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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Neuropharmacology. 2020 Sep 17;180:108310. doi: 10.1016/j.neuropharm.2020.108310

Prenatal Alcohol Exposure Reduces Posterior Dorsomedial Striatum Excitability and Motivation in a Sex- and Age-Dependent Fashion

Victoria Roselli 1,2, Changyong Guo 1, Donald Huang 1, Di Wen 1, Daniel Zona 2, Tiebing Liang 3, Yao-Ying Ma 1,4
PMCID: PMC7773417  NIHMSID: NIHMS1631973  PMID: 32950559

Abstract

Prenatal alcohol exposure (PAE)-induced clinical symptoms have been widely reported but effective treatments are not yet available due to our limited knowledge of the neuronal mechanisms underlying behavioral outputs. Operant behaviors, including both goal-directed and habitual actions, are essential for everyday life. The dorsomedial striatum (DMS) and the dorsolateral striatum (DLS) have been identified as mediating each type of instrumental behavior, respectively. The current studies were designed to evaluate the effects of PAE (i.e., 3 g/kg, twice a day on gestational days 17–20) on goal-directed vs. habitual behaviors in both females and males during their adolescent and adult stages. We found that PAE-treated adult, but not adolescent, males display similar habitual oral sucrose self-administration but reduced goal-directed sucrose self-administration, compared to those treated by prenatal control (water) exposure (PCE). There were no differences in either habitual or goal-directed sucrose taking between PCE- vs. PAE-treated adolescent and adult females. These results indicate sex- and age-specific effects of PAE on operant behaviors. Further, whole-cell patch clamp recordings showed that the excitability of medium-sized spiny neurons (MSNs) in the posterior DMS (pDMS), but not the anterior DMS (aDMS), was significantly decreased in PAE-treated adult male rats. Notably, chemogenetic enhancement of MSN excitability in the pDMS by the DREADD agonist, compound 21, rescued the motivation of PAE-treated male adult rats. These data suggest that the pDMS may be a key neuronal substrate mediating the PAE-induced low motivation in male adults.

Keywords: Prenatal Alcohol Exposure, Motivation, Dorsomedial Striatum, Whole-Cell Patch Clamp, Adulthood, Sex

1. Introduction

Analysis of the 2015–2017 Behavioral Risk Factor Surveillance System data indicated that 11.5% of pregnant women reported current drinking, and 3.9% reported binge drinking during the past 30 days (Alati et al., 2006; Watts et al., 2018). Women who were not married were more likely to drink alcohol and binge drink during pregnancy compared to married women (Watts et al., 2018). Together with the low rate of marriage, prenatal alcohol exposure (PAE) is highly prevalent. The behavioral consequences of PAE, especially the prolonged effects not only during adolescent but also adult stages, cause substantial morbidity (Lebel et al., 2012; Merrick et al., 2006), but available treatments are limited. However, more emphasis has been placed on the early developmental outcomes of PAE (Subramoney et al., 2018). For example, in infant laboratory animals, PAE affected operant behaviors by increasing the reinforcing effects of sucrose pellet taking (Cullere et al., 2014), but less is known about the long-term consequences of PAE on operant outputs in both males and females. This knowledge is critical as operant behaviors to gain access to rewards are essential for survival. Thus, there is an urgent need to explore the neuronal mechanisms related to PAE-induced instrumental behavioral consequences in both adolescents and adults of both sexes.

The reward-driven operant action consists, in large part, of goal-directed and habitual processes. Governed by the association between responses and outcomes (R–O) and controlled by the outcome, the goal-directly behavior is a flexible, deliberate action that can be inhibited when the expected outcome is no longer desirable after devaluation (Burton et al., 2015; Rescorla, 1992a). Determined by the association between stimulus and response (S–R) and driven by the stimulus, the acquired habit is an inflexible and automatic behavior, which cannot be impacted by the outcome devaluation (Rescorla, 1992b). In the current study, similar to previous research (Yin et al., 2004; Yin et al., 2005), the devaluable goal-directed vs. non-devaluable habitual behaviors were dissected by pre-feeding sessions before progressive ratio (PR) test of sucrose pellet taking following active lever presses.

