl. Table S2). Of these 10, seven were different phosphopeptides from the neurofilament heavy and medium chain proteins.
Figure 3A shows the quantitative difference between groups from one of these neurofilament phosphopeptides, which was representative of the pattern of results observed for all of the neurofilament phosphopeptides. Tukey’s post-hoc analysis revealed that phosphorylation of the neurofilament proteins was highest in the adolescent control group that self-administered sucrose (
p < 0.0001 relative to all other groups). A similar pattern was observed for two of the other phosphopeptides identified, synaptotagmin 2 and Map 1a (all
p < 0.0001 comparing H
2O–sucrose to all other groups;
Figure 3B,C). Therefore, either prior adolescent CORT exposure or ethanol self-administration resulted in reduced phosphorylation of these peptides relative to controls. The only phosphopeptide to show a different pattern of results was IPP2 (protein phosphatase inhibitor 2, PPP1R2). Phosphorylation of IPP2 on serines 121 and 122 was reduced in the adolescent CORT-exposed rats that self-administered sucrose relative to H
2O–sucrose controls (
p = 0.012), but CORT-exposed rats that self-administered ethanol showed a significant reversal of this effect (
p = 0.035;
Figure 3D). Thus, with the exception of IPP2, all significant interactions between adolescent exposure groups and reinforcer types indicated that self-administration of ethanol could reduce protein phosphorylation in the adolescent H
2O-exposed group to the levels of adolescent CORT-exposed rats, while ethanol produced no further effects beyond the CORT exposure.
These results were further supported by a principal component analysis (PCA) and a hierarchical clustering analysis.
Figure 4 illustrates that the first principal component explained the majority of the variance, with the H
2O–sucrose group showing a concentration ellipse that did not overlap with the other three groups. Overlaying the PCA plot is a biplot indicating that the H
2O–sucrose group generally had higher values for each phosphopeptide relative to the other groups, suggestive of reduced phosphorylation in the experimental groups. In addition, hierarchical clustering analysis based on the abundance of the 10 phosphopeptides was significantly different among groups, showing that the adolescent H
2O- and CORT-exposed groups largely clustered separately, independent of the reinforcer self-administered, with the exception of some of the rats in the H
2O–ethanol group, which clustered more closely with the CORT groups (
Figure 5).
Next, due to the large effect of adolescent CORT exposure on the amygdala phosphoproteome, independent of self-administration condition, we focused our analysis on the 16 significantly regulated phosphopeptides shown in red in
Figure 2A. The identity of each of the phosphopeptides is listed in
Table 1, where the protein, modified peptide sequence, log2 magnitude of change, and
p-value from the ANOVA are given. The phosphorylated residues are shown in red.
Overall, adolescent CORT exposure appeared to produce increased phosphorylation of the microtubule-associated protein MAP2, particularly in the N-terminal domain, while the phosphorylation of neurofilament proteins was decreased. These data are suggestive of CORT-induced structural changes in the amygdala, though the exact functions of the phosphorylation sites identified are currently unknown. Of interest for alcohol use and other psychiatric disorders, adolescent CORT exposure also regulated the gap junction protein, connexin43, the protein phosphatase 1 regulatory subunit 1a (PPP1R1a), which is also known as inhibitor 1 (I-1), and the α
2AAR. In addition, the most highly statistically significant change in phosphopeptide abundance between groups was for the metabotropic glutamate receptor 5 (mGluR5), though the magnitude of effect was slightly less than 4-fold. Given the known relevance of mGluR5, particularly in the amygdala, for alcohol-motivated behaviors [
24,
25,
26,
27,
28], we further inspected the two-way interaction between adolescent treatment and reinforcer self-administered for this receptor and the other highly regulated phosphopeptides. A Two-way ANOVA revealed the main effects of CORT in increasing the abundance of each of these phosphopeptides (connexin 43: (F
(1, 33) = 14.44,
p < 0.001); I-1: (F
(1, 33)=15.5,
p < 0.001); α
2AAR: (F
(1, 33) = 24.17,
p < 0.001); mGluR5: (F
(1, 33)=26.64,
p < 0.001)), with no effect of reinforcer consumed during self-administration (all
p > 0.25;
Figure 6A–D).
We next determined the potential functional implications of the phosphorylation events observed. The function of the phosphorylation sites on I-1 (Ser43, Ser46, and Ser47) and mGluR5 (Ser1014 and Ser1016) are unknown. On the other hand, increased phosphorylation of connexin43 was found on Ser365, Ser368, and Ser369, which have been described previously [
29]. In particular, phosphorylation of Ser368 is known to decrease the permeability of gap junctions, and it is thought to be mediated by protein kinase C (PKC) [
30,
31]. Thus, adolescent CORT exposure may lead to long-lasting changes in neural signaling via gap junctions in the amygdala.
Finally, increased phosphorylation of the α
2AAR was found on four consecutive serines (366–369), which are a known substrate of the G-protein coupled receptor kinase 2 (GRK2) [
19,
32]. Phosphorylation at these sites mediates agonist-stimulated receptor desensitization, association with arrestin, decoupling from the G-protein, and clathrin-mediated endocytosis [
19,
32]. Thus, we predicted that the adolescent CORT-treated rats would have a reduced sensitivity to α
2AR-mediated signaling, which could result in an increase in norepinephrine release and post-synaptic signaling in the brain, as the normal autoreceptor-mediated brake on noradrenergic-transmission would be impaired. Given the large literature on the involvement of heightened noradrenergic signaling, particularly in the amygdala, for both stress- and alcohol-related behaviors, including the potential clinical use of α
2AAR agonists as a treatment for alcohol use disorders [
33,
34,
35], GRK2-mediated phosphorylation of α
2AAR after adolescent CORT exposure is a strong candidate as a mediator of increased motivation for alcohol. Therefore, we decided to directly test if inhibition of GRK2 in the amygdala could reduce alcohol-motivated behaviors in adolescent CORT- or H
2O-exposed rats.