Perinatal Exposure to Delta-9-tetrahydrocannabinol (THC) Alters Goal-Directed Behavior and Dopamine Functioning in Wistar Rats

Perinatal Exposure to Delta-9-tetrahydrocannabinol (THC) Alters Goal-Directed Behavior and Dopamine Functioning in Wistar Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Perinatal Exposure to Delta-9-tetrahydrocannabinol (THC) Alters Goal-Directed Behavior and Dopamine Functioning in Wistar Rats Helen Sable, Monica Carbajal, Victoria Williams, Rebecca Crenshaw, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6207382/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cannabis use during pregnancy is common as many pregnant women consider cannabis as a safe way to alleviate symptoms associated with pregnancy because it is “natural”. However, clinical evidence links perinatal exposure to cannabis to externalizing behavior in offspring including impulsivity, hyperactivity, and substance use. In preclinical research, most studies focus on exposure to the psychoactive constituent of cannabis, delta-9-tetrahydrocannabinol (THC). THC is lipophilic allowing it to cross the placental barrier and be secreted in maternal milk, thereby exposing the fetus/neonate. We used operant procedures to measure motivation to work for rewards, impulsive action, and impulsive choice in adult offspring perinatally exposed to 0 or 5 mg/kg/day THC. Differential reinforcement of high rates (DRH) was used to assess motivation, differential reinforcement of low rates (DRL) was used to examine impulsive action and delay discounting (DD) was used to measure impulsive choice. We also measured dopamine (DA) functioning in the medial prefrontal cortex (mPFC) and in the nucleus accumbens (NAc) via in vivo fixed potential amperometry in littermates of rats that completed behavioral testing. Perinatal exposure to THC dramatically decreased responding for reinforcers during DRH in offspring of both sexes, decreased reinforcers earned and trials completed during DRL, but had no effect on impulsive choice as measured during DD. In addition, perinatal THC exposure did not alter baseline DA release in the NAc or mPFC, but did attenuate the dopaminergic response to cocaine in the NAc. These results suggest perinatal exposure to THC may decrease motivation to work for reinforcers and provide neurochemical support for the “amotivational state” resulting from perinatal THC exposure. Biological sciences/Neuroscience/Motivation Biological sciences/Neuroscience/Synaptic transmission/Neurotransmitters Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In the United States, cannabis (aka marijuana) use for medical and recreational purposes is increasing. Since legalization, the perception that cannabis use produces little to no harm has increased [ 1 , 2 ] and this increased perception of safety is also present in pregnant women [ 3 ], many of whom view cannabis as a “natural” way to alleviate symptoms associated with pregnancy [ 4 ]. Women who use cannabis three months before becoming pregnant experienced more severe morning sickness compared to those who did not and increased severity of morning sickness was associated with higher cannabis use during pregnancy suggesting prior use may worsen morning sickness and promote continued use during pregnancy [ 5 ]. Cannabis's primary psychoactive constituent, delta-9-tetrahydrocannabinol (THC), crosses the placenta and is found in breastmilk, thereby exposing the fetus during gestation and the neonate during lactation [ 6 , 7 ]. Children of women who used cannabis during pregnancy demonstrate an increase in impulsivity, hyperactivity, and substance use and diagnoses of attention-deficit hyperactivity disorder (ADHD) and substance use disorder (SUD) [ 8 , 9 ]. In response to these concerns, the National Institutes of Health (NIH) prioritized better characterization of cannabis-associated developmental neurotoxicology, alongside recommendations from the Surgeon General of the United States to discourage cannabis use during pregnancy [ 10 ]. Perinatal THC exposure can result in long-term, neurobiological changes, due in part to THC’s activation of CB1 cannabinoid receptors (CB1R) [ 11 ]. CB1R has been shown to play a crucial role in mediating the effects of cannabinoids on dopamine- (DA) related behaviors including motivation to engage in goal-directed behavior and executive functions [ 12 , 13 ] which are regulated by DA in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) [ 14 – 17 ]. Dopaminergic terminals in the mPFC and NAc do not contain CB1R [ 18 , 19 ], but cannabinoids appear to modulate DA transmission indirectly via CB1R activation on GABAergic and glutamatergic projections in close proximity to DA neurons in the ventral tegmental area (VTA) which project to the mPFC and NAc [ 20 – 22 ]. Thus, the purpose of this study was to examine the long-term effects of perinatal THC exposure on goal-directed behavior and related DA functioning. We used a differential reinforcement of high rates (DRH) task to assess motivation, a differential reinforcement of low rates (DRL) task to examine impulsive action, and a delay discounting (DD) task to measure impulsive choice. In addition, we employed in vivo fixed potential amperometry (FPA) to measure DA dynamics in the mPFC and NAc at baseline and in the NAc after a systemic cocaine challenge. We hypothesized adult offspring perinatally exposed to THC would exhibit a) deficits in impulsive action and impulsive choice, and b) disruptions in DA dynamics (i.e., release, half-life) in the NAc and mPFC at baseline and in the NAc after a systemic cocaine challenge. Method Subjects Twenty, nulliparous, female Wister rats were purchased from Envigo (Indianapolis) and shipped to the University of Memphis at 60 days old. They were maintained in a temperature- and humidity-controlled room (22° C, 40–55% humidity) on a 12-hr reverse light/dark cycle (lights off 0700 hr) with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Memphis and were in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals [ 23 ] and the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research [ 24 ]. THC Exposure Each female rat was weighed and received an undosed vanilla wafer covered with Nutella to promote eating this novel food item for a few days before dosing. Each female was randomly assigned to either the THC or vehicle exposure group. The THC (suspended in 5 ml of 200-proof ethanol) was obtained from NIH and was diluted with sesame oil to make a dosing solution of 11.0 mg/ml. The vehicle solution had 5 ml of 200-proof ethanol added to the unadulterated sesame oil. Each day, either the THC or vehicle solution was pipetted at a volume of 0.45 mL/kg onto one-half of a vanilla wafer cookie smothered in Nutella to yield final doses of 5.0 mg/kg/day (n = 10) or 0 mg/kg/day (n = 10) for the THC and vehicle dams, respectively. An oral dose of 5 mg/kg THC in rats corresponds to a moderate level of exposure in humans while correcting for differences in route of administration and body surface area [ 25 , 26 ]. Each cookie was placed under a fume hood for several hours to allow the ethanol to evaporate. Females received their cookie daily beginning 14 days prior to breeding and ending when a litter was 14 days old to ensure pups did not directly consume the cookie. During breeding, two females were paired with an undosed male for eight days. Each female was removed to consume its daily cookie which was visually confirmed before placing the female back with the same male. Three control dams failed to get pregnant resulting in 10 THC-exposed litters and 7 vehicle-exposed litters. The litter was culled to 10 pups on postnatal day (PND) 2 and additional pups of the same age and exposure group were fostered to smaller litters to ensure all litters had 10 pups. Fostered pups were marked by ear clip and not used for behavioral or neurochemistry testing. Pups were weaned on PND 21, group-housed by litter and sex, and allowed to age to adulthood. Separate male/female littermate pairs were used for DRH/DRL, DD, and FPA testing. DRH/DRL and DD each included 10 THC and 7 vehicle littermate pairs. Some litters were heavily male or female, so FPA only included 8 THC and 6 vehicle littermate pairs. Neurotoxicological Measures Gestational weight gain, litter size, percent live births, and percent male was determined at birth. Lactational weight gain, implantation sites (at necropsy), dam brain:body weight, dam liver:body weight, male pup brain:body weight, male pup liver:body weight, female pup brain:body weight, and female pup liver:body weight was determined at weaning. Postnatal weight gain was measured separately for each sex on PND 0, 7, 14, and the time it took for the dam to retrieve a single pup from the nest was measured as an indicator of maternal behavior on PND 2, 4, 6, and 8. Behavioral Measures When the offspring reached PND 60 ± 10, mild food restriction (i.e., 85–90% of free-feeding weight) was implemented in offspring assigned to operant testing to ensure the rats would work for food reinforcers. Operant testing occurred at the same time of day, seven days a week (one session/day). Body weights were recorded daily throughout food restriction, and supplemental food was provided daily after operant testing to maintain rats at their target body weights. Apparatus. Operant testing was performed in 18 automated, rat operant chambers (Med Associates Inc., St. Albans, VT, USA) housed in sound-attenuating cubicles equipped with a fan for ventilation. The exact specifications of the operant chambers are described elsewhere [ 27 , 28 ]. Dustless grain-based precision pellets (45 mg; Bio-Serv, Flemington, NJ, USA) were used as reinforcers. Med-PC V software (Med Associates) was used to present the operant programs and record data. Autoshaping and fixed ratio training. Details about these programs, implemented to train the rats to press both response levers, have been previously published [ 27 , 28 ]. Delay-discounting (DD). Both the right and left levers were presented. The rat received one pellet delivered immediately for a press on one lever, but three pellets delivered after a 0, 4, 8, 12, or 16 s delay for a press on the other lever (counterbalanced across rats). The delay for the larger reward increased every 10 trials, based on the sequence above, for a total of 50 trials per session. If the rat pressed the immediate reward lever, the