Many studies have suggested that the dorsal striatum plays distinct roles in instrumental conditioning. There is evidence that the dorsomedial or associative striatum (DMS, roughly homologous to the caudate in primates) is preferentially involved in goal-directed actions, whereas the dorsolateral or sensorimotor striatum (DLS, roughly homologous to the putamen in primates) is significantly involved in habit processes. The functional output of a brain region, by definition, is the action potential of the projecting neurons in that region (Hille, 2001), which is directly associated with the intrinsic excitability of these neurons. However, the effects of PAE on the intrinsic excitability of striatal medium-sized spiny neurons (MSNs) in the dorsal striatum, as well as their contributions to the operant action, have not been explored.

In the current studies, after acquisition of the oral sucrose self-administration operant behavior, goal-directed and habitual components of the instrumental output were evaluated specifically in both male and female rats at their adolescent and adult stages, respectively. Interestingly, we found that, relative to data from those with a history of prenatal control exposure (PCE; water controls), PAE decreased goal-directed instrumental output specifically in adult male rats, but had no effects on habitual processes. Thus, the intrinsic excitability of MSNs in the DMS, but not the DLS, particularly along the anterior-posterior axis (the anterior DMS, aDMS; posterior DMS, pDMS) in these rats was quantified too. Thereafter, we attempted to reverse PAE-induced changes in operant performance by manipulating MSN excitability chemogenetically at a specific sub-region of the DMS.

2. Materials and methods

2.1. Animals

All procedures were performed in accordance with the United States Public Health Service Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use committees at the Indiana University School of Medicine and State University of New York, Binghamton. Similar to previous publications (Gore-Langton and Spear, 2019), experiments were conducted on Sprague-Dawley rats, bred in-house using breeders originally derived from Envigo, USA. The day after birth, all litters were culled to up to 10 pups with a balanced number of female vs. male pups, and housed with their dams until weaning on postnatal day (P) 21. At weaning, each animal was pair-housed with a same sex littermate from the same prenatal exposure (ethanol vs. water) group. Rats were maintained on a 7 AM / 7 PM light / dark schedule with ad libitum access to food and water. One day before sucrose self-administration training, rats were limited to 1 hr daily feeding session from the moment they were single housed in order to measure the amount of individual food intake and to avoid any potential interruptions of food taking from roommates. A total of 480 rats, including breeders and experimental pups, were used. However, 21 rats were excluded from data collection because of complications after gavage intubation (2 dams), mislocation of AAV expression in the DMS (13 rats, found by checking the slices during recordings or after behavioral testing), or not within two standard deviations of the group mean value of the behavioral test (6 rats).

2.2. Prenatal ethanol exposure

The breeding and PAE procedures were adapted from previous publications (Chappell et al., 2007; Hausknecht et al., 2017; Hausknecht et al., 2005). In brief, two adult female Sprague-Dawley rats (8–10 weeks of age) were mated with 1 male Sprague-Dawley rat (10–20 weeks of age) for 4–6 days during which a daily smear test was performed at 9 AM until the pregnancy was confirmed by the presence of sperm in the vaginal smear, recorded as gestational day (G) 0. Then, the pregnant rats were single housed and randomly assigned to PCE vs. PAE treatment. From G17 to G20, these female rats received twice a day (i.e., 9 AM and 3 PM, respectively) intragastric gavage of ethanol (20%v/v, 3 g/kg) or water in the same volume.

