Adolescent Alcohol Drinking Renders Adult Drinking BLA-Dependent: BLA Hyper-Activity as Contributor to Comorbid Alcohol Use Disorder and Anxiety Disorders

Abstract

Adolescent alcohol drinking increases the risk for alcohol-use disorder in adulthood. Yet, the changes in adult neural function resulting from adolescent alcohol drinking remain poorly understood. We hypothesized that adolescent alcohol drinking alters basolateral amygdala (BLA) function, making alcohol drinking BLA-dependent in adulthood. Male, Long Evans rats were given voluntary, intermittent access to alcohol (20% ethanol) or a bitter, isocaloric control solution, across adolescence. Half of the rats in each group received neurotoxic BLA lesions. In adulthood, all rats were given voluntary, intermittent access to alcohol. BLA lesions reduced adult alcohol drinking in rats receiving adolescent access to alcohol, but not in rats receiving adolescent access to the control solution. The effect of the BLA lesion was most apparent in high alcohol drinking adolescent rats. The BLA is essential for fear learning and is hyper-active in anxiety disorders. The results are consistent with adolescent heavy alcohol drinking inducing BLA hyper-activity, providing a neural mechanism for comorbid alcohol use disorder and anxiety disorders.

Keywords: amygdala; chronic; ethanol; fear; intermittent; rat; voluntary.

Figure 1

Histology. (A) The extent of neurotoxic basolateral amygdala (BLAx) lesions across five coronal planes is shown, and the posterior distance from bregma (millimeters) indicated. Each individual’s lesion was drawn (n = 24), shaded at 5% opacity using Adobe Photoshop (CS6) and stacked. Darker areas indicate greater overlap between individual lesions, and thus, greater damage. Representative Nissl images showing (B) Control with BLA intact and (C) BLAx with a neurotoxic lesion. BLA, basolateral amygdaloid nucleus, anterior part; BLP, basolateral amygdaloid nucleus, posterior part; BLV, basolateral amygdaloid nucleus, ventral part; BMA, basomedial amygdaloid nucleus, anterior part; BMP, basomedial amygdaloid nucleus, posterior part; CeC, central amygdaloid nucleus, capsular part; CeL, central amygdaloid nucleus, lateral part; CeM, central amygdaloid nucleus, medial part; IM, intercalated amygdaloid nucleus, main part; LaDL, lateral amygdaloid nucleus, dorsolateral part; LaVL, lateral amygdaloid nucleus, ventrolateral part; LaVM, lateral amygdaloid nucleus, ventromedial part.

Figure 2

Alcohol drinking pre- and post-surgery. (A) Mean + SEM alcohol drinking (g/kg/24 h) for each voluntary access session is shown for control (Con—black) and mean − SEM for BLAx rats (red). Time of surgery is indicated by an arrow. (B) Mean + SEM alcohol drinking (g/kg/24 h) for the eight sessions prior to surgery (pre) and the eight sessions following surgery (post). Asterisks indicate the significance of the two-tailed, paired samples t-test (p < 0.05). NS indicates non-significance of the two-tailed, paired samples t-test (p > 0.1).

Figure 3

Relationship between alcohol drinking pre- and post-surgery. (A) Scatter plot compares pre-surgery drinking (mean of eight sessions prior to surgery) and post-surgery drinking (mean of eight sessions following surgery) for all control rats (Con). (B) Same data but zoomed in to visualize low drinking individuals. (C) Scatter plot compares pre-surgery drinking (mean of eight sessions prior to surgery) and post-surgery drinking (mean of eight sessions following surgery) for all BLAx rats. (D) Same data but zoomed in to visualize low drinking individuals. For each group, the square of Pearson’s correlation coefficient (R²) and its associated p value are reported. The p-value for a sign test comparing alcohol drinking (post – pre) to zero is reported. Note: scatter data points fully correspond to pre- and post-surgery alcohol drinking, shown in Figure 2B.

Figure 4

Alcohol drinking pre- and post-surgery for high and low drinkers. (A) Mean + SEM alcohol drinking (g/kg/24 h) for each voluntary access session is shown for the high drinking control (Con indicated in black) and mean − SEM shown for high drinking BLAx rats (red). The time of surgery is indicated by an arrow. (B) Mean + SEM alcohol drinking (g/kg/24 h) for the eight sessions prior to surgery (pre) and the eight sessions following surgery (post). (C) Mean + SEM alcohol drinking (g/kg/24 h) for each voluntary access session is shown for low drinking controls (Con indicated in black) and mean − SEM for low drinking BLAx rats (red). The time of surgery is indicated by an arrow. (D) Mean + SEM alcohol drinking (g/kg/24 h) for the eight sessions prior to surgery (pre) and the eight sessions following surgery (post). Pound sign indicates two-tailed, paired samples t-test (p = 0.09). NS indicates non-significance of the paired samples t-test (p > 0.1). Note: data are those exactly shown in Figure 2 (n = 12 per group), only separated with respect to high (n = 4 per group) or low (n = 8 per group) adolescent drinking.

Figure 5

QuAD drinking pre-surgery and alcohol drinking post-surgery. (A) Mean ± SEM QuAD drinking (g/kg/24 h) for each voluntary access session prior to surgery is shown for controls (Con indicated in black) and BLAx rats (BLAx indicated in red). The time of surgery is indicated by an arrow. (B) Mean ± SEM alcohol drinking (g/kg/24 h) for each voluntary access session following surgery is shown for controls and BLAx rats. (C) Mean ± SEM alcohol drinking (g/kg/24 h) for the eight sessions following surgery (post). NS indicates non-significance of the two-tailed, independent samples t-test (p > 0.1).

Figure 6

Working hypotheses for the alteration of BLA function by adolescent alcohol drinking. BLA is represented by rounded rectangles. Black circles indicate GABA interneurons, while all other circle colors (grey, orange, green) indicate pyramidal projection neurons. (A) In adult rats that are alcohol-naïve or were adolescent moderate alcohol drinkers, one class of pyramidal neuron signals fear (orange) while a second class is uninvolved in fear or alcohol (grey). GABA interneurons provide strong inhibitory input onto pyramidal neurons to modulate fear output, resulting in appropriate fear responses. (B) In scenario #1, adolescent heavy alcohol drinking recruits a new pyramidal population (green) whose activity is necessary to promote alcohol drinking. Simultaneously, adolescent drinking enhances the output of existing pyramidal neurons controlling fear (orange), and weakens GABAergic input onto each population. (C) In scenario #2, adolescent heavy alcohol drinking co-opts the pyramidal population controlling fear, whose activity now promotes alcohol drinking and fear. GABAergic input onto this population is simultaneously weakened. In both scenarios #1 and #2 the BLA is hyper-active, increasing fear and driving alcohol drinking.