Importance of sex and trauma context on circulating cytokines and amygdalar GABAergic signaling in a comorbid model of posttraumatic stress and alcohol use disorders

Abstract

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share mechanisms that could be therapeutic targets. To facilitate mechanistic studies, we adapted an inhibitory avoidance-based “2-hit” rat model of posttraumatic stress disorder (PTSD) and identified predictors and biomarkers of comorbid alcohol (ethanol)/PTSD-like symptoms in these animals. Stressed Wistar rats received a single footshock on two occasions. The first footshock occurred when rats crossed into the dark chamber of a shuttle box. Forty-eight hours later, rats received the second footshock in a familiar (FAM) or novel context (NOV). Rats then received 4 weeks of two-bottle choice (2BC) ethanol access. During subsequent abstinence, PTSD-like behavior responses, GABAergic synaptic transmission in the central amygdala (CeA), and circulating cytokine levels were measured. FAM and NOV stress more effectively increased 2BC drinking in males and females, respectively. Stressed male rats, especially drinking-Vulnerable individuals (≥0.8g/kg average 2-hr ethanol intake with >50% ethanol preference), showed higher fear overgeneralization in novel contexts, increased GABAergic transmission in the CeA, and a profile of increased G-CSF, GM-CSF, IL-13, IL-6, IL-17a, leptin and IL-4 that discriminated between stress context (NOV>FAM>Control). However, drinking-resilient males showed the highest G-CSF, IL-13, and leptin levels. Stressed females showed increased acoustic startle and decreased sleep maintenance, indicative of hyperarousal, with increased CeA GABAergic transmission in NOV females. This paradigm promotes key features of PTSD, including hyperarousal, fear generalization, avoidance, and sleep disturbance, with comorbid ethanol intake, in a sex-specific fashion that approximates clinical comorbidities better than existing models, and identifies increased CeA GABAergic signaling and a distinct pro-hematopoietic, proinflammatory, and pro-atopic cytokine profile that may aid in treatment.

Glucocorticoid Receptor Antagonist Mifepristone Does Not Alter Innate Anxiety-Like Behavior in Genetically-Selected Marchigian Sardinian (msP) Rats

“…Rats were housed on a 12 h reverse light/dark cycle (lights off at 8:00 a.m.), with food and water available ad libitum . The rats were pair-housed, separated by a perforated clear plexiglass divider to habituate them to the behavioral test conditions while also reducing isolation stress [ 44 ]. The experimental groups consisted of rats that were randomly selected, and simple randomization for treatment groups condition occurred prior to the start of the experiments via a number labeling system for each rat.…”

“…Since stress and anxiety are often accompanied by sleep disturbances [ 49 ], we assessed sleep patterns in a comprehensive lab animal monitoring system (CLAMS, Columbus Instruments, Columbus, OH, USA). Such noninvasive, activity-based measurements correlate well with EEG-defined sleep [ 32 , 44 ]. We used OXYmax–CLAMS units to interpret each rat’s sleep parameters from photocell-defined motor activity across the first 11 h of their 12 h light phase.…”

“…Exaggerated acoustic startle responses are present in patients with PTSD and indicate hyperarousal [ 44 ]. Startle reflexes were measured in four identical startle response systems (SR-LAB, San Diego Instruments, San Diego, CA, USA) consisting of a nonrestrictive Plexiglas cylinder (13 cm inner diameter, 25 cm length for males; 9 cm inner diameter, 20 cm length for females) mounted on a Plexiglas platform and placed in a dark, ventilated, sound-attenuated chamber.…”

Self-Administration of Entactogen Psychostimulants Dysregulates Gamma-Aminobutyric Acid (GABA) and Kappa Opioid Receptor Signaling in the Central Nucleus of the Amygdala of Female Wistar Rats

“…Preparation of acute brain slices containing the central nucleus of the amygdala (CeA) and electrophysiological recordings were performed as previously described ( Varodayan et al, 2018 ; Suárez et al, 2019 ; Khom et al, 2020a , b ; Steinman et al, 2020 ). Briefly, deeply anesthetized rats (3–5% isoflurane anesthesia) were quickly decapitated, and their brains placed in an ice-cold oxygenated high-sucrose cutting solution composed of 206 mM sucrose, 2.5 mM KCl, 0.5 mM CaCl 2 , 7 mM MgCl 2 , 1.2 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 5 mM glucose, and 5 mM HEPES.…”

Self-Administration of entactogen psychostimulants dysregulates GABA and Kappa Opioid Receptor signaling in the central nucleus of the amygdala of female Wistar rats

“…Preparation of acute brain slices containing the central nucleus of the amygdala (CeA) and electrophysiological recordings were performed as previously described (Khom et al, 2020a,b;Steinman et al, 2020;Suárez et al, 2019;Varodayan et al, 2018). Briefly, deeply anesthetized rats (3-5% isoflurane anesthesia) were quickly decapitated, and their brains placed in an ice-cold oxygenated high-sucrose cutting solution composed of 206 mM sucrose, 2.5 mM KCl, 0.5 mM CaCl2, 7 mM MgCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, 5 mM glucose, and 5 mM HEPES.…”