2.3. Evaluation of blood ethanol concentration

Ethanol concentration was evaluated as described in our previous publication (Shan et al., 2019). Briefly, blood samples were taken from the tail vein in a separate cohort for the analysis of blood ethanol concentrations (BECs) 30 min after the 2nd alcohol administration on G18 and G20, respectively. Blood samples were maintained at −80 °C until analysis. BECs were assessed via headspace gas chromatography using a Hewlett Packard (HP) 5890 series II Gas Chromatograph (GC) and procedures in standard use in the Developmental Exposure Alcohol Research Center at Binghamton University (Saalfield and Spear, 2015; Willey et al., 2012). Our data show that the average concentrations of BECs in ethanol-treated female dams (i.e., 143±9.4 and 168±10.1 mg/dL on G18 and G20, respectively) were in the binge range (defined as >80 mg/dL by the NIAAA).

2.4. Operant conditioning

2.4.1. Apparatus

Each operant chamber (ENV-007CT, Med Associates) contained a house-light in one lateral wall and in the other wall, two retractable levers (an active lever, AL, and an inactive lever, IAL) with a cue light positioned above the center of each lever, as well as a food dispenser between the two levers. Chambers were located in sound-attenuating containers (ENV-018MD, Med Associates) with a fan that always was on during the training sessions. No food or water was provided in the chambers during the training or testing sessions.

2.4.2. Sucrose self-administration

Our operant training procedure was adapted from previously used sucrose or drug self-administration procedures/standards in our studies (Ma et al., 2013; Ma et al., 2014) as well as others (Yin et al., 2004; Yin et al., 2005). After an overnight session, rats were trained / tested sequentially by (1) fixed ratio (FR) 1, (2) FR5, (3) progressive ratio (PR) without 1-hr pre-feeding, and (4) PR with 1-hr pre-feeding. The criterion for moving from one phase to the next was either no more than 20% variation in the number of pellet-taking between 2 consecutive sessions or reaching the maximum days for each phase (i.e., 4 days), which ever occurred first. There is one exception to this criterion. i.e., a prolonged FR training schedule was performed, including 2 weeks of FR1 and 3 weeks of FR5 (Fig. 5A). The data presented in Figs. 1, 3–4, S1, and S2C,D were averaged from the measurement of daily sessions for 2–4 days. 1-hr daily feeding session started 1 day before the overnight session until the end of the experiments (including both in vivo and in vitro procedures). In general, the daily training/testing sessions, including FR or PR sucrose taking sessions and extinction test as sucrose seeking sessions, started at ~11AM, except the overnight session, which started at 7 PM until 7 AM on the following day. The time of daily feeding sessions was variable (anytime between 1–5 PM, to avoid associations between behavioral performance in operant chamber and the expected feeding session) except during the devaluated PR test when the 1-hr feeding sessions were performed right before PR test (i.e., ~10AM).

Figure 5. Prolonged training sessions in adult rats.

Figure 5.

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A, Experimental timeline of data collection in B–D. Extended training sessions, including 2 weeks of FR1 schedule and 3 weeks of FR5 schedule, were used before PR test, which were devaluated by 1 hr pre-feeding session of sucrose pellets. Bold lines indicate the procedures used in D only. W indicates the age in weeks.

B, C, No effects of 1 hr sucrose pre-feeding, the prenatal treatment, or the interactions between pre-feeding procedure and prenatal treatment detected in adult female (B, no pre-feeding/sucrose pellet pre-feeding × PCE/PAE interaction F1,14=0.1, p=0.78; no pre-feeding/sucrose pellet pre-feeding F1,14=1.6, p=0.23; PCE/PAE F1,14<0.1, p=0.88) or male (C, no pre-feeding/sucrose pellet pre-feeding × PCE/PAE interaction F1,14=1.7, p=0.21; no pre-feeding/sucrose pellet pre-feeding F1,14=0.88, p=0.37; PCE/PAE F1,14=3.1, p=0.10) rats.

