Chemogenetic Inhibition of the Cortical Amygdala Reduces Alcohol Intake and Restores Thalamic Connectivity in Dependent Female Mice

bioRxiv (Cold Spring Harbor Laboratory) · 2026-05-12

Abstract Background Alcohol use disorder is a chronic relapsing condition characterized by excessive drinking and withdrawal symptoms. Alcohol dependence disrupts function across multiple brain regions, and recent evidence implicates the cortical amygdala (CoA) as a critical node in alcohol-related circuits. However, how CoA activity influences alcohol intake and brain-wide network function during withdrawal remains unclear. Methods Alcohol dependence was induced using chronic intermittent ethanol vapor (CIE). In one cohort, electrophysiological activity of CoA neurons was assessed during withdrawal. In a second cohort, mice underwent CIE paired with two-bottle choice drinking, and inhibitory DREADDs (hM4Di) were used to suppress CoA activity during drinking and withdrawal while behavioral outcomes were measured. Brains were then collected for Fos immunolabeling and iDISCO+ based whole-brain activity mapping to determine how CoA inhibition during withdrawal altered network organization. Results Repeated CIE increased alcohol sensitivity in CoA neurons during withdrawal. Chemogenetic inhibition of the CoA reduced alcohol intake in dependent mice without affecting withdrawal-related behaviors. Whole-brain Fos mapping showed that CoA inhibition reduced activity within the CoA while enhancing functional connectivity across multiple brain regions, particularly in the isocortex, thalamus, and anterior hypothalamic nucleus. During withdrawal without CoA inhibition, thalamic regions exhibited negative connectivity, consistent with disrupted network function; CoA inhibition reversed this pattern, producing strongly positive thalamic and medial prefrontal cortex connectivity. Conclusions These findings demonstrate that alcohol dependence alters CoA sensitivity, alcohol dependence-induced drinking and brain-wide network organization during withdrawal. The CoA appears to selectively regulate withdrawal-associated alcohol drinking, and its inhibition may reduce intake by restoring thalamic and cortical connectivity. Highlights This study identifies the cortical amygdala as a previously underexplored brain region involved in alcohol-related behaviors. By integrating chemogenetic inhibition with brain-wide network analysis, the study reveals candidate circuit connections through which the CoA may regulate alcohol dependence-related brain activity. This study establishes the CoA as a potential driver of excessive alcohol drinking and alcohol-related network dysfunction.

Large-scale behavioral characterization of oxycodone self-administration in heterogeneous stock rats reveals initial analgesic effects are associated with addiction-like behaviors

Neuropsychopharmacology · 2026-01-30 · 1 citations

Family and twin studies indicate that 20-60% of the vulnerability to opioid use disorder (OUD) is influenced by genetic factors, but the specific genes driving addiction-like behaviors, including sensitivity to opioid analgesia, tolerance, dependence, and escalation of oxycodone self-administration, remain unidentified, limiting precision medicine approaches. To address this, we phenotyped over 500 heterogeneous stock (HS) rats, an outbred population with high genetic diversity, to characterize traits associated with OUD vulnerability and resilience. Rats self-administered oxycodone (150 µg/kg/infusion) in short-access (2 h/day, 4 days) followed by long-access (12 h/day, 14 days) sessions. We assessed motivation for oxycodone using progressive ratio testing, withdrawal-induced hyperalgesia with von Frey tests, and tolerance to oxycodone's analgesic effects via tail immersion tests. Large cohorts (n = 46-60) and Z-score normalization minimized cohort-specific effects. An Addiction Index, derived from averaging Z-scores of escalation, motivation, tolerance, and hyperalgesia, revealed significant individual variability. Rats with severe addiction-like behaviors displayed higher initial analgesia, greater escalation, and more pronounced tolerance compared to resilient rats. Females showed increased escalation and motivation compared to males, but similar tolerance and hyperalgesia. Principal component analysis confirmed the Addiction Index's validity, accounting for 40% of behavioral variance. This high-throughput phenotyping in HS rats, leveraging their genetic diversity, provides a robust framework for genome-wide association studies to identify gene variants linked to OUD vulnerability, offering translational potential for discovering novel therapeutic targets and advancing pharmacogenetic strategies for OUD treatment.

Disrupted theta synchronization and synaptic connectivity in the visual cortex of Fmr1 KO mice

Nature Communications · 2025-11-26

Fragile X syndrome (FX) is a leading inherited cause of autism spectrum disorder, characterized by sensory hypersensitivity and impaired visual learning. Visual experience induces synchronized theta oscillations in the primary visual cortex (V1) and lateromedial area (LM), supporting inter-areal sensory binding. Using the Fmr1 knockout (KO) mouse model of FX, we quantify experience-dependent c-Fos expression in V1 and LM via iDISCO whole-brain clearing. Simultaneous in vivo recordings and channelrhodopsin-2-assisted circuit mapping (CRACM) reveal synchronized V1–LM theta oscillations and strengthened feedforward V1 → LM connectivity in wild-type (WT) mice, but attenuated LM oscillations and impaired connectivity in FX mice. Using 4Pi single-molecule localization microscopy, we identify experience-driven dendritic spine remodeling in layer 5 pyramidal cells of V1 and LM in WT mice, which is absent in FX mice. FX mice also show elevated baseline spine density and length. Our findings demonstrate that visual experience drives inter-areal synchronization and synaptic plasticity, which are disrupted in FX. Visual experience synchronizes V1–LM theta oscillations, strengthens V1 → LM connectivity, and drives dendritic spine remodeling in WT mice, the processes disrupted in Fmr1 KO mice, revealing impaired inter-areal binding in Fragile X syndrome.