scheduled delay for the larger reward lever was implemented after reinforcer delivery to ensure overall session length was the same for all rats. Rats completed 25 sessions. Differential reinforcement of high rates (DRH) . For DRH, only the left response lever was extended. To earn a reinforcer, the rat was required to make a minimum number of lever presses within a specific time interval. Three sessions of DRH testing were conducted (1 sessions/day), including DRH 2:1 (2 lever presses within 1 sec), DRH 4:2 (four lever presses in 2 seconds), and DRH 8:4 (eight lever presses within four seconds). Each session terminated after 200 reinforcers were delivered or 90 min elapsed, whichever occurred first. Differential reinforcement of low response task (DRL). For DRL only the right lever was extended. Rats had to press the lever to start a trial and then needed to wait 5 (DRL 5), 10 (DRL 10), or 15 (DRL 15) sec before pressing the lever again to earn a reinforcer. A premature response during the required inter-response time (IRT) ended the trial with no reinforcer delivered. DRL 5 and DRL 10 were transitional phases lasting two sessions, while DRL 15 lasted 30 sessions. Only one session occurred/day and terminated after 90 min. Dopamine Measures Fixed potential amperometry occurred when the offspring reached 60 ± 10 days of age and is also known as continuous amperometry. When coupled with carbon fiber recording microelectrodes, FPA has been confirmed as a valid technique for real-time, in vivo monitoring of stimulation‐evoked DA release [ 29 – 33 ]. Pharmacological studies have confirmed the measured current changes in the NAc and mPFC of both mice and rats to be DA-dependent [ 32 , 34 , 35 ]. Surgical set-up. Rats were anesthetized with urethane (1.5 g/kg, IP), placed on a heating pad with a temperature monitor (37° C ± 1°), and put into a stereotaxic frame inside a Faraday cage. Stereotaxic coordinates were determined according to the rat atlas of Paxinos and Watson [ 36 ] and measured in mm from bregma, midline, and dura. First, a stimulating electrode was placed into the left medial forebrain bundle (MFB) (AP -4.2, ML + 1.8, and DV -7.8) followed by a stainless-steel auxiliary and Ag/AgCl reference combination electrode placed contralaterally on the surface of the cortex, -2.0 mm from bregma. The recording electrode (active recording surface of 500 µm length x 7 µm o.d.) was placed into the left mPFC (AP -2.7, ML + 0.8, DV -4.0) or NAc core (AP -1.6, ML + 1.5, and DV -7.4). An auxiliary electrode applied a fixed potential of + 0.8V, and DA efflux was measured using an electrometer (ED401 e-corder 401 and EA162 Picostat, eDAQ Inc.) filtered at 50 Hz, allowing for continuous monitoring of DA oxidation (10,000 samples/sec). Stimulation parameters. Cathodal pulses were delivered to the stimulating electrode via an optical isolator and programmable pulse generator (Iso-flex/Master-8, AMPI). Stimulation parameters were altered as the experiment progressed to assess different aspects of DA transmission. Initially, 20 monophasic stimulation pulses (800 µA intensity, 0.5 ms pulse duration) were delivered every 30 sec while adjusting electrode depths to establish optimal baseline responses in the NAc. These stimulation parameters were chosen to mimic phasic firing of dopaminergic neurons [ 37 , 38 ]. As in our previous studies [ 32 , 33 , 39 ], an optimal response was first found in the NAc to ensure proper placement of the stimulating electrode in the MFB. Recording electrodes were then moved to the mPFC, and stimulation parameters consisted of 50 monophasic 0.5 ms duration pulses (800 µA) at 50 Hz every 30 s for 10 min. DA release was defined as the magnitude of the stimulation-evoked response (from the pre-stimulation baseline to peak-stimulated response). The synaptic half-life of DA was defined as the time required for evoked DA to clear from the synapse (from peak DA release and restoration to 50% of baseline). Once baseline DA release and synaptic half-life were measured in the mPFC, we moved the recording electrode back to the NAc and assessed DA autoreceptor functioning thereby applying a pair of test stimuli (T1 and T2, each 10 pulses at 50 Hz separated by 10 s) every 60 s [ 34 , 35 ]. A set of conditioning pre-pulses (0, 1, 5, 10, 20, 40, or 80 pulses at 15 Hz) was delivered prior to T2 with 0.3s between the end of the conditioning pulse train and T2. The percent autoreceptor inhibition was defined as percent change in amplitude of T2 stimulations with respect to T1 (T2/T1 x 100) for each set of conditioning pre-pulses. After autoreceptor testing, stimulation parameters were returned to 20 pulses at 50 Hz every 30 sec, and after a 5-min baseline recording, rats received a cocaine injection (10/mg/kg, IP). Stimulations and amperometric recordings continued for 60 min post-cocaine. Measurements of DA release and synaptic half-life were converted to percent change following cocaine with pre-drug responses being 100%. Following the drug challenge, a continuous 3-minute MFB stimulation (9000 pulses at 50Hz) was used to determine available presynaptic DA supply stores. After amperometric recordings, recording electrodes were calibrated using an in vitro flow injection system and standard solutions of DA (0.2–1.2 µM). Histology. To verify electrode placements, a direct anodic current (100 µAmps for 10 sec) was applied through the stimulating electrode. Rats were euthanized using a 0.25-ml intracardiac injection of urethane (0.345 g/ml). Brains were removed and placed in a solution of 10% formalin and 0.1% potassium ferricyanide for at least one week, then 30% sucrose/10% formalin for another week. Brains were sliced into 30 µm coronal sections using a cryostat at − 20°C and electrode placements assessed under a light microscope and recorded on coronal diagrams [ 36 ]. Drugs Delta-9-tetrahydrocannabinol (THC) was donated from the NIH Drug Supply Program. Urethane (U2500) and cocaine (C5776) were obtained from Sigma-Aldrich and mixed with 0.9% saline. Data Analyses Data were analyzed using SPSS (IBM) version 29.0. The criterion for statistical significance for omnibus analyses was p p > .05) accompanied by a large (η p ² > .14) effect size [ 40 ], post hoc analyses were conducted to reduce the risk of Type II error. We also used a nested design with sex nested within litter, as it offers greater power, controls for litter effects, and also reduces the risk of Type II error. If a sphericity violation occurred for a within-subjects effect, a Greenhouse-Geisser correction using adjusted degrees of freedom was used to reduce the risk of Type I error because ε < 0.75 in all cases [ 41 ]. All neurotoxicological, behavioral, and DA neurochemistry dependent variables analyzed, along with the omnibus analyses conducted, are presented in Supplemental Table 1. In the interest of brevity, only effects related to exposure or sex are presented in the results. For DRL 15 we also conducted peak deviation analyses to quantify burst ratio (i.e., < 2.5 sec IRT responses) peak area, and peak location. Individuals with high impulsivity tend to produce a large number of burst responses characterized by rapidly occurring non-reinforced responses occurring close together in time [ 42 ]. Peak deviation analysis compares the obtained IRT response distribution to a corresponding random IRT distribution. The latter is a negative exponential probability function that predicts the relative distribution of IRT responses verses a distribution based on the same number of responses categorized randomly with respect to time [ 43 ]. The random IRT distribution shows that based on chance, shorter IRTs will occur more often than longer ones. Peak deviation analysis provides a quantitative measure of how much the actual peak location and/or peak area differ versus random performance [ 43 – 45 ]. Results Neurotoxicology Outcomes. See Supplemental Table 1. There was an effect of exposure on lactational weight gain [ t (15) =-2.322, p = .035], dam brain:body weight [ t (15) = 2.309, p = .036], and male pup brain:body weight [ t (15) = 1.999, p = .064, η p ²=.210]. The lactational weight gain in THC -exposed dams was lower compared to vehicle-exposed dams, and brain:body weight ratio in THC-exposed dams and male pups was higher than vehicle-exposed dams and male pups, respectively. There was also a significant main effect of exposure on postnatal weight gain, [ F (1,15) = 6.351, p = .024] as well as a significant exposure × day interaction, [ F (1.246,18.693) = 7.342, p = 0.010]. Post hoc analysis revealed a significant difference between the exposure groups on PND 14 ( p = .009) and PND 21 ( p = .019). There were no other exposure- or sex-related differences at birth or weaning. Maternal Behavior. There was not a main effect of exposure, nor an exposure × day interaction on the latency of pup retrieval (Supplemental Table 1). Behavioral Measures. DD. No effects related to exposure or sex were found for any dependent measure. Data from the last testing block (days 21–25) are presented in Supplemental Fig. 1. DRH. The main effect of exposure was significant, F (1,15) = 4.618, p = 0.048. In addition, while the interaction of exposure × phase did not reach the criterion for significance, a large effect size was present, F (1.45,17.180) = 3.854, p = .061, η p ²=.220. Post hoc analysis revealed a difference between the exposure groups during DRH 2:1 ( p = .031) and DRH 8:4 ( p = .036). As seen in Fig. 1 , rats perinatally exposed to THC elicited significantly fewer lever presses during these two phases. DRL 15. Data across the 30 days of DRL 15 were averaged into 6, 5-day blocks. Reinforced:Non-reinforced responses . The exposure × block interaction only approached the criterion for significance [ F (3.090,46.343) = 2.686, p = 0.056] even though a large effect size was present (η p ²=.152). Post hoc analysis revealed vehicle rats had a higher ratio than THC-exposed rats in block 2 ( p = .064, η p ²=.210, Fig. 2 A). Reinforcers earned. There was a significant main effect of exposure [ F (1,15) = 5.476, p = .034] and significant exposure × sex interaction [ F (1,15) = 19.266, p = .001]. The THC-exposure group had fewer reinforcers overall compared to the vehicle-exposure group, an effect driven by the high number of reinforcers earned in vehicle males compared to THC-exposed males ( p = .001) and vehicle females ( p = .001) (Fig. 2 B). Total