D, In adult male rats, activation of pDMS-hM3D (Gq) by DREADD agonist C21 has no detectable effects on breakpoint values in the PR test with or without 1-hr sucrose feeding session in PAE-treated adult male rats after a prolonged training period (no pre-feeding/sucrose pellet pre-feeding × vehicle/C21 interaction F1,6=3.4, p=0.11; vehicle/C21 F1,6=1.2, p=0.32; nopre-feeding/sucrose pellet pre-feeding F1,6=4.1, p=0.09).

The litter number is shown in B–D in parentheses. Data were analyzed by two-way ANOVA with repeated measures on no pre-feeding/pre-feeding (B, C) and two-way ANOVA with repeated measures on no pre-feeding/pre-feeding and vehicle/C21 (D), followed by Bonferroni post-test.

*p<0.05, **p<0.01.

Figure 1. Break point values in progressive ratio (PR) test with no pre-feeding session vs. lab chow pre-feeding session.

Figure 1.

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A, Experimental timeline for Figs. 1 and S1.

B, In adolescent female rats, there were neither significant effects of interactions between devaluation procedure and prenatal treatment (no-pre-feeding/pre-feeding × PCE/PAE interaction F1,12=0.1, p=0.75) nor effects of the prenatal treatment (PCE/PAE F1,12=0.7, p=0.43), although the devaluation effects of 1 hr pre-feeding is significant (no-pre-feeding/pre-feeding F1,12=39.4, p<0.01)

C, In adolescent male rats, there were neither significant effects of interactions between devaluation procedure and prenatal treatment (no-pre-feeding/pre-feeding × PCE/PAE interaction F1,13=0.1, p=0.75) nor effects of the prenatal treatment (PCE/PAE F1,13=0.7, p=0.41), although the devaluation effects of 1 hr pre-feeding is significant (no-pre-feeding/pre-feeding F1,13=25.1, p<0.01)

D, In adult female rats, there were neither significant effects of interactions between devaluation procedure and prenatal treatment (no-pre-feeding/pre-feeding × PCE/PAE interaction F1,18=0.2, p=0.66) nor effects of the prenatal treatment (PCE/PAE F1,18=0.4, p=0.53), although the devaluation effects of 1 hr pre-feeding is significant (no-pre-feeding/pre-feeding F1,18=20.0, p<0.01)

E, In adult male rats, there were significant effects of interactions between devaluation procedure and prenatal treatment (no-pre-feeding/pre-feeding × PCE/PAE interaction F1,18=5.4, p=0.03) and significant effects of 1 hr pre-feeding (no-pre-feeding/pre-feeding F1,18=20.6, p<0.01) as well, but no effects of the prenatal treatment (PCE/PAE F1,18=1.8, p=0.19).

F, H, In female rats, both the resilience index (F, calculated by BPpre-feeding / BPno-pre-feeding) and the sensitive index (H, calculated by (BPno-pre-feeding − BPpre-feeding) / BPno-pre-feeding) were not affected by the prenatal treatment (PCE/PAE F1,30<0.01, p=0.95), developmental stage (Ado/Adu F1,30=0.4, p=0.51), or the interactions between prenatal treatment and developmental stage (PCE/PAE × Ado/Adu interaction F1,30=0.8, p=0.37).

G, I, In male rats, both the resilience index (G) and the sensitive index (I) were significantly affected by the interactions between prenatal treatment and developmental stage (PCE/PAE × Ado/Adu interaction F1,31=4.9, p=0.03), although there were no significant effects of prenatal treatment (PCE/PAE F1,31=1.8, p=0.19) or developmental stage (Ado/Adu F1,31=2.9, p=0.10). The litter number is shown in parentheses. Data were analyzed by two-way ANOVA (F–I) or two-way ANOVA with repeated measurements on pre-feeding procedure (B–E), followed by Bonferroni post-test. *p<0.05, **p<0.01.

Figure 3. Effects of DREADD agonist C21 at the pDMS vs. aDMS on oral sucrose self-administration after lab chow pre-feeding session in male adult rats treated by PAE.

Figure 3.

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A, Experimental timeline for data collected in B–E. W indicates the age in weeks.