Genome-wide association study of cocaine self-administration behavior in Heterogeneous Stock rats

bioRxiv (Cold Spring Harbor Laboratory) · 2025-07-18 · 1 citations

Abstract Background Cocaine use disorder (CUD) is a major public health crisis with detrimental individual and societal effects. The specific genes mediating CUD remain largely unknown. Methods We conducted a genome-wide association study (GWAS) using outbred N/NIH Heterogeneous Stock (HS; n = 836, female = 415, male = 421) rats. We examined multiple CUD-related phenotypes that captured acquisition of self-administration, escalation of intake, and compulsive-like responding. Results Consistent with the existing literature, these traits were phenotypically and genetically correlated and exhibited modest heritability (h 2 = 0.07 – 0.16). We identified six genome-wide significant associations. One locus on chromosome 19 was associated with the variable time between cocaine infusions (post infusion interval) and contains several carboxylesterase genes that are orthologous to the human CES1 gene; notably, carboxylesterases metabolize cocaine. Three non-synonymous coding variants in the genes Ces1c and Ces1d were in perfect linkage disequilibrium with this locus, suggesting that one or more of them might be the causal SNP. The other 5 loci also contained promising coding and expression variants, including Trak2, a gene previously associated with CUD in human GWAS and Slc10a7 , Plcl1 , and Satb2 which have been associated with alcohol and tobacco use disorder. Conclusions This is the largest genetic study of cocaine self-administration ever conducted in any species. Our results replicate previous loci associated with CUD in humans and provide several novel biological insights including the potential of pharmacological strategies targeting carboxylesterases for the treatment of CUD.

Author response: Large-scale characterization of cocaine addiction-like behaviors reveals that escalation of intake, aversion-resistant responding, and breaking-points are highly correlated measures of the same construct

2024-11-01

Genetically diverse rats reveal a single underlying construct linking key addiction-like behaviors, challenging compulsivity as an independent measure, and offering a new perspective on vulnerability and resilience to addiction.

Sex Differences in Neural Networks Recruited by Frontloaded Binge Alcohol Drinking

bioRxiv (Cold Spring Harbor Laboratory) · 2024-02-09 · 9 citations

Abstract Frontloading is an alcohol drinking pattern where intake is skewed toward the onset of access. The goal of the current study was to identify brain regions involved in frontloading. Whole brain imaging was performed in 63 C57Bl/6J (32 female and 31 male) mice that underwent 8 days of binge drinking using the drinking-in-the-dark (DID) model. On days 1-7, three hours into the dark cycle, mice received 20% (v/v) alcohol or water for two hours. Intake was measured in 1-minute bins using volumetric sippers, which facilitated analyses of drinking patterns. On day 8 mice were perfused 80 minutes into the DID session and brains were extracted. Brains were then processed to stain for Fos protein using iDISCO+. Following light sheet imaging, ClearMap2.1 was used to register brains to the Allen Brain Atlas and detect Fos+ cells. For brain network analyses, day 8 drinking patterns were used to characterize mice as frontloaders or non-frontloaders using a recently developed change-point analysis. Based on this analysis the groups were female frontloaders (n = 20), female non-frontloaders (n = 2), male frontloaders (n = 13) and male non-frontloaders (n = 8). There were no differences in total alcohol intake in animals that frontloaded versus those that did not. Only two female mice were characterized as non-frontloaders, thus preventing brain network analysis of this group. Functional correlation matrices were calculated for each group from log 10 Fos values. Euclidean distances were calculated from these R values and hierarchical clustering was used to determine modules (highly connected groups of brain regions). In males, alcohol access decreased modularity (3 modules in both frontloaders and non-frontloaders) as compared to water drinkers (7 modules). In females, an opposite effect was observed. Alcohol access (9 modules for frontloaders) increased modularity as compared to water drinkers (5 modules). These results suggest sex differences in how alcohol consumption reorganizes the functional architecture of neural networks. Next, key brain regions in each network were identified. Connector hubs, which primarily facilitate communication between modules, and provincial hubs, which facilitate communication within modules, were of specific interest for their important and differing roles. In males, 4 connector hubs and 17 provincial hubs were uniquely identified in frontloaders (i.e., were brain regions that did not have this status in male non-frontloaders or water drinkers). These represented a group of hindbrain regions (e.g., locus coeruleus and the pontine gray) functionally connected to striatal/cortical regions (e.g., cortical amygdalar area) by the paraventricular nucleus of the thalamus. In females, 16 connector and 17 provincial hubs were uniquely identified which were distributed across 8 of the 9 modules in the female frontloader alcohol drinker network. Only one brain region (the nucleus raphe pontis) was a connector hub in both sexes, suggesting that frontloading in males and females may be driven by different brain regions. In conclusion, alcohol consumption led to fewer, but more densely connected, groups of brain regions in males but not females, and recruited different hub brain regions between the sexes. These results suggest that alcohol frontloading leads to a reduction in network efficiency in male mice.