trials. There were significant exposure × block [ F (2.855,42.821) = 3.165, p = .036] and sex × block [ F (1.720, 25.796) = 5.794, p = .011] interactions. Post hoc analysis revealed a difference between exposure groups in block 4 ( p = .059, η p ²=.218), block 5 ( p = .016), and block 6 ( p = .055, η p ²=.224; Fig. 2 C), and a sex difference in block 1 ( p = .033) and block 6 ( p = .002). Females completed more trials in block 1, while males completed more in block 6. Peak Deviation Analysis . Analyses of burst ratios revealed a main effect of exposure in block 1 [ F (1,15) = 4.058, p = .062, η p ²=.203], block 2 [ F (1,15) = 4.238, p = .057, η p ²=.220], and block 6 [ F (1,15) = 6.630, p = .021], as well as an exposure × sex interaction in block 2 [ F (1,15) = 3.383, p = .086, η p ²=.184]. In all three blocks, the burst ratio was lower for the THC-exposed compared to vehicle-exposed rats (Fig. 3 ). In addition, in block 2, the effect of exposure was sex-specific, as it was present in the males ( p = .006) but not females (Fig. 3 middle). We calculated the difference between the negative exponential curves and the “debursted” relative proportion of responses for the pause IRTs (i.e., IRTs > 2.5s) [ 43 , 45 ]. The difference curves were used to determine peak location and peak area within each testing block. For peak area, there was a significant main effect of exposure in block 1 only [ F (1,15) = 5.392, p = .035], with a higher peak area in the THC-exposed rats (Fig. 4A, B). For peak location, there was a significant main effect of exposure only in block 2, [ F (1,15) = 5.719, p = .030]. As seen in Fig. 4C and 4D, the peak location occurred earlier in the THC-exposed rats. Dopamine measures mPFC and NAc DA Release and Half-life. Analysis of baseline stimulation-evoked DA release and the synaptic half-life of DA did not reveal a main effect of exposure, sex, or an exposure x sex interaction in either the mPFC (Supplemental Fig. 2A, B, C) or the NAc (Supplemental Fig. 2D, E, F). Histological placements of the stimulating and recording electrodes are shown in Supplemental Fig. 2G. NAc DA Autoreceptor Functioning. There was not a main effect of exposure or sex, nor interactions of exposure × sex, exposure × prepulse, sex × prepulse, or exposure × sex × prepulse (Supplemental Fig. 3A,B). NAc DA Release and Half-life After Cocaine Challenge. Peak DA release and DA half-life were converted into percent change with pre-drug responses being 100%. Analyses of the percent change in release post-cocaine revealed a significant main effect of exposure [ F (1,12) = 7.499, p = .018] and a significant exposure x time interaction [ F (2.755,33.058) = 4.035, p = .019]. Rats perinatally exposed to THC exhibited an attenuated increase in DA release post-cocaine compared to controls (Fig. 5 A), at all-time points starting at 10 minutes post-cocaine (Fig. 5 B). Likewise, a significant main effect of exposure was also found for the percent change in the synaptic half-life of DA post-cocaine [ F (1,12) = 7.830, p = .016]. As seen in Fig. 5 C, the percent change in DA half-life post-cocaine was reduced in rats perinatally exposed to THC. NAc DA Supply. Analysis did not reveal a main effect of exposure or sex. The exposure × sex interaction approached the criterion for significance [ F (1,12) = 3.345, p = .092] with a large effect size (η p ²=.218). However, post hoc simple effects analysis did not reveal an effect of exposure in either sex or an effect of sex in the vehicle or THC-exposed rats (Supplemental Fig. 3C,D). Discussion Does Perinatal THC Exposure Increase Impulsivity? Our results demonstrate perinatal exposure to THC produces deficits in goal-directed behavior. There appeared to be a deficit in impulsive action in rats perinatally exposed to THC, particularly during the early testing blocks of DRL 15. Offspring exposed to THC had an increase in the peak area, decrease in peak latency, and lower reinforced:nonreinforced response ratio during these early testing blocks, which suggests an impulsivity problem. However, surprisingly, DRL 15 burst responses (IRTs < 2.5 sec) were lower in THC-exposed rats. High impulsivity is typically associated with a larger number of burst responses [ 42 ], so the decrease in THC-exposed rats was inconsistent with an increase in impulsivity and with the increase in peak area and location observed in the same animals. Notably, the degree of burst responding observed in our vehicle controls was similar to controls we have examined in other studies [ 46 – 49 ] and the decrease observed in THC-exposed rats was lower than we have seen previously. This led us to consider alternative explanations for the discrepancy in these outcomes. Recall we found no evidence of a deficit in impulsive choice between vehicle- and THC-exposed rats. While it has been shown there is a neural dissociation between impulsive choice and impulsive action [ 50 ], our DD results further support our theory that the effects in THC-exposed rats during DRL 15 were not entirely reflective of an impulsivity problem. During DRL 15, THC-exposed rats earned significantly fewer reinforcers overall (across all testing blocks) and completed fewer trials during later testing blocks. Likewise, during DRH, THC-exposed rats had a significantly lower number of lever presses during two phases of DRH (2:1 and 8:4). Taken together, these results suggest THC-exposed rats were less motivated to work to earn reinforcers. While THC-exposed rats did exhibit a lower reinforced:nonreinforced response ratio indicative of an impulsivity problem, this effect occurred only in block 2. Further evidence of an amotivational state is seen when looking at the burst responses, which reflect lever presses occurring close together in time. A lack of motivation in the THC-exposed rats could also explain the decrease in burst responses relative to vehicle controls. While children of women who use cannabis during pregnancy demonstrate an increase in impulsivity and diagnoses of ADHD [ 8 , 9 , 51 , 52 ], there is also increasing evidence that ADHD individuals have deficits in motivation [ 53 ]. Children diagnosed with ADHD require stronger incentives to motivate them to modify their behavior than children not diagnosed with ADHD [ 54 ]. Does Perinatal THC Exposure Alter Dopamine Release? Dopamine release in the NAc has a direct influence on instrumental goal-directed behavior [ 55 – 58 ], especially when engaging in high-effort reward-seeking [ 59 – 61 ]. NAc disruption leads to engagement in behaviors requiring less effort, even when the outcome is less valuable [ 62 – 65 ]. In this study, perinatal THC exposure did not alter baseline stimulation-evoked DA release in the NAc, but THC-exposed rats displayed an attenuated dopaminergic response to cocaine. Our measures of DA neuronal supply did not differ between THC-exposed rats and controls. Thus, our findings suggest perinatal THC exposure altered the presynaptic functioning of DA neurons related to the mechanisms of cocaine (DAT inhibition), and/or magnified it to an observable level during the cocaine challenge. Others have similarly shown animals exposed to perinatal THC exposure display normal indices of baseline DA activity but different responses to drugs targeting D1, D2, and DAT [ 66 ], while chronic cannabis users have displayed baseline measures of DA receptor availability similar to controls but markedly blunted responses when challenged with the DAT inhibiter methylphenidate [ 67 ]. As DA has been argued to mediate the “elasticity of demand” (i.e. sensitivity to increase in price) of a reinforcer [ 68 ], a behavioral economics approach suggests the attenuated DA response to cocaine in THC-exposured rats should devalue this reinforcer and reduce the effort these rats are willing to exert for it. By extension, the decreased lever pressing during DRH and DRL15 burst responding we observed in THC-exposured rats may represent a similar DA-driven devaluation of the food reinforcer. Future research is planned to examine this question more directly. Summary This project demonstrated perinatal THC exposure impaired goal-directed behavior, as exposed rats exhibited disengagement during high effort responding, earned fewer reinforcers, and completed fewer trials, indicative of a motivational deficit. The attenuated NAc DA response to cocaine suggests reinforcer devaluation may underlie this reduced effort. These findings highlight the potential neurodevelopmental risks of maternal cannabis use, helping to inform public health policy on its safety during pregnancy and lactation. Declarations Acknowledgements: Appreciation is extended to Donny Ray for assistance with lab animal care, Dr. Timothy Mandrell for his veterinary support, and Dr. Randy Floyd for his suggested revisions to the manuscript. Data Availability Statement: The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Conflict of Interest Statement: All authors declare no conflict of interest. Funding Sources: This work was supported by the California Doctoral Incentive Program (MSC), NSF 2051105 (LGB, CMD), and a Dunavant professorship (HJKS). References Nathan DL, Clark HW, Elders J. The Physicians' Case for Marijuana Legalization. Am J Public Health. 2017;107(11):1746-47. Miech RA, Johnston L, O'Malley PM, Bachman JG, Schulenberg J, Patrick ME. Trends in use of marijuana and attitudes toward marijuana among youth before and after decriminalization: the case of California 2007-2013. 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Behav Brain Res. 2019;362:140-51. Holloway ZR, Freels TG, Comstock JF, Nolen HG, Sable HJ, Lester DB. Comparing phasic dopamine dynamics in the striatum, nucleus accumbens, amygdala, and medial prefrontal cortex. Synapse. 2018:e22074. Sable HJK, Paige NB, Nalan PA, Pace RL, Hicks CB, Regan SL, et al. Phasic dopamine release in two different rat models of attention-deficit/hyperactivity disorder: Spontaneously hypertensive rats (SHR) versus Lphn3 knockout rats. Neuroscience. 2025;567:150-62. Mittleman G, Call SB, Cockroft JL, Goldowitz D, Matthews DB, Blaha CD. Dopamine dynamics associated with, and resulting from, schedule-induced alcohol self-administration: analyses in dopamine transporter knockout mice. Alcohol. 2011;45(4):325-39. Tye KM, Mirzabekov JJ, Warden MR, Ferenczi EA, Tsai HC, Finkelstein J, et al. Dopamine neurons modulate neural encoding and expression of depression-related behaviour. Nature. 