B, Activation of pDMS-hM3D (Gq) by DREADD agonist C21 significantly increased the break point values in the PR test with no pre-feeding session in PAE-treated adult male rats (Veh/C21 × pre-feeding/no-pre-feeding Interaction F1,6=11.9, p=0.01; Veh/C21 F1,6=12.9, p=0.01; pre-feeding/no-pre-feeding F1,6=7.5, p=0.03).

C, Activation of aDMS-hM3D(Gq) by DREADD agonist C21 did not affect the break point values of PR test in oral sucrose self-administration in PAE-treated adult male rats (Veh/C21 × pre-feeding/no pre-feeding Interaction F1,7=0.05, p=0.83; Veh/C21 F1,7=1.5, p=0.26; pre-feeding/no pre-feeding F1,7=3.6, p=0.10).

D, Example traces (upper panel) and summarized data showing no effects of agonist C21 to PAE-treated adult male rats, expressing hM3D(Gq) in either the aDMS or pDMS, on the total distance traveled in 5-min open field (OF) test (Veh/C21 × pDMS / aDMS Interaction F1,7=0.02, p=0.90; Veh/C21 F1,7=0.2, p=0.68; pDMS / aDMS F1,7=0.2, p=0.68).

E, Diagrams of coronal slices showing the injection sites of AAV-CaMKIIa-hM3D(Gq)-mCherry expressed in the aDMS and pDMS.

The litter number is shown in B–D in parentheses. Data were analyzed by two-way ANOVA with repeated measures on no pre-feeding/pre-feeding and vehicle/C21 (B, C) and two-way ANOVA with repeated measures on vehicle/C21 (D), followed by Bonferroni post-test. *p<0.05, **p<0.01.

Figure 4. Sucrose pellet devaluation in adult male rats.

Figure 4.

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A, Experimental timeline of data collection in C–F. 1-hr sucrose pellet feeding was used as the devaluation procedure. The bold lines indicate the procedures used in F only. W indicates the age in weeks.

B, Diagrams of coronal slices showing the injection sites of AAV-CaMKIIa-hM3D(Gq)-mCherry expressed in the pDMS for data collection in Figs. 4F and 5D.

C, Significant effects of interactions between pre-feeding procedure and prenatal treatment, and significant effects of 1 hr sucrose pre-feeding as well, but no effects of the prenatal treatment itself (no-pre-feeding/sucrose pre-feeding × PCE/PAE interaction F1,16=56.8, p<0.01; no-pre-feeding/sucrose pre-feeding F1,18=99.2, p<0.01; PCE/PAE F1,16=2.3, p=0.15).

D, E, In PCE- or PAE- treated adult male rats, neither the resilience index (D) nor the sensitivity index (E) was affected by lab chow vs. sucrose pellet pre-feeding (lab chow /sucrose pellet pre-feeding × PCE/PAE interaction F1,34=1.9, p=0.18; lab chow /sucrose pellet pre-feeding F1,34=2.4, p=0.13; although PCE/PAE F1,34=28.4, p<0.01). Lab chow data were the same as in Fig. 1G, I. F, Activation of pDMS-hM3D (Gq) by DREADD agonist C21 significantly increased the break point values in the PR test with no pre-feeding, relative to either the vehicle group with no pre-feeding or the C21 group with sucrose pre-feeding, in PAE-treated adult male rats (no-pre-feeding/sucrose pellet pre-feeding × vehicle/C21 interaction F1,7=20.8, p<0.01; vehicle/C21 F1,7=27.6, p<0.01; no-pre-feeding/sucrose pellet pre-feeding F1,7=9.6, p=0.02).

The litter number is shown in C–F in parentheses. Data were analyzed by two-way ANOVA with repeated measures on no pre-feeding/pre-feeding (C), two-way ANOVA (D, E), and two-way ANOVA with repeated measures on no pre-feeding/pre-feeding and vehicle/C21 (F), followed by Bonferroni post-test. *p<0.05, **p<0.01.