Sex differences in neural networks recruited by frontloaded binge alcohol drinking

Addiction Biology · 2024-09-01 · 11 citations

Abstract Frontloading is an alcohol drinking pattern where intake is skewed towards the onset of access. This study aimed to identify brain regions involved in frontloading. Whole brain imaging was performed in 63 C57Bl/6J (32 female, 31 male) mice that underwent 8 days of binge drinking using drinking‐in‐the‐dark (DID). On Days 1–7 mice received 20% (v/v) alcohol or water for 2 h. Intake was measured in 1‐min bins using volumetric sippers. On Day 8 mice were perfused 80 min into the DID session and brains were extracted. Brains were processed to stain for Fos protein using iDISCO+. Following light sheet imaging, ClearMap2.1 was used to register brains to the Allen Brain Atlas and detect Fos+ cells. For network analyses, Day 8 drinking patterns were used to characterize mice as frontloaders or non‐frontloaders using a change‐point analysis. Functional correlation matrices were calculated for each group from log 10 Fos values. Euclidean distances were calculated from these R values and clustering was used to determine modules (highly connected groups of brain regions). In males, alcohol access decreased modularity (three modules in both frontloaders and non‐frontloaders) as compared to water (seven modules). In females, an opposite effect was observed. Alcohol access (nine modules for frontloaders) increased modularity as compared to water (five modules). Further, different brain regions served as hubs in frontloaders as compared to control groups. In conclusion, alcohol consumption led to fewer, but more densely connected, groups of brain regions in males but not females and we identify several brain‐wide signatures of frontloading.

Large-scale characterization of cocaine addiction-like behaviors reveals that escalation of intake, aversion-resistant responding, and breaking-points are highly correlated measures of the same construct

eLife · 2024-10-07 · 2 citations

Abstract Addiction is commonly characterized by escalation of drug intake, compulsive drug seeking, and continued use despite harmful consequences. However, the factors contributing to the transition from moderate drug use to these problematic patterns remain unclear, particularly regarding the role of sex. Many preclinical studies have been limited by small sample sizes, low genetic diversity, and restricted drug access, making it challenging to model significant levels of intoxication or dependence and translate findings to humans. To address these limitations, we characterized addiction-like behaviors in a large sample of >500 outbred heterogeneous stock (HS) rats using an extended cocaine self-administration paradigm (6 h/daily). We analyzed individual differences in escalation of intake, progressive-ratio (PR) responding, continued use despite adverse consequences (contingent foot shocks), and irritability-like behavior during withdrawal. Principal component analysis showed that escalation of intake, progressive ratio responding, and continued use despite adverse consequences loaded onto a single factor that was distinct from irritability-like behaviors. Categorizing rats into resilient, mild, moderate, and severe addiction-like phenotypes showed that females exhibited higher addiction-like behaviors, with a lower proportion of resilient individuals compared to males. These findings suggest that, in genetically diverse rats with extended drug access, escalation of intake, continued use despite adverse consequences, and PR responding are highly correlated measures of a shared underlying construct. Furthermore, our results highlight sex differences in resilience to addiction-like behaviors.

Author response: Large-scale characterization of cocaine addiction-like behaviors reveals that escalation of intake, aversion-resistant responding, and breaking-points are highly correlated measures of the same construct

2024-10-07

Addiction is commonly characterized by escalation of drug intake, compulsive drug seeking, and continued use despite harmful consequences. However, the factors contributing to the transition from moderate drug use to these problematic patterns remain unclear, particularly regarding the role of sex. Many preclinical studies have been limited by small sample sizes, low genetic diversity, and restricted drug access, making it challenging to model significant levels of intoxication or dependence and translate findings to humans. To address these limitations, we characterized addiction-like behaviors in a large sample of >500 outbred heterogeneous stock (HS) rats using an extended cocaine self-administration paradigm (6 h/daily). We analyzed individual differences in escalation of intake, progressive-ratio (PR) responding, continued use despite adverse consequences (contingent foot shocks), and irritability-like behavior during withdrawal. Principal component analysis showed that escalation of intake, progressive ratio responding, and continued use despite adverse consequences loaded onto a single factor that was distinct from irritability-like behaviors. Categorizing rats into resilient, mild, moderate, and severe addiction-like phenotypes showed that females exhibited higher addiction-like behaviors, with a lower proportion of resilient individuals compared to males. These findings suggest that, in genetically diverse rats with extended drug access, escalation of intake, continued use despite adverse consequences, and PR responding are highly correlated measures of a shared underlying construct. Furthermore, our results highlight sex differences in resilience to addiction-like behaviors.

Temporal differential effects of post-injury alcohol consumption in a mouse model of blast-induced traumatic brain injury

Neuroscience · 2024-10-05 · 4 citations