2013;493(7433):537-41. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates . 4th ed. ed. Academic Press: San Diego, CA; 1998. Hyland BI, Reynolds JN, Hay J, Perk CG, Miller R. Firing modes of midbrain dopamine cells in the freely moving rat. Neuroscience. 2002;114(2):475-92. Dreyer JK, Herrik KF, Berg RW, Hounsgaard JD. Influence of phasic and tonic dopamine release on receptor activation. J Neurosci. 2010;30(42):14273-83. Freels TG, Gabriel DBK, Lester DB, Simon NW. Risky decision-making predicts dopamine release dynamics in nucleus accumbens shell. Neuropsychopharmacology. 2020;45(2):266-75. Richardson J. Eta squared and partial eta squared as measures of effect size in educational research. Educational Research Review. 2011;6:135-47. Maxwell SE, Delaney HD. Designing experiments and analyzing data: A model comparison perspective . 2nd ed. Lawrence Erlbaum Associates Publishers: Mahwah, NJ; 1999. Pattij T, Broersen LM, Peter S, Olivier B. Impulsive-like behavior in differential-reinforcement-of-low-rate 36 s responding in mice depends on training history. Neurosci Lett. 2004;354(2):169-71. Richards JB, Seiden LS. A quantitative interresponse-time analysis of DRL performance differentiates similar effects of the antidepressant desipramine and the novel anxiolytic gepirone. J Exp Anal Behav. 1991;56(2):173-92. Barthelemy OJ, Richardson MA, Cabral HJ, Frank DA. Prenatal, perinatal, and adolescent exposure to marijuana: Relationships with aggressive behavior. Neurotoxicology and Teratology. 2016;58:60-77. Richards JB, Sabol KE, Seiden LS. DRL interresponse-time distributions: quantification by peak deviation analysis. J Exp Anal Behav. 1993;60(2):361-85. Sable HJ, MacDonnchadh JJ, Lee HW, Butawan M, Simpson RN, Krueger KM, et al. Working memory and hippocampal expression of BDNF, ARC, and P-STAT3 in rats: effects of diet and exercise. Nutr Neurosci. 2021:1-14. Sable HJ, Powers BE, Wang VC, Widholm JJ, Schantz SL. Alterations in DRH and DRL performance in rats developmentally exposed to an environmental PCB mixture. Neurotoxicol Teratol. 2006;28(5):548-56. Meyer AE, Miller MM, Nelms Sprowles JL, Levine LR, Sable HJ. A comparison of presynaptic and postsynaptic dopaminergic agonists on inhibitory control performance in rats perinatally exposed to PCBs. Neurotoxicol Teratol. 2015;50:11-22. Sable HJ, Eubig PA, Powers BE, Wang VC, Schantz SL. Developmental exposure to PCBs and/or MeHg: effects on a differential reinforcement of low rates (DRL) operant task before and after amphetamine drug challenge. Neurotoxicol Teratol. 2009;31(3):149-58. Wang Q, Chen C, Cai Y, Li S, Zhao X, Zheng L, et al. Dissociated neural substrates underlying impulsive choice and impulsive action. Neuroimage. 2016;134:540-49. Andrade C. Maternal Cannabis Use During Pregnancy and Neuropsychiatric Adverse Outcomes During Childhood and Early Adult Life. J Clin Psychiatry. 2025;86(1). Bassalov H, Yakirevich-Amir N, Reuveni I, Monk C, Florentin S, Bonne O, et al. Prenatal cannabis exposure and the risk for neuropsychiatric anomalies in the offspring: a systematic review and meta-analysis. Am J Obstet Gynecol. 2024;231(6):574-88.e8. Volkow ND, Wang GJ, Newcorn JH, Kollins SH, Wigal TL, Telang F, et al. Motivation deficit in ADHD is associated with dysfunction of the dopamine reward pathway. Mol Psychiatry. 2011;16(11):1147-54. Kollins SH, Newland MC, Critchfield TS. Human sensitivity to reinforcement in operant choice: How much do consequences matter? Psychon Bull Rev. 1997;4(2):208-20. Cacciapaglia F, Saddoris MP, Wightman RM, Carelli RM. Differential dopamine release dynamics in the nucleus accumbens core and shell track distinct aspects of goal-directed behavior for sucrose. Neuropharmacology. 2012;62(5-6):2050-6. Roitman MF, Stuber GD, Phillips PE, Wightman RM, Carelli RM. Dopamine operates as a subsecond modulator of food seeking. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2004;24(6):1265-71. Carelli RM. Nucleus accumbens cell firing and rapid dopamine signaling during goal-directed behaviors in rats. Neuropharmacology. 2004;47 Suppl 1:180-9. Cameron CM, Wightman RM, Carelli RM. Dynamics of rapid dopamine release in the nucleus accumbens during goal-directed behaviors for cocaine versus natural rewards. Neuropharmacology. 2014;86:319-28. Phillips PE, Walton ME, Jhou TC. Calculating utility: preclinical evidence for cost-benefit analysis by mesolimbic dopamine. Psychopharmacology (Berl). 2007;191(3):483-95. Salamone JD, Correa M, Farrar A, Mingote SM. Effort-related functions of nucleus accumbens dopamine and associated forebrain circuits. Psychopharmacology (Berl). 2007;191(3):461-82. Floresco SB, St Onge JR, Ghods-Sharifi S, Winstanley CA. Cortico-limbic-striatal circuits subserving different forms of cost-benefit decision making. Cogn Affect Behav Neurosci. 2008;8(4):375-89. Sokolowski JD, Salamone JD. The role of accumbens dopamine in lever pressing and response allocation: effects of 6-OHDA injected into core and dorsomedial shell. Pharmacol Biochem Behav. 1998;59(3):557-66. Cousins MS, Atherton A, Turner L, Salamone JD. Nucleus accumbens dopamine depletions alter relative response allocation in a T-maze cost/benefit task. Behav Brain Res. 1996;74(1-2):189-97. Salamone JD, Steinpreis RE, McCullough LD, Smith P, Grebel D, Mahan K. Haloperidol and nucleus accumbens dopamine depletion suppress lever pressing for food but increase free food consumption in a novel food choice procedure. Psychopharmacology (Berl). 1991;104(4):515-21. Salamone JD, Cousins MS, Snyder BJ. Behavioral functions of nucleus accumbens dopamine: empirical and conceptual problems with the anhedonia hypothesis. Neurosci Biobehav Rev. 1997;21(3):341-59. Ramos JA, De Miguel R, Cebeira M, Hernandez M, Fernández-Ruiz J. Exposure to cannabinoids in the development of endogenous cannabinoid system. Neurotox Res. 2002;4(4):363-72. Volkow ND, Wang GJ, Telang F, Fowler JS, Alexoff D, Logan J, et al. Decreased dopamine brain reactivity in marijuana abusers is associated with negative emotionality and addiction severity. Proc Natl Acad Sci U S A. 2014;111(30):E3149-56. Salamone JD, Correa M. Motivational views of reinforcement: implications for understanding the behavioral functions of nucleus accumbens dopamine. Behav Brain Res. 2002;137(1-2):3-25. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementalMaterials.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6207382","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":428942667,"identity":"84db98a0-4222-461a-86bd-ff0989c6cb5a","order_by":0,"name":"Helen Sable","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACA4YDDMwMBQxyIA4zkAsECcRoMWAwJkULRGViA5jBQIQWc8bjDz8XGNikr23vTntcUFDHwM+eY4BXi2XDGWPpGQZpudvOnN1uPMPgMINkzxv8WgwOnGGQ5jE4nLvtRu42IOMAg8ENArYYHDj++DePwf90s/tvQVrqGOwJazlgBjI8wewGL0gLMCgkCGo5Y2Y9wyDZcNuZ3O3GQBfySJx5VoBfy43jj28XVNjJmx0/u+0xz586Of725A14tTBIHIAz2UAED37lIMDfgKplFIyCUTAKRgEGAAA7akkABxXjfwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0768-9544","institution":"University of Memphis","correspondingAuthor":true,"prefix":"","firstName":"Helen","middleName":"","lastName":"Sable","suffix":""},{"id":428942668,"identity":"66f6c56b-5f93-4fd3-a53b-e8cb297456a7","order_by":1,"name":"Monica Carbajal","email":"","orcid":"","institution":"University of Memphis","correspondingAuthor":false,"prefix":"","firstName":"Monica","middleName":"","lastName":"Carbajal","suffix":""},{"id":428942669,"identity":"bab1686c-72a7-42e9-a446-681104c99cc3","order_by":2,"name":"Victoria Williams","email":"","orcid":"","institution":"University of Memphis","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"","lastName":"Williams","suffix":""},{"id":428942670,"identity":"28725910-9cf6-403e-a5bc-a4bbc62c340a","order_by":3,"name":"Rebecca Crenshaw","email":"","orcid":"","institution":"University of Memphis","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Crenshaw","suffix":""},{"id":428942671,"identity":"43e0e99e-cae0-408f-923a-d5760cf6164a","order_by":4,"name":"Laura Billings","email":"","orcid":"","institution":"Christian Brothers University","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Billings","suffix":""},{"id":428942672,"identity":"dca78605-6ce7-495b-a837-286253788747","order_by":5,"name":"Chelsea Dixon","email":"","orcid":"","institution":"University of Memphis","correspondingAuthor":false,"prefix":"","firstName":"Chelsea","middleName":"","lastName":"Dixon","suffix":""},{"id":428942673,"identity":"396e4eae-286f-4310-add5-ac44ca2c6d86","order_by":6,"name":"Deranda Lester","email":"","orcid":"","institution":"University of Memphis","correspondingAuthor":false,"prefix":"","firstName":"Deranda","middleName":"","lastName":"Lester","suffix":""}],"badges":[],"createdAt":"2025-03-12 00:35:35","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6207382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6207382/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78664226,"identity":"39a34aa9-9aa7-4cfa-b276-0fed314749ad","added_by":"auto","created_at":"2025-03-17 10:52:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53227,"visible":true,"origin":"","legend":"\u003cp\u003eVehicle-exposed rats pressed the lever significantly more overall (\u003cem\u003ep\u003c/em\u003e=.048), but particularly when completing DRH 2:1 and DRH 8:4. THC = delta-9-tetrahydrocannabinol; SEM = standard error of the mean\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6207382/v1/cc24505d33811a9c24414288.png"},{"id":78665159,"identity":"5a0fee41-8ce0-4e27-8994-0424ad58d5fe","added_by":"auto","created_at":"2025-03-17 11:00:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":294888,"visible":true,"origin":"","legend":"\u003cp\u003e(A)\u003cem\u003e \u003c/em\u003eTHC-exposed rats had a higher ratio of reinforced (rein) to nonreinforced (nonrein) responses than vehicle-exposed rats in block 2. (B) The vehicle-exposed rats earned more reinforcers overall (\u003cem\u003ep\u003c/em\u003e=.034) and vehicle-exposed males earned more reinforcers than males perinatally exposed to THC and females exposed to vehicle. (C) THC-exposed rats completed fewer trials than the vehicle group in blocks 4, 5, and 6. THC = delta-9-tetrahydrocannabinol; SEM = standard error of the mean\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6207382/v1/cf1b063f47287182cdfb19fb.png"},{"id":78664233,"identity":"b1bc47d9-fc49-4391-a31f-0b7766708c6f","added_by":"auto","created_at":"2025-03-17 