Adolescent Alcohol Exposure Produces Sex-Specific Long-term Hyperalgesia via Changes in Central Amygdala Circuit Function

Abstract

Background: Exposure to alcohol during adolescence produces many effects that last well into adulthood. Acute alcohol use is analgesic, and people living with pain report drinking alcohol to reduce pain, but chronic alcohol use produces increases in pain sensitivity.

Methods: We tested the acute and lasting effects of chronic adolescent intermittent ethanol (AIE) exposure on pain-related behavioral and brain changes in male and female rats. We also tested the long-term effects of AIE on synaptic transmission in midbrain (ventrolateral periaqueductal gray [vlPAG])-projecting central amygdala (CeA) neurons using whole-cell electrophysiology. Finally, we used circuit-based approaches (DREADDs [designer receptors exclusively activated by designer drugs]) to test the role of vlPAG-projecting CeA neurons in mediating AIE effects on pain-related outcomes.

Results: AIE produced long-lasting hyperalgesia in male, but not female, rats. Similarly, AIE led to a reduction in synaptic strength of medial CeA cells that project to the vlPAG in male, but not female, rats. Challenge with an acute painful stimulus (i.e., formalin) in adulthood produced expected increases in pain reactivity, and this effect was exaggerated in male rats with a history of AIE. Finally, CeA-vlPAG circuit activation rescued AIE-induced hypersensitivity in male rats.

Conclusions: Our findings are the first, to our knowledge, to show long-lasting sex-dependent effects of adolescent alcohol exposure on pain-related behaviors and brain circuits in adult animals. This work has implications for understanding the long-term effects of underage alcohol drinking on pain-related behaviors in humans.

Keywords: Adolescents; Alcohol; Central amygdala; Pain; Rats; Ventral lateral periaqueductal gray.

Figure 1.. Intermittent alcohol exposure during adolescence produces sex-dependent long-lasting changes in pain sensitivity.

A) Timeline of the experiment including adolescence intermittent alcohol exposure (AIE), chronic withdrawal and pain-like behaviors from adolescent to early adulthood. Hargreaves (B) and Von Frey (C) thresholds in male rats during acute (~7 hours after each daily exposure) and chronic alcohol withdrawal intervals. Control animals (AIR) are shown in open circles while exposed animals (AIE) are shown in closed purple circles. Hargreaves (D) and Von Frey (E) thresholds in female rats during acute (~7 hours after each daily exposure) and chronic alcohol withdrawal intervals. Control animals (AIR) are shown in open circles while exposed animals (AIE) are shown in closed green circles. The data are represented as the mean ± SEM (males: n=96; females: n=36): *p <0.05 AIR vs. AIE was assessed by two-way analysis of variance with repeated measures followed by Sidak’s post hoc test.

Figure 2.. CeA-vlPAG cells show decreased synaptic drive in adult male rats exposed to alcohol vapor during adolescence.

A) Timeline of the experiment including adolescence intermittent alcohol exposure (AIE), chronic withdrawal, retrograde tracer injections, pain-like behaviors from adolescent to adulthood and whole-cell electrophysiology recordings. B-E) sIPSC and sEPSC amplitude and frequency measured in CeM to vlPAG projecting cells in control and AIE male rats three to five weeks after the end of adolescent alcohol vapor exposure. F-G) Excitatory vs inhibitory ratio and synaptic drive in CeM to vlPAG projecting cells from whole-cell electrophysiology recordings in control and AIE male rats. J-M) sIPSC and sEPSC amplitude and frequency measured in CeM to vlPAG projecting cells in control and AIE male rats three to five weeks after the end of adolescent alcohol vapor exposure. N-O) Excitatory vs inhibitory ratio and synaptic drive in CeM to vlPAG projecting cells from whole-cell electrophysiology recordings in control and AIE male rats. The data are represented as the mean ± SEM (males: n_cells= 17; females: n_cells= 27): *p <0.05 AIR vs. AIE was assessed Welch’s correction was used for t-tests with unequal variance.

Figure 3.. Behavioral effects following formalin challenge in adult rats exposed to alcohol vapor during adolescence.

A) Timeline of the experiment including adolescence intermittent alcohol exposure (AIE), chronic withdrawal, retrograde tracer injections, formalin test, brain collections post-formalin and molecular biology assays to measure expression of c-fos in the central amygdala. B) Schematics of retrograde tracer injections in vlPAG (cholera toxin subunit B or green retrobeads) useful to trace the pathway backwards the central amygdala. One to three weeks after tracer injections, male and female rats underwent to a dorsal hindpaw formalin injection (50 ul at 5%) and number of flinches and lickings recorded for 60 minutes. C) Formalin curve in male rats show a significant difference between AIE and AIR animals in the behavior following the chemical stimulus injection. D) Cumulative responses in the biphasic behavior show no differences between groups (AIE vs AIE) during the acute phase (I) while a significant difference in the tonic phase (II). E) Formalin curve in female rats does not show any significant difference between AIE and AIR animals in the behavior following the chemical stimulus injection. F) Cumulative responses in the biphasic behavior show no differences between groups (AIE vs AIE) during the acute phase (I) and tonic phase (II) in female rats. The data are represented as the mean ± SEM (males: n=22; females: n=23): *p <0.05 AIR vs. AIE was assessed by two-way analysis of variance with repeated measures followed by Sidak’s post hoc test for the formalin curve or unpaired t-test for the cumulative responses. Schematics displayed in B were created with BioRender.com.

Figure 4.. Effect of formalin injection on the expression of c-fos in the CeA in adult rats exposed to alcohol vapor during adolescence.

