Substance Use Disorder (SUD) is characterized by drug seeking, increased drug taking, and relapse. Females are particularly vulnerable to the debilitating effects of SUD and show increases in drug taking, a faster transition from first drug use to the development of SUD, and enhanced relapse. The mesolimbic dopamine pathway, which connects the ventral tegmental area (VTA) to the nucleus accumbens (NAc), is an essential component of the neural circuit that promotes reward‐seeking behavior. This is because nearly all drugs of abuse increase levels of the neurotransmitter dopamine in the NAc. The ability of stimulant substances of abuse, like cocaine, to drive drug taking/seeking has been linked to their ability to inhibit the function of the dopamine transporter (DAT) ‐ thus increasing dopamine levels in the synaptic cleft in the NAc. A major factor that could be contributing to sex differences in psychostimulant effects on dopamine neurotransmission and DAT kinetics, is estrous cycle dependent ovarian hormone fluctuations. Taking this into consideration, we took a multifaceted approach to understand how sex differences in psychostimulant effects at the DAT could be driven by estrous cycle‐dependent neurochemical mechanisms. First, using positive reinforcement operant conditioning to measure sex differences in reward‐seeking behavior, we found that female mice consumed more sucrose than males, suggesting that female mice are more motivated to work for non‐drug rewards. Next, using fast‐scan cyclic voltammetry (FSCV) paired with pharmacology, we measured the effect of cocaine on sub‐second dopamine release and DAT kinetics in male and female mice in either diestrus (low circulating hormones) or estrus (high circulating hormones). We found that cocaine potency was increased specifically during estrus (high circulating hormones), compared to males and females in diestrus (low circulating hormones). Together, these data suggest that psychostimulant effects on DAT in females during estrus increases dopamine levels in the NAc and likely drives enhanced reward seeking behavior in females. Moving forward it will be critical to determine whether this effect in females is unique to cocaine or generalizes across all stimulant drugs and further determine how these mechanisms play a key role in increasing reward seeking behavior in female subjects compared to males.

ID 18982 Poster Board 572 Substance use disorder (SUD) is a chronic neuropsychiatric disease characterized by long-lasting changes in behavior in response to drugs of abuse. These behavioral changes have previously been linked to drug-induced alternations in dopamine signaling. Although women are more susceptible to developing SUD and are more likely to relapse following periods of abstinence, a majority of work on the behavior basis of SUD has focused on male subjects. Emerging data has identified that estrous cycle-dependent hormone fluctuations play a key role in dopamine release regulation and may also play a role in sex differences in motor behavior in response to stimulant administration. Previous studies have shown that females display enhanced locomotor activity after cocaine exposure, but it is unclear if this increase is cycle-stage dependent and if this effect is similar across structurally dissimilar psychostimulants. Here we investigated a range of doses of each stimulant – cocaine (3, 10, or 30 mg/kg), amphetamine (1, 3, or 10 mg/kg), and methylphenidate (3, 10, or 30 mg/kg) – in males and freely cycling females to analyze the psychostimulant’s effect on locomotor activity. For cocaine, we observed a dose-dependent effect of cycle stage, where females in estrus (high circulating hormones) showed elevated motor responses as compared to females in diestrus (low circulating hormones) and males at the high doses. In contrast, for amphetamine, females were more responsive than males at the lower doses, but these effects were less sensitive to changes in estrous cycle. Finally, for methylphenidate, there was an upward shift in the dose-response curve in males but not females. Interestingly, there was also an estrous-cycle effect in females with diestrus females showing more motor activity than males. Together, these data suggest each psychostimulant displays unique sex- and cycle stage-dependent differences depending on its dose. In the future, it will be critical to directly link these differences to reward processing for the development of sex-specific pharmacotherapies for SUD. Acknowledgements: Funding was provided by startup funds from V.U. School of Medicine, Department of Pharmacology and the National Institutes of Health (NIH) (to E.S.C.). Funds from the National Institute of Drug Abuse (NIDA) DA042111 and DA048931, the Brain and Behavior Research Foundation, Whitehall Foundation, and the Edward J Mallinckrodt Jr. Foundation (E.S.C) also supported this work. Personal funding was provided by NIH’s MARC START program.