10:52:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":414937,"visible":true,"origin":"","legend":"\u003cp\u003eThere was a main effect of exposure in block 1 (A), block 2 (B), and block 6 (C). In all three cases, the burst ratio was lower for the THC-exposed rats compared to the vehicle exposed rats. In block 2, the effect of exposure was sex specific, as the effect was present in the males but not the females (Figure 3 middle). \u0026nbsp;THC = delta-9-tetrahydrocannabinol; SEM = standard error of the mean\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6207382/v1/948a498776e9150a23ee597f.png"},{"id":78665160,"identity":"60d37ee8-31eb-432a-adf0-1ea6c4e0f0a8","added_by":"auto","created_at":"2025-03-17 11:00:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":575577,"visible":true,"origin":"","legend":"\u003cp\u003eThere was a main effect of exposure in block 1 only, with a higher peak area in the THC-exposed rats (A, B). For peak location, there was a main effect of exposure only in block 2, with the peak location occurring earlier in the THC-exposed rats (C, D). THC = delta-9-tetrahydrocannabinol; SEM = standard error of the mean\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6207382/v1/be2ab45cd9a1745384d41c38.png"},{"id":78665753,"identity":"2acfdcdf-d992-4226-b2cd-112a882c3b01","added_by":"auto","created_at":"2025-03-17 11:08:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":519540,"visible":true,"origin":"","legend":"\u003cp\u003eDopamine (DA) release in the NAc following cocaine administration. Example responses show the difference between baseline (dotted line) and post-cocaine (solid line) for both exposure groups (A). Rats perinatally exposed to THC exhibited a smaller percent change in DA release (B) and half-life (C) following cocaine. THC = delta-9-tetrahydrocannabinol; Error bars represent standard error of the mean (SEM)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6207382/v1/276bda959d9d32bf06f682b9.png"},{"id":80152924,"identity":"ec7bb93f-1841-4558-885d-ea64d58afb11","added_by":"auto","created_at":"2025-04-08 13:51:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2717019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6207382/v1/e1c25818-17fc-4a5b-8012-09e7e99fa192.pdf"},{"id":78664228,"identity":"6a8c1278-ed86-480d-b3ff-6c809b02d288","added_by":"auto","created_at":"2025-03-17 10:52:42","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":507837,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6207382/v1/a99c9f8dc4b7bb7332812630.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Perinatal Exposure to Delta-9-tetrahydrocannabinol (THC) Alters Goal-Directed Behavior and Dopamine Functioning in Wistar Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the United States, cannabis (aka marijuana) use for medical and recreational purposes is increasing. Since legalization, the perception that cannabis use produces little to no harm has increased [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and this increased perception of safety is also present in pregnant women [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], many of whom view cannabis as a \u0026ldquo;natural\u0026rdquo; way to alleviate symptoms associated with pregnancy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Women who use cannabis three months \u003cem\u003ebefore\u003c/em\u003e becoming pregnant experienced more severe morning sickness compared to those who did not and increased severity of morning sickness was associated with higher cannabis use \u003cem\u003eduring\u003c/em\u003e pregnancy suggesting prior use may worsen morning sickness and promote continued use during pregnancy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cannabis's primary psychoactive constituent, delta-9-tetrahydrocannabinol (THC), crosses the placenta and is found in breastmilk, thereby exposing the fetus during gestation and the neonate during lactation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Children of women who used cannabis during pregnancy demonstrate an increase in impulsivity, hyperactivity, and substance use and diagnoses of attention-deficit hyperactivity disorder (ADHD) and substance use disorder (SUD) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In response to these concerns, the National Institutes of Health (NIH) prioritized better characterization of cannabis-associated developmental neurotoxicology, alongside recommendations from the Surgeon General of the United States to discourage cannabis use during pregnancy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePerinatal THC exposure can result in long-term, neurobiological changes, due in part to THC\u0026rsquo;s activation of CB1 cannabinoid receptors (CB1R) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CB1R has been shown to play a crucial role in mediating the effects of cannabinoids on dopamine- (DA) related behaviors including motivation to engage in goal-directed behavior and executive functions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] which are regulated by DA in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Dopaminergic terminals in the mPFC and NAc do not contain CB1R [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], but cannabinoids appear to modulate DA transmission indirectly via CB1R activation on GABAergic and glutamatergic projections in close proximity to DA neurons in the ventral tegmental area (VTA) which project to the mPFC and NAc [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, the purpose of this study was to examine the long-term effects of perinatal THC exposure on goal-directed behavior and related DA functioning. We used a differential reinforcement of high rates (DRH) task to assess motivation, a differential reinforcement of low rates (DRL) task to examine impulsive action, and a delay discounting (DD) task to measure impulsive choice. In addition, we employed \u003cem\u003ein vivo\u003c/em\u003e fixed potential amperometry (FPA) to measure DA dynamics in the mPFC and NAc at baseline and in the NAc after a systemic cocaine challenge. We hypothesized adult offspring perinatally exposed to THC would exhibit a) deficits in impulsive action and impulsive choice, and b) disruptions in DA dynamics (i.e., release, half-life) in the NAc and mPFC at baseline and in the NAc after a systemic cocaine challenge.\u003c/p\u003e"},{"header":"Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003eTwenty, nulliparous, female Wister rats were purchased from Envigo (Indianapolis) and shipped to the University of Memphis at 60 days old. They were maintained in a temperature- and humidity-controlled room (22\u0026deg; C, 40\u0026ndash;55% humidity) on a 12-hr reverse light/dark cycle (lights off 0700 hr) with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Memphis and were in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTHC Exposure\u003c/h3\u003e\n\u003cp\u003eEach female rat was weighed and received an undosed vanilla wafer covered with Nutella to promote eating this novel food item for a few days before dosing. Each female was randomly assigned to either the THC or vehicle exposure group. The THC (suspended in 5 ml of 200-proof ethanol) was obtained from NIH and was diluted with sesame oil to make a dosing solution of 11.0 mg/ml. The vehicle solution had 5 ml of 200-proof ethanol added to the unadulterated sesame oil. Each day, either the THC or vehicle solution was pipetted at a volume of 0.45 mL/kg onto one-half of a vanilla wafer cookie smothered in Nutella to yield final doses of 5.0 mg/kg/day (n\u0026thinsp;=\u0026thinsp;10) or 0 mg/kg/day (n\u0026thinsp;=\u0026thinsp;10) for the THC and vehicle dams, respectively. An oral dose of 5 mg/kg THC in rats corresponds to a moderate level of exposure in humans while correcting for differences in route of administration and body surface area [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Each cookie was placed under a fume hood for several hours to allow the ethanol to evaporate. Females received their cookie daily beginning 14 days prior to breeding and ending when a litter was 14 days old to ensure pups did not directly consume the cookie.\u003c/p\u003e \u003cp\u003eDuring breeding, two females were paired with an undosed male for eight days. Each female was removed to consume its daily cookie which was visually confirmed before placing the female back with the same male. Three control dams failed to get pregnant resulting in 10 THC-exposed litters and 7 vehicle-exposed litters. The litter was culled to 10 pups on postnatal day (PND) 2 and additional pups of the same age and exposure group were fostered to smaller litters to ensure all litters had 10 pups. Fostered pups were marked by ear clip and not used for behavioral or neurochemistry testing. Pups were weaned on PND 21, group-housed by litter and sex, and allowed to age to adulthood. Separate male/female littermate pairs were used for DRH/DRL, DD, and FPA testing. DRH/DRL and DD each included 10 THC and 7 vehicle littermate pairs. Some litters were heavily male or female, so FPA only included 8 THC and 6 vehicle littermate pairs.\u003c/p\u003e\n\u003ch3\u003eNeurotoxicological Measures\u003c/h3\u003e\n\u003cp\u003eGestational weight gain, litter size, percent live births, and percent male was determined at birth. Lactational weight gain, implantation sites (at necropsy), dam brain:body weight, dam liver:body weight, male pup brain:body weight, male pup liver:body weight, female pup brain:body weight, and female pup liver:body weight was determined at weaning. Postnatal weight gain was measured separately for each sex on PND 0, 7, 14, and the time it took for the dam to retrieve a single pup from the nest was measured as an indicator of maternal behavior on PND 2, 4, 6, and 8.\u003c/p\u003e\n\u003ch3\u003eBehavioral Measures\u003c/h3\u003e\n\u003cp\u003eWhen the offspring reached PND 60\u0026thinsp;\u0026plusmn;\u0026thinsp;10, mild food restriction (i.e., 85\u0026ndash;90% of free-feeding weight) was implemented in offspring assigned to operant testing to ensure the rats would work for food reinforcers. Operant testing occurred at the same time of day, seven days a week (one session/day). Body weights were recorded daily throughout food restriction, and supplemental food was provided daily after operant testing to maintain rats at their target body weights.\u003c/p\u003e \u003cp\u003e \u003cem\u003eApparatus.