2.4.3. Acquisition by FR schedule

Oral sucrose self-administration training began in rats on P28 or P80 with an overnight training session on a FR1 reinforcement schedule. Lever presses on the AL resulted in delivery of a sucrose pellet (45 mg, Banana Flavor, BioServe Cat # F0024) and 3 sec of the house light off and the cue light above the AL on, during which AL presses were counted but resulted in no sucrose pellet delivery. House light turned on after the 3-sec time-out. IAL presses had no reinforced consequences but were recorded. One (>95% of rats) or 2 (<5% of rats) overnight sessions were performed during which rats used in this project received the pre-set maximum number (i.e., 100) of sucrose pellets triggered by AL presses. Then, rats were trained by 2–4 days of 30-min daily sessions under FR1 reinforcement schedule, followed by 2–4 days of 30-min daily sessions under FR5 reinforcement schedule.

2.4.4. Operant behavior test by PR schedule

After acquisition of oral self-administration of sucrose pellets by operant conditioning under FR1 and FR5 schedules, rats were tested for their sucrose taking behavior under PR schedules. The response ratio schedule during PR testing was calculated as per Richardson and Roberts (1996)(Richardson and Roberts, 1996; Sharma et al., 2012) using the formula [5e (R*0.2)]- 5, where R is equal to the number of sucrose pellets already earned plus 1 (i.e., next reinforcer). Thus, the number of responses required to earn a reward follow the order: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95 and so on. The PR test session ended when reaching either the maximal session duration (i.e., 6 hrs) or the maximal between-pellet interval (i.e., 30 min). The final ratio completed is the breakpoint. PR test was performed with 1 hr post-training feeding (no pre-feeding) or 1 hr pre-training feeding (pre-feeding) session. The resilience index was calculated by BPpre-feeding / BPno-pre-feeding and the sensitivity index was calculated by (BPno-pre-feeding - BPpre-feeding) / BPno-pre-feeding, in which BP represents the break point value. A higher resilience index (usually associated with a lower sensitivity index) indicates an increase in the ratio of the habitual components and a decreased ratio of the goal-directed components, respectively.

2.5. Open field test

The test chamber was 60 cm for both side-widths × 40cm in height (Maze Engineers) with a floor illumination at ~60–80 lux. Rats were placed in the peripheral area facing the middle of one of the randomly assigned chamber sidewalls and then allowed to freely explore the chamber. Total testing time was 5 min. The videos were tracked and analyzed by EthoVision XT14 Software (Noldus).

2.6. Brain slice whole-cell patch clamp recordings

2.6.1. Slice preparation

Standard procedures were used for preparing slices and whole-cell patch clamp recordings as detailed in our previous publications (Ma et al., 2013; Ma et al., 2014). Before sacrifice, the rats were anesthetized with isoflurane and subsequently transcardially perfused with 4°C cutting solution (in mM: 135 N-methyl-D-glutamine, 1 KCl, 1.2 KH2PO4, 0.5 CaCl2, 1.5 MgCl2, 20 choline-HCO3, 11 glucose, pH adjusted to 7.4 with HCl, and saturated with 95% O2 /5% CO2). The rat was decapitated, and then the brain was removed and glued to a block before slicing using a Leica VT1200s vibratome in 4°C cutting solution. Coronal slices of 250-μm thickness were cut such that the preparation contained the signature anatomical landmarks (e.g., the anterior commissure, third ventricle and the corpus callosum) that clearly delineate the striatal subregions. After allowing at least 1 hr for recovery, slices were transferred from a holding chamber to a submerged recording chamber where it was continuously perfused with oxygenated artificial cerebrospinal fluid (ACSF) maintained at 30 ± 1°C.