ID 15167 Poster Board 541 Substance use disorder (SUD) is a chronic, relapsing brain disease characterized by irregularities in motivation and reward processing. In humans, women transition from recreational to chronic substance abuse more rapidly than men and are more likely to relapse following abstinence. Thus, understanding the biological basis of these differences is critical for improved SUD treatment in women. The reinforcing properties of nearly all drugs of abuse are dependent on their ability to increase dopamine levels in the nucleus accumbens (NAc) – a brain region involved in reward processing and motivation. For stimulant drugs of abuse, this occurs via their ability to alter the function of the dopamine transporter (DAT). Three notable stimulants - cocaine, amphetamine, and methylphenidate - all can bind to the DAT and thus decrease dopamine clearance and increase the magnitude and time course of synaptic dopamine levels. However, unlike cocaine and methylphenidate, amphetamine can also drive active dopamine release through reversal of the DAT. The goal of this study was to understand exactly how biological sex alters the ability of these drugs to alter dopamine dynamics in the NAc depending on their mechanism of action. There are a variety of factors that could contribute to sex differences in stimulant effects on reward circuitry, one being estrous cycle-dependent ovarian hormone fluctuations. Previous studies have demonstrated that cocaine has an enhanced ability to inhibit the DAT in females during estrus (high circulating hormones) as compared to females in diestrus (low circulating hormones) and males, but it is unclear as to whether this effect is unique to cocaine or generalizes across all stimulant drugs. To this end, ex vivo fast-scan cyclic voltammetry (FSCV) combined with site-specific pharmacology was utilized to measure the effect of cocaine, amphetamine, and methylphenidate on sub-second dopamine release in males and freely-cycling females. We found that amphetamine’s ability to inhibit dopamine uptake was higher in estrus females than diestrus females and males, suggesting that the effects are hormone-mediated. However, methylphenidate’s ability to inhibit dopamine uptake was higher in females than males, regardless of cycle stage. Together, these data suggest that sex differences in the primary mechanism of action of stimulant drugs exist and may play a key role in SUD development in males and females. Funding was provided by startup funds from V.U. School of Medicine, Department of Pharmacology and the National Institutes of Health (NIH) (to E.S.C.). Funds from the National Institute of Drug Abuse (NIDA) DA042111 and DA048931, the Brain and Behavior Research Foundation, Whitehall Foundation, and the Edward J Mallinckrodt Jr. Foundation (E.S.C) also supported this work.

Background/Objectives: Dopamine is well known for its role in reward learning, where phasic activity encodes prediction errors and supports the formation of cue–outcome associations. More recently, dopamine has been implicated in encoding salience, novelty, and aversive events, suggesting a broader function in shaping learning and memory across motivational contexts. However, how accumbal dopamine contributes to the recovery of extinguished fear, a process central to relapse in anxiety and trauma-related disorders, remains unclear. Methods: Here, we examined NAc core dopamine dynamics during shock-induced reinstatement of an extinguished fear memory. Using dLight fiber photometry, we found that a sustained decrease in baseline dopamine marked extinction recall. In contrast, an unexpected reminder footshock prevented this reduction, maintaining dopamine levels near baseline as freezing behavior re-emerged. Results: The reminder shock also evoked a transient dopamine peak, and optogenetic manipulations demonstrated that dopamine signaling during this period bidirectionally modulated reinstatement, with enhancement of dopamine release increasing reinstatement and inhibition of dopamine terminals markedly attenuating it. Together, these results demonstrate that unexpected aversive events reset NAc core dopamine levels and gate the return of extinguished fear. Conclusions: By revealing that accumbal dopamine contributes to fear recovery, this work broadens current models of dopamine function and identifies a neural mechanism through which surprising events may promote relapse of aversive memories in anxiety- and stress-related disorders.