\u003c/em\u003e Operant testing was performed in 18 automated, rat operant chambers (Med Associates Inc., St. Albans, VT, USA) housed in sound-attenuating cubicles equipped with a fan for ventilation. The exact specifications of the operant chambers are described elsewhere [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Dustless grain-based precision pellets (45 mg; Bio-Serv, Flemington, NJ, USA) were used as reinforcers. Med-PC V software (Med Associates) was used to present the operant programs and record data.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAutoshaping and fixed ratio training.\u003c/em\u003e Details about these programs, implemented to train the rats to press both response levers, have been previously published [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eDelay-discounting (DD).\u003c/em\u003e Both the right and left levers were presented. The rat received one pellet delivered immediately for a press on one lever, but three pellets delivered after a 0, 4, 8, 12, or 16 s delay for a press on the other lever (counterbalanced across rats). The delay for the larger reward increased every 10 trials, based on the sequence above, for a total of 50 trials per session. If the rat pressed the immediate reward lever, the scheduled delay for the larger reward lever was implemented after reinforcer delivery to ensure overall session length was the same for all rats. Rats completed 25 sessions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferential reinforcement of high rates (DRH)\u003c/em\u003e. For DRH, only the left response lever was extended. To earn a reinforcer, the rat was required to make a minimum number of lever presses within a specific time interval. Three sessions of DRH testing were conducted (1 sessions/day), including DRH 2:1 (2 lever presses within 1 sec), DRH 4:2 (four lever presses in 2 seconds), and DRH 8:4 (eight lever presses within four seconds). Each session terminated after 200 reinforcers were delivered or 90 min elapsed, whichever occurred first.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferential reinforcement of low response task (DRL).\u003c/em\u003e For DRL only the right lever was extended. Rats had to press the lever to start a trial and then needed to wait 5 (DRL 5), 10 (DRL 10), or 15 (DRL 15) sec before pressing the lever again to earn a reinforcer. A premature response during the required inter-response time (IRT) ended the trial with no reinforcer delivered. DRL 5 and DRL 10 were transitional phases lasting two sessions, while DRL 15 lasted 30 sessions. Only one session occurred/day and terminated after 90 min.\u003c/p\u003e\n\u003ch3\u003eDopamine Measures\u003c/h3\u003e\n\u003cp\u003eFixed potential amperometry occurred when the offspring reached 60\u0026thinsp;\u0026plusmn;\u0026thinsp;10 days of age and is also known as continuous amperometry. When coupled with carbon fiber recording microelectrodes, FPA has been confirmed as a valid technique for real-time, in vivo monitoring of stimulation‐evoked DA release [\u003cspan additionalcitationids=\"CR30 CR31 CR32\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Pharmacological studies have confirmed the measured current changes in the NAc and mPFC of both mice and rats to be DA-dependent [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eSurgical set-up.\u003c/em\u003e Rats were anesthetized with urethane (1.5 g/kg, IP), placed on a heating pad with a temperature monitor (37\u0026deg; C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;), and put into a stereotaxic frame inside a Faraday cage. Stereotaxic coordinates were determined according to the rat atlas of Paxinos and Watson [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and measured in mm from bregma, midline, and dura. First, a stimulating electrode was placed into the left medial forebrain bundle (MFB) (AP -4.2, ML\u0026thinsp;+\u0026thinsp;1.8, and DV -7.8) followed by a stainless-steel auxiliary and Ag/AgCl reference combination electrode placed contralaterally on the surface of the cortex, -2.0 mm from bregma. The recording electrode (active recording surface of 500 \u0026micro;m length x 7 \u0026micro;m o.d.) was placed into the left mPFC (AP -2.7, ML\u0026thinsp;+\u0026thinsp;0.8, DV -4.0) or NAc core (AP -1.6, ML\u0026thinsp;+\u0026thinsp;1.5, and DV -7.4). An auxiliary electrode applied a fixed potential of +\u0026thinsp;0.8V, and DA efflux was measured using an electrometer (ED401 e-corder 401 and EA162 Picostat, eDAQ Inc.) filtered at 50 Hz, allowing for continuous monitoring of DA oxidation (10,000 samples/sec).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStimulation parameters.\u003c/em\u003e Cathodal pulses were delivered to the stimulating electrode via an optical isolator and programmable pulse generator (Iso-flex/Master-8, AMPI). Stimulation parameters were altered as the experiment progressed to assess different aspects of DA transmission. Initially, 20 monophasic stimulation pulses (800 \u0026micro;A intensity, 0.5 ms pulse duration) were delivered every 30 sec while adjusting electrode depths to establish optimal baseline responses in the NAc. These stimulation parameters were chosen to mimic phasic firing of dopaminergic neurons [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. As in our previous studies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], an optimal response was first found in the NAc to ensure proper placement of the stimulating electrode in the MFB. Recording electrodes were then moved to the mPFC, and stimulation parameters consisted of 50 monophasic 0.5 ms duration pulses (800 \u0026micro;A) at 50 Hz every 30 s for 10 min. DA release was defined as the magnitude of the stimulation-evoked response (from the pre-stimulation baseline to peak-stimulated response). The synaptic half-life of DA was defined as the time required for evoked DA to clear from the synapse (from peak DA release and restoration to 50% of baseline).\u003c/p\u003e \u003cp\u003eOnce baseline DA release and synaptic half-life were measured in the mPFC, we moved the recording electrode back to the NAc and assessed DA autoreceptor functioning thereby applying a pair of test stimuli (T1 and T2, each 10 pulses at 50 Hz separated by 10 s) every 60 s [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A set of conditioning pre-pulses (0, 1, 5, 10, 20, 40, or 80 pulses at 15 Hz) was delivered prior to T2 with 0.3s between the end of the conditioning pulse train and T2. The percent autoreceptor inhibition was defined as percent change in amplitude of T2 stimulations with respect to T1 (T2/T1 x 100) for each set of conditioning pre-pulses. After autoreceptor testing, stimulation parameters were returned to 20 pulses at 50 Hz every 30 sec, and after a 5-min baseline recording, rats received a cocaine injection (10/mg/kg, IP). Stimulations and amperometric recordings continued for 60 min post-cocaine. Measurements of DA release and synaptic half-life were converted to percent change following cocaine with pre-drug responses being 100%. Following the drug challenge, a continuous 3-minute MFB stimulation (9000 pulses at 50Hz) was used to determine available presynaptic DA supply stores. After amperometric recordings, recording electrodes were calibrated using an \u003cem\u003ein vitro\u003c/em\u003e flow injection system and standard solutions of DA (0.2\u0026ndash;1.2 \u0026micro;M).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHistology.\u003c/em\u003e To verify electrode placements, a direct anodic current (100 \u0026micro;Amps for 10 sec) was applied through the stimulating electrode. Rats were euthanized using a 0.25-ml intracardiac injection of urethane (0.345 g/ml). Brains were removed and placed in a solution of 10% formalin and 0.1% potassium ferricyanide for at least one week, then 30% sucrose/10% formalin for another week. Brains were sliced into 30 \u0026micro;m coronal sections using a cryostat at \u0026minus;\u0026thinsp;20\u0026deg;C and electrode placements assessed under a light microscope and recorded on coronal diagrams [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDrugs\u003c/h2\u003e \u003cp\u003eDelta-9-tetrahydrocannabinol (THC) was donated from the NIH Drug Supply Program. Urethane (U2500) and cocaine (C5776) were obtained from Sigma-Aldrich and mixed with 0.9% saline.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData Analyses\u003c/h3\u003e\n\u003cp\u003eData were analyzed using SPSS (IBM) version 29.0. The criterion for statistical significance for omnibus analyses was \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05. If this criterion was not met but showed a trend (i.e., .10\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;.05) accompanied by a large (η\u003csub\u003ep\u003c/sub\u003e\u0026sup2; \u0026gt; .14) effect size [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], post hoc analyses were conducted to reduce the risk of Type II error. We also used a nested design with sex nested within litter, as it offers greater power, controls for litter effects, and also reduces the risk of Type II error. If a sphericity violation occurred for a within-subjects effect, a Greenhouse-Geisser correction using adjusted degrees of freedom was used to reduce the risk of Type I error because ε\u0026thinsp;\u0026lt;\u0026thinsp;0.75 in all cases [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. All neurotoxicological, behavioral, and DA neurochemistry dependent variables analyzed, along with the omnibus analyses conducted, are presented in Supplemental Table\u0026nbsp;1. In the interest of brevity, only effects related to exposure or sex are presented in the results.\u003c/p\u003e \u003cp\u003eFor DRL 15 we also conducted peak deviation analyses to quantify burst ratio (i.e., \u0026lt; 2.5 sec IRT responses) peak area, and peak location. Individuals with high impulsivity tend to produce a large number of burst responses characterized by rapidly occurring non-reinforced responses occurring close together in time [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Peak deviation analysis compares the obtained IRT response distribution to a corresponding random IRT distribution. The latter is a negative exponential probability function that predicts the relative distribution of IRT responses verses a distribution based on the same number of responses categorized randomly with respect to time [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The random IRT distribution shows that based on chance, shorter IRTs will occur more often than longer ones. Peak deviation analysis provides a quantitative measure of how much the actual peak location and/or peak area differ versus random performance [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eNeurotoxicology Outcomes.