Marsida Kallupi

Cocaine use disorder (CUD) is a major public health crisis. The specific genes mediating CUD remain largely unknown. We conducted a genome-wide association study (GWAS) using outbred N/NIH Heterogeneous Stock (HS; n = 836, female = 415, male = 421) rats. We examined CUD-related phenotypes including acquisition of self-administration, escalation of intake, and compulsive-like responding. These traits were phenotypically correlated and exhibited modest SNP heritability (h ² = 0.07 – 0.16). We identified six genome-wide significant associations (>-log ₁₀ (p)=5.58; α = 0.05 by permutation). One locus on chromosome 19 was associated with 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 Ces1c and Ces1d were in perfect linkage disequilibrium with this locus. The other five loci 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. This is the largest genetic study of cocaine self-administration ever conducted in rats. Our results replicate previous loci associated with CUD in humans and provide several novel biological insights including the potential of pharmacological strategies targeting carboxylesterases.

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.

Alcohol use disorder (AUD) remains a pervasive public health issue with limited effective treatments. Cannabidiol (CBD), a non-psychotropic constituent of cannabis, shows promise in modulating addictive behaviors. This study investigated the effects of chronic CBD administration on alcohol dependence, withdrawal symptoms, and neurodegeneration using two complementary rodent models: chronic intermittent ethanol (CIE) exposure, which models established alcohol dependence, and ethanol vapor self-administration (EVSA), which captures the volitional aspects of alcohol intake. In the CIE model, CBD reduced alcohol self-administration during acute withdrawal without affecting alcohol metabolism or locomotor activity. CBD decreased motivation for alcohol, somatic withdrawal signs, withdrawal-induced anxiety-like behaviors, and mechanical sensitivity. During extinction, CBD attenuated alcohol-seeking behavior and stress-induced reinstatement. Electrophysiological recordings revealed that CBD reversed alcohol-induced decreases in neuronal excitability in the basolateral amygdala, suggesting a mechanism involving normalization of neural function. In the EVSA model, CBD reduced voluntary alcohol intake during the escalation phase, impacting voluntary alcohol intake. This effect was specific to alcohol-related behaviors, as it did not affect saccharin self-administration. Immunohistochemical analyses showed that CBD prevented alcohol-induced neurodegeneration in the nucleus accumbens shell and dorsomedial striatum, regions implicated in the volitional control of alcohol consumption. These findings indicate that chronic CBD administration attenuates both behavioral and neurobiological facets of alcohol dependence by modulating neuronal excitability and preventing neurodegeneration, supporting its therapeutic potential for AUD and providing mechanistic insights for future research.

Hormonal fluctuations throughout the estrous cycle have been hypothesized to influence drug-related behaviors. Preclinical models show that some cocaine-related behaviors are influenced by the estrous cycle. However, the extent to which the estrous cycle modulates cocaine self-administration in outbred heterogeneous stock (HS) rats, a population that captures human genetic diversity, is unknown. This study aimed to examine the relationship between estrous phases and cocaine self-administration behavior in HS rats using a model of extended access to cocaine self-administration. We focused on the escalation of intake, breaking point, and resistance to foot shock. Using vaginal swabbing and lavage techniques, we first characterized the relationship between estrous phase and cocaine intake. We then comprehensively evaluated estrous cycling patterns in young adult and adult HS rats, comparing them with Wistar rats. Contrary to our hypothesis, estrous phase showed no association with cocaine self-administration in HS rats. HS rats exhibited irregular estrous cycling with variability to the phase length, even in the absence of drug exposure, a phenomenon not observed in the Wistar strain. Irregular estrous cycle was associated with high cocaine-related behaviors. This study provides the first evidence that some female HS rats exhibit irregular estrous cycling. Moreover, rats with severe addiction-like behaviors had more instances of irregular cycling. These results demonstrate that, in HS rats, the estrous phase per se has no major influence on cocaine self-administration, but that the severity of addiction-like behaviors are associated with more irregularity of the estrus cycle. As HS rats gain popularity in behavioral and genome-wide studies, understanding these cycle disruptions is crucial as they may reveal genetic links into female vulnerability to drugs.

Genome-wide association studies typically evaluate the autosomes and sometimes the X Chromosome, but seldom consider the Y or mitochondrial Chromosomes. We genotyped the Y and mitochondrial Chromosomes in heterogeneous stock rats (Rattus norvegicus), an outbred population created from eight inbred strains. We identified 8 distinct Y and 4 distinct mitochondrial Chromosomes among the 8 founders. However, only two types of each nonrecombinant chromosome were observed in our modern heterogeneous stock rat population (generations 81-97). Despite the relatively large sample size, there were virtually no significant associations for behavioral, physiological, metabolome, or microbiome traits after correcting for multiple comparisons. However, both Y and mitochondrial Chromosomes were strongly associated with expression of a few genes located on those chromosomes, which provided a positive control. Our results suggest that within modern heterogeneous stock rats there are no Y and mitochondrial Chromosomes differences that strongly influence behavioral or physiological traits. These results do not address other ancestral Y and mitochondrial Chromosomes that do not appear in modern heterogeneous stock rats, nor do they address effects that may exist in other rat populations, or in other species.

The amygdala processes positive and negative valence and contributes to addiction, but the cell-type-specific gene regulatory programs involved are unknown. We generated an atlas of single-nucleus gene expression and chromatin accessibility in the amygdala of outbred rats with high and low cocaine addiction-like behaviors following prolonged abstinence. Differentially expressed genes between the high and low groups were enriched for energy metabolism across cell types. Rats with high addiction index (AI) showed increased relapse-like behaviors and GABAergic transmission in the amygdala. Both phenotypes were reversed by pharmacological inhibition of the glyoxalase 1 enzyme, which metabolizes methylglyoxal—a GABA_A receptor agonist produced by glycolysis. Differences in chromatin accessibility between high and low AI rats implicated pioneer transcription factors in the basic helix-loop-helix, FOX, SOX and activator protein 1 families. We observed opposite regulation of chromatin accessibility across many cell types. Most notably, excitatory neurons had greater accessibility in high AI rats and inhibitory neurons had greater accessibility in low AI rats.