2.6.2. Whole-cell patch clamp recording

Standard whole-cell current- or voltage-clamp recordings were obtained with a MultiClamp 700B amplifier (Molecular Devices), filtered at 3 kHz, amplified 5 times, and then digitized at 20 kHz with a Digidata 1550A analog-to-digital converter (Molecular Devices). The recording electrodes (3–5 MΩ) were filled with (in mM): 108 KMeSO3, 20 KCl, 0.4 K-EGTA, 10 HEPES, 2.5 Mg-ATP, 0.25 Na-GTP, 7.5 phosphocreatine (Na2), 1 L-glutathione, 2 MgCl2, pH 7.3. The recording bath solution (i.e., ACSF) contained (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose, saturated with 95% O2 / 5% CO2 at 30 ± 1°C. Details for whole-cell patch clamp recordings can be found in one of our previous publications (Ma et al., 2013; Ma et al., 2014; Shan et al., 2019). In brief, MSNs in the pDMS and aDMS were located using the corpus callosum and the third ventricle as landmarks and recorded in coronal slices. Cells were patched in voltage clamp mode and held at −70 mV. Cell membrane capacitance (Cm), input resistance (Rm) and decay time constant (τ) were calculated by applying a depolarizing step voltage command (5 mV) and using the membrane test function integrated in the pClamp11 software. Then recordings were switched to current clamp mode. Resting membrane potential was adjusted to −80 mV through injection of positive current (50 – 100 pA) and then intrinsic excitability was examined using a series of depolarizing current pulses and by constructing input-output (I–O) functions.

2.7. Chemogenetic procedures

2.7.1. Microinjection of AAV

A 28-gauge injection needle was used to bilaterally inject 1 μl/site (0.2 μl/min) of the hM3Dq-mCherry-AAV5 solution via a Hamilton syringe into the pDMS (IL) (in mm: AP, +0.0; ML, ±2.1; DV, −4.6), or the aDMS (in mm: AP, +2.0; ML, ±1.7; DV, −4.6), using the Pump 11 Elite Syringe Pumps (Harvard Apparatus). Injection needles were left in place for 5 min following injection. AAV injections were given no less than 3 weeks before any hM3Dq-targeting manipulations to ensure sufficient infection.

2.7.2. Chemogenetic inhibition of MSNs in the pDMS or Adms

Compound 21 (C21) at 1 mg/kg (Jendryka et al., 2019; Thompson et al., 2018) (purchased from Tocris and HelloBio; controlled by same volume of vehicle, i.e., injectable 0.9% saline) was used for in vivo chemogentic manipulations. Rats received IP injection of C21 or vehicle 10 min before 1 hr pre-feeding session in the devaluated PR test, 70 min before the non-devaluated PR test, or 30 min before open field test. In vitro chemogenetic manipulations were performed on hM3Dq-mCherry-AAV5-expressing DMS-containing coronal slices by evaluating the spike number of patch-clamped MSNs 3–5 min after bath application of C21, following the recordings of spikes in current clamp mode in ACSF.

2.8. Data Analysis

Behavioral tests (Figs.1, 3–5, and S1–2) and brain slice recordings (Figs. 2 and S2) were collected from adolescent rats at 4–6 weeks (denoted as “Ado”) or adult (10–12 weeks, denoted as “Adu”) stages. All results are shown as mean ± SEM. Each experiment was replicated in at least 6 litters, with 1.7±0.9 male rats and 3.1±0.6 female rats per litter, for behavioral tests, and at least 4 litters for electrophysiological analysis. A larger sample size in females was aimed at collecting enough data to investigate estrous cycle specificity. Our data showed no effects of female cycle in PCE- or PAE-treated rats. Sample size in in vivo experiments (Figs.1, 3–5, S1, and S2) is presented as litter number (i.e., “n”). Sample size in electrophysiology experiments (Figs. 2 and S2) is presented as m/n, where “m” refers to the number of cells examined and “n” refers to the number of litters. The plotted dots in Figs. 1B–I, 3B–D, 4C–F, 5B–D, S1A–H, and S2C,D represent litter-based individual values. The plotted dots in Figs. 2I,N,R, S2B represent neuron-based individual values. Statistical significance was assessed using paired Student’s t test (Fig. S2B,D), one-way ANOVA (Fig. 2N,R), and two-way ANOVA (Figs. 1E–H, 2I, 4D,E, and S1A–H), two-way ANOVA with repeated measures (Figs. 1B–E, 2D,H,M,Q, 3B–D, 4C,F, 5B–D, and S2A,C), followed by Bonferroni post-hoc tests. Three-way ANOVA (data from Figs. S1A and B; S1C and D), or three-way ANOVA with repeated measures (data from Figs. 1B and C; 1D and 1E; 1F and 1G; 1H and 1I; 5B and 5C) was used to determine the behavioral effects of sex.