\u003c/b\u003e See Supplemental Table\u0026nbsp;1. There was an effect of exposure on lactational weight gain [\u003cem\u003et\u003c/em\u003e(15) =-2.322, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.035], dam brain:body weight [\u003cem\u003et\u003c/em\u003e(15)\u0026thinsp;=\u0026thinsp;2.309, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.036], and male pup brain:body weight [\u003cem\u003et\u003c/em\u003e(15)\u0026thinsp;=\u0026thinsp;1.999, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.064, η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.210]. The lactational weight gain in THC -exposed dams was lower compared to vehicle-exposed dams, and brain:body weight ratio in THC-exposed dams and male pups was higher than vehicle-exposed dams and male pups, respectively. There was also a significant main effect of exposure on postnatal weight gain, [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;6.351, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.024] as well as a significant exposure \u0026times; day interaction, [\u003cem\u003eF\u003c/em\u003e(1.246,18.693)\u0026thinsp;=\u0026thinsp;7.342, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010]. Post hoc analysis revealed a significant difference between the exposure groups on PND 14 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.009) and PND 21 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.019). There were no other exposure- or sex-related differences at birth or weaning.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMaternal Behavior.\u003c/b\u003e There was not a main effect of exposure, nor an exposure \u0026times; day interaction on the latency of pup retrieval (Supplemental Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBehavioral Measures.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDD.\u003c/em\u003e No effects related to exposure or sex were found for any dependent measure. Data from the last testing block (days 21\u0026ndash;25) are presented in Supplemental Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDRH.\u003c/em\u003e The main effect of exposure was significant, \u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;4.618, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048. In addition, while the interaction of exposure \u0026times; phase did not reach the criterion for significance, a large effect size was present, \u003cem\u003eF\u003c/em\u003e(1.45,17.180)\u0026thinsp;=\u0026thinsp;3.854, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.061, η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.220. Post hoc analysis revealed a difference between the exposure groups during DRH 2:1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.031) and DRH 8:4 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.036). As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, rats perinatally exposed to THC elicited significantly fewer lever presses during these two phases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDRL 15.\u003c/em\u003e Data across the 30 days of DRL 15 were averaged into 6, 5-day blocks.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eReinforced:Non-reinforced responses\u003c/span\u003e. The exposure \u0026times; block interaction only approached the criterion for significance [\u003cem\u003eF\u003c/em\u003e(3.090,46.343)\u0026thinsp;=\u0026thinsp;2.686, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.056] even though a large effect size was present (η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.152). Post hoc analysis revealed vehicle rats had a higher ratio than THC-exposed rats in block 2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.064, η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.210, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eReinforcers earned.\u003c/span\u003e There was a significant main effect of exposure [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;5.476, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.034] and significant exposure \u0026times; sex interaction [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;19.266, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.001]. The THC-exposure group had fewer reinforcers overall compared to the vehicle-exposure group, an effect driven by the high number of reinforcers earned in vehicle males compared to THC-exposed males (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.001) and vehicle females (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTotal trials.\u003c/span\u003e There were significant exposure \u0026times; block [\u003cem\u003eF\u003c/em\u003e(2.855,42.821)\u0026thinsp;=\u0026thinsp;3.165, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.036] and sex \u0026times; block [\u003cem\u003eF\u003c/em\u003e(1.720, 25.796)\u0026thinsp;=\u0026thinsp;5.794, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.011] interactions. Post hoc analysis revealed a difference between exposure groups in block 4 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.059, η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.218), block 5 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.016), and block 6 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.055, η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.224; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and a sex difference in block 1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.033) and block 6 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.002). Females completed more trials in block 1, while males completed more in block 6.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePeak Deviation Analysis\u003c/span\u003e. Analyses of burst ratios revealed a main effect of exposure in block 1 [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;4.058, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.062, η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.203], block 2 [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;4.238, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.057, η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.220], and block 6 [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;6.630, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.021], as well as an exposure \u0026times; sex interaction in block 2 [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;3.383, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.086, η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.184]. In all three blocks, the burst ratio was lower for the THC-exposed compared to vehicle-exposed rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, in block 2, the effect of exposure was sex-specific, as it was present in the males (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.006) but not females (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e middle).\u003c/p\u003e \u003cp\u003eWe calculated the difference between the negative exponential curves and the \u0026ldquo;debursted\u0026rdquo; relative proportion of responses for the pause IRTs (i.e., IRTs\u0026thinsp;\u0026gt;\u0026thinsp;2.5s) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The difference curves were used to determine peak location and peak area within each testing block. For peak area, there was a significant main effect of exposure in block 1 only [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;5.392, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.035], with a higher peak area in the THC-exposed rats (Fig.\u0026nbsp;4A, B). For peak location, there was a significant main effect of exposure only in block 2, [\u003cem\u003eF\u003c/em\u003e(1,15)\u0026thinsp;=\u0026thinsp;5.719, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.030]. As seen in Fig.\u0026nbsp;4C and 4D, the peak location occurred earlier in the THC-exposed rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDopamine measures\u003c/h2\u003e \u003cp\u003e \u003cem\u003emPFC and NAc DA Release and Half-life.\u003c/em\u003e Analysis of baseline stimulation-evoked DA release and the synaptic half-life of DA did not reveal a main effect of exposure, sex, or an exposure x sex interaction in either the mPFC (Supplemental Fig.\u0026nbsp;2A, B, C) or the NAc (Supplemental Fig.\u0026nbsp;2D, E, F). Histological placements of the stimulating and recording electrodes are shown in Supplemental Fig.\u0026nbsp;2G.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNAc DA Autoreceptor Functioning.\u003c/em\u003e There was not a main effect of exposure or sex, nor interactions of exposure \u0026times; sex, exposure \u0026times; prepulse, sex \u0026times; prepulse, or exposure \u0026times; sex \u0026times; prepulse (Supplemental Fig.\u0026nbsp;3A,B).\u003c/p\u003e \u003cp\u003e \u003cem\u003eNAc DA Release and Half-life After Cocaine Challenge.\u003c/em\u003e Peak DA release and DA half-life were converted into percent change with pre-drug responses being 100%. Analyses of the percent change in release post-cocaine revealed a significant main effect of exposure [\u003cem\u003eF\u003c/em\u003e(1,12)\u0026thinsp;=\u0026thinsp;7.499, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.018] and a significant exposure x time interaction [\u003cem\u003eF\u003c/em\u003e(2.755,33.058)\u0026thinsp;=\u0026thinsp;4.035, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.019]. Rats perinatally exposed to THC exhibited an attenuated increase in DA release post-cocaine compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), at all-time points starting at 10 minutes post-cocaine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Likewise, a significant main effect of exposure was also found for the percent change in the synaptic half-life of DA post-cocaine [\u003cem\u003eF\u003c/em\u003e(1,12)\u0026thinsp;=\u0026thinsp;7.830, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.016]. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, the percent change in DA half-life post-cocaine was reduced in rats perinatally exposed to THC.