A) Timeline of the experiment including adolescence intermittent alcohol exposure (AIE), chronic withdrawal, retrograde tracer injections, formalin test, brain collections post-formalin and molecular biology assays to measure expression of c-fos in the central amygdala. B) c-fos mRNA expression in the central amygdala subdivisions in the marked cells 90 minutes after formalin test of male rats. Formalin blunted expression of the immediate early gene in AIE but not AIE rats. C) c-fos mRNA expression in the central amygdala subdivisions in the marked cells 90 minutes after formalin test of female rats. Formalin did not produce any differences in c-fos expression in the central amygdala subdivisions. D) Representative image of the three markers used in the RNAscope (DAPI in blue, cholera toxin subunit B in green, and c-fos in red). Image was taken at 20 um using a fluorescence microscope. E) c-fos protein expression in the central amygdala subdivisions in the marked cells 90 minutes after formalin test of male rats. Formalin blunted expression of the immediate early gene in AIE but not AIE rats. F) c-fos protein expression in the central amygdala subdivisions in the marked cells 90 minutes after formalin test of female rats. Formalin did not produce any differences in c-fos expression in the central amygdala subdivisions. G) Representative image of the three markers used in the IHC (retrobeads in green, and c-fos in white). Images were taken at 20x magnification using a fluorescence microscope. The data are represented as the mean ± SEM (males IHC: n=10 and RNAscope: n=12; females IHC: n=12 and RNAscope n=11): *p <0.05 AIR vs. AIE was assessed by unpaired t-test analysis.

Figure 5.. Chemogenetic activation of the CeA-vlPAG pathway in adult male rats exposed to alcohol vapor during adolescence.

A) Timeline of the experiment including adolescence intermittent alcohol exposure (AIE), chronic withdrawal, DREADDs viral injections, recovery, behavioral baselines for pain-like behaviors (thermal and mechanical nociception) and chemogenetic activation using the actuator JHU37160 (J60), histology and imaging. B) Schematic representation of the hM3D(Gq)-DREADD (designer receptor exclusively activated by designer drugs) viral and cannula delivery in the behavioral experiments on male Wistar rats and representative merge image of the CeM cells stained with EGFP (Cre-dependent retrograde tracer - green) and mCherry [DREADD- hM3D(Gq)-red] (orange). C) The effects of activation the CeA-vlPAG pathway in thermal nociception assays (Hargreaves test) following J60 system injection (Active Virus - 30 minutes before session). D) The effects of activation the CeA-vlPAG in thermal nociception assays (Hargreaves test) following J60 systemic injection (Control Virus – 30 minutes before session). E) The effects of activation the CeA-vlPAG pathway in mechanical nociception assays (Von Frey test) following J60 system injection (Active Virus - 30 minutes before session). F) The effects of activation the CeA-vlPAG pathway in mechanical nociception assays (Von Frey test) following J60 system injection (Control Virus - 30 minutes before session). The data are represented as the mean ± SEM (males: n=45): *p <0.05 AIR vs. AIE was assessed by two-way analysis of variance with repeated measures followed by Sidak’s post hoc test. Images were taken at 20x magnification using a fluorescence microscope. Schematics displayed in B were created with BioRender.com.

Figure 6.. Chemogenetic inhibition of the CeA-vlPAG in female Wistar rats during chronic withdrawal.

A) Timeline of the experiment including adolescence intermittent alcohol exposure (AIE), chronic withdrawal, DREADDs viral injections, recovery, behavioral baselines for pain-like behaviors (thermal and mechanical nociception) and chemogenetic inhibition using the actuator JHU37160 (J60), histology and imaging. B) Schematic representation of the hM4D(Gi)-DREADD (designer receptor exclusively activated by designer drugs) viral and cannula delivery in the behavioral experiments on female Wistar rats. C) The effects of inhibition of the CeA-vlPAG pathway in thermal nociception assays (Hargreaves test) following J60 system injection (Active Virus - 30 minutes before session). D) The effects of inhibition the CeA-vlPAG in thermal nociception assays (Hargreaves test) following J60 systemic injection (Control Virus – 30 minutes before session). E) The effects of inhibition the CeA-vlPAG pathway in mechanical nociception assays (Von Frey test) following J60 system injection (Active Virus - 30 minutes before session). F) The effects of inhibition the CeA-vlPAG pathway in mechanical nociception assays (Von Frey test) following J60 system injection (Control Virus - 30 minutes before session). The data are represented as the mean ± SEM (females: n=28): *p <0.05 AIR vs. AIE was assessed by two-way analysis of variance with repeated measures followed by Sidak’s post hoc test. Schematics displayed in B were created with BioRender.com.

Figure 7.. Schematic depicting the effects of AIE and chemogenetic interventions on pain-like behaviors.

CeM GABAergic neurons project to vlPAG and synapse on neurons in that brain region. Repeated cycles of adolescent alcohol exposure and withdrawal leads to a reduction in excitation of CeA projections to vlPAG in male rats but not female rats. We hypothesize that the net result of this weakened CeA input is disinhibition of vlPAG interneurons and increased inhibition of vlPAG output neurons that project to RVM, ultimately contribute to hyperalgesia in chronic alcohol-exposed adult male rats. This hyperalgesia is reversed (normal nociception) by activating vlPAG-projecting CeA neurons with chemogenetic tools (AAV-Gq). In females, although alcohol dependence did not induce hyperalgesia, chemogenetic inhibition (AAV-Gi) of vlPAG-projecting CeA neurons did produce hyperalgesia, suggesting that the pathway is relevant for mediating nociception in female rats, but that either 1) its activity is not altered by adolescent alcohol exposure or 2) baseline activity is so low that it cannot be further weakened by adolescent alcohol exposure. Schematics was created with BioRender.com.