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNAc DA Supply.\u003c/em\u003e Analysis did not reveal a main effect of exposure or sex. The exposure \u0026times; sex interaction approached the criterion for significance [\u003cem\u003eF\u003c/em\u003e(1,12)\u0026thinsp;=\u0026thinsp;3.345, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.092] with a large effect size (η\u003csub\u003ep\u003c/sub\u003e\u0026sup2;=.218). However, post hoc simple effects analysis did not reveal an effect of exposure in either sex or an effect of sex in the vehicle or THC-exposed rats (Supplemental Fig.\u0026nbsp;3C,D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDoes Perinatal THC Exposure Increase Impulsivity?\u003c/h2\u003e \u003cp\u003eOur results demonstrate perinatal exposure to THC produces deficits in goal-directed behavior. There appeared to be a deficit in impulsive action in rats perinatally exposed to THC, particularly during the early testing blocks of DRL 15. Offspring exposed to THC had an increase in the peak area, decrease in peak latency, and lower reinforced:nonreinforced response ratio during these early testing blocks, which suggests an impulsivity problem. However, surprisingly, DRL 15 burst responses (IRTs \u0026lt; 2.5 sec) were \u003cem\u003elower\u003c/em\u003e in THC-exposed rats. High impulsivity is typically associated with a larger number of burst responses [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], so the decrease in THC-exposed rats was inconsistent with an increase in impulsivity and with the increase in peak area and location observed in the same animals. Notably, the degree of burst responding observed in our vehicle controls was similar to controls we have examined in other studies [\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e–\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and the decrease observed in THC-exposed rats was lower than we have seen previously. This led us to consider alternative explanations for the discrepancy in these outcomes.\u003c/p\u003e \u003cp\u003eRecall we found no evidence of a deficit in impulsive choice between vehicle- and THC-exposed rats. While it has been shown there is a neural dissociation between impulsive choice and impulsive action [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], our DD results further support our theory that the effects in THC-exposed rats during DRL 15 were not entirely reflective of an impulsivity problem. During DRL 15, THC-exposed rats earned significantly fewer reinforcers overall (across all testing blocks) and completed fewer trials during later testing blocks. Likewise, during DRH, THC-exposed rats had a significantly lower number of lever presses during two phases of DRH (2:1 and 8:4). Taken together, these results suggest THC-exposed rats were less motivated to work to earn reinforcers. While THC-exposed rats did exhibit a lower reinforced:nonreinforced response ratio indicative of an impulsivity problem, this effect occurred only in block 2. Further evidence of an amotivational state is seen when looking at the burst responses, which reflect lever presses occurring close together in time. A lack of motivation in the THC-exposed rats could also explain the decrease in burst responses relative to vehicle controls. While children of women who use cannabis during pregnancy demonstrate an increase in impulsivity and diagnoses of ADHD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], there is also increasing evidence that ADHD individuals have deficits in motivation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Children diagnosed with ADHD require stronger incentives to motivate them to modify their behavior than children not diagnosed with ADHD [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDoes Perinatal THC Exposure Alter Dopamine Release?\u003c/h2\u003e \u003cp\u003eDopamine release in the NAc has a direct influence on instrumental goal-directed behavior [\u003cspan additionalcitationids=\"CR56 CR57\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e–\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], especially when engaging in high-effort reward-seeking [\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e–\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. NAc disruption leads to engagement in behaviors requiring less effort, even when the outcome is less valuable [\u003cspan additionalcitationids=\"CR63 CR64\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e–\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In this study, perinatal THC exposure did not alter baseline stimulation-evoked DA release in the NAc, but THC-exposed rats displayed an attenuated dopaminergic response to cocaine. Our measures of DA neuronal supply did not differ between THC-exposed rats and controls. Thus, our findings suggest perinatal THC exposure altered the presynaptic functioning of DA neurons related to the mechanisms of cocaine (DAT inhibition), and/or magnified it to an observable level during the cocaine challenge. Others have similarly shown animals exposed to perinatal THC exposure display normal indices of baseline DA activity but different responses to drugs targeting D1, D2, and DAT [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], while chronic cannabis users have displayed baseline measures of DA receptor availability similar to controls but markedly blunted responses when challenged with the DAT inhibiter methylphenidate [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs DA has been argued to mediate the “elasticity of demand” (i.e. sensitivity to increase in price) of a reinforcer [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], a behavioral economics approach suggests the attenuated DA response to cocaine in THC-exposured rats should devalue this reinforcer and reduce the effort these rats are willing to exert for it. By extension, the decreased lever pressing during DRH and DRL15 burst responding we observed in THC-exposured rats may represent a similar DA-driven devaluation of the food reinforcer. Future research is planned to examine this question more directly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSummary\u003c/h3\u003e\n\u003cp\u003eThis project demonstrated perinatal THC exposure impaired goal-directed behavior, as exposed rats exhibited disengagement during high effort responding, earned fewer reinforcers, and completed fewer trials, indicative of a motivational deficit. The attenuated NAc DA response to cocaine suggests reinforcer devaluation may underlie this reduced effort. These findings highlight the potential neurodevelopmental risks of maternal cannabis use, helping to inform public health policy on its safety during pregnancy and lactation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eAppreciation is extended to Donny Ray for assistance with lab animal care, Dr. Timothy Mandrell for his veterinary support, and Dr. Randy Floyd for his suggested revisions to the manuscript.\u0026nbsp;\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement:\u0026nbsp;\u003c/strong\u003eAll authors declare no conflict of interest.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eFunding Sources:\u0026nbsp;\u003c/strong\u003eThis work was supported by the California Doctoral Incentive Program (MSC), NSF 2051105 (LGB, CMD), and a Dunavant professorship (HJKS).\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNathan DL, Clark HW, Elders J. 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Behav Brain Res. 2002;137(1-2):3-25.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6207382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6207382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCannabis use during pregnancy is common as many pregnant women consider cannabis as a safe way to alleviate symptoms associated with pregnancy because it is \u0026ldquo;natural\u0026rdquo;. However, clinical evidence links perinatal exposure to cannabis to externalizing behavior in offspring including impulsivity, hyperactivity, and substance use. In preclinical research, most studies focus on exposure to the psychoactive constituent of cannabis, delta-9-tetrahydrocannabinol (THC). THC is lipophilic allowing it to cross the placental barrier and be secreted in maternal milk, thereby exposing the fetus/neonate. We used operant procedures to measure motivation to work for rewards, impulsive action, and impulsive choice in adult offspring perinatally exposed to 0 or 5 mg/kg/day THC. Differential reinforcement of high rates (DRH) was used to assess motivation, differential reinforcement of low rates (DRL) was used to examine impulsive action and delay discounting (DD) was used to measure impulsive choice. We also measured dopamine (DA) functioning in the medial prefrontal cortex (mPFC) and in the nucleus accumbens (NAc) via \u003cem\u003ein vivo\u003c/em\u003e fixed potential amperometry in littermates of rats that completed behavioral testing. Perinatal exposure to THC dramatically decreased responding for reinforcers during DRH in offspring of both sexes, decreased reinforcers earned and trials completed during DRL, but had no effect on impulsive choice as measured during DD. In addition, perinatal THC exposure did not alter baseline DA release in the NAc or mPFC, but did attenuate the dopaminergic response to cocaine in the NAc. These results suggest perinatal exposure to THC may decrease motivation to work for reinforcers and provide neurochemical support for the \u0026ldquo;amotivational state\u0026rdquo; resulting from perinatal THC exposure.\u003c/p\u003e","manuscriptTitle":"Perinatal Exposure to Delta-9-tetrahydrocannabinol (THC) Alters Goal-Directed Behavior and Dopamine Functioning in Wistar Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 10:52:37","doi":"10.21203/rs.3.rs-6207382/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fb222d14-6f3b-429c-b8e0-3582fc2f237a","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45698874,"name":"Biological sciences/Neuroscience/Motivation"},{"id":45698875,"name":"Biological sciences/Neuroscience/Synaptic transmission/Neurotransmitters"}],"tags":[],"updatedAt":"2025-10-27T18:03:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-17 10:52:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6207382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6207382","identity":"rs-6207382","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}