Figure 2. Excitability of MSNs in the pDMS vs. aDMS from male adult rats treated by PAE or PCE (A–I) and effects of DREADD agonist C21 on the excitability of MSNs in the pDMS vs. aDMS from male adult rats treated by PAE (J–R).

Figure 2.

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A, Timeline of data collection in B–I. ~50% of data were collected from rats 1–3 days after the last session of sucrose self-administration in PCE/PAE-treated rats (i.e., rats done with data collection for Figs. 1 and S1; solid circles in I); another ~50% of data, collected at the same development stage of rats treated by only PCE or PAE but no postnatal operant training/testing nor limited food supplies (open circles in I). Data were pooled together in D, H and the columns in I. W indicates the age in weeks.

B, E, F, Example DIC images of coronal section at a low magnification (4X) showing the anatomical boundaries used to collect data from the pDMS (B) and aDMS (F), and a DIC image at high magnification (40x) showing the patch clamp electrode attached to a MSN (E). C, D, Example traces (C) and summarized data (D) showing that PAE, relatively to PCE, induced injection current-dependent decreases in the excitability of pDMS MSNs (cell-based analyses, PCE/PAE × Iinj Interaction F10,200=3.3, p<0.01; PCE/PAE F1,20=2.8, p=0.11; Iinj F10,200=166.0, p<0.01; litter-based analyses, PCE/PAE × Iinj Interaction F10,70=2.9, p<0.01; PCE/PAE F1,7=2.5, p=0.16; Iinj F10,70=133.0, p<0.01).

G, H, Example traces (G) and summarized data (H) showing that no changes occurred in the excitability of aDMS MSNs between PCE- vs. PAE-treated rats (cell-based analyses, PCE/PAE × Iinj Interaction F10,250=1.6, p=0.11; PCE/PAE F1,25=0.11, p=0.74; Iinj F10,250=294.5, p<0.01; litter-based analyses, PCE/PAE × Iinj Interaction F10,80=1.3, p=0.24; PCE/PAE F1,8=0.18, p=0.68; Iinj F10,80=278.1, p<0.01).

I, Summarized data of the spike number induced by Iinj=500pA showing that PAE decreased MSN excitability in the pDMS but not aDMS (cell-based analyses, PCE/PAE × pDMS/aDMS F1,45=4.2, p=0.047; PCE/PAE F1,45=1.9, p=0.17; aDMS/pDMS F1,45=1.2, p=0.28; litter-based analyses, PCE/PAE × aDMS/pDMS F1,14=4.9, p=0.043; PCE/PAE F1,15=0.9, p=0.35; aDMS/pDMS F1,14=1.3, p=0.27).

J, Experimental timeline for data collected in K–R. W indicates the age in weeks. K, O, Example image of a coronal section showing the viral expression of AAV-CaMKIIahM3D(Gq)-mCherry at the pDMS (K) and aDMS (O).

L, M, Example traces (L) and summarized data (M) showing that C21 dose-dependently increased the excitability of pDMS MSNs from male rats treated by PAE (cell-based analyses, C21 dose × Iinj Interaction F20,100=15.5, p<0.01; C21 dose F2,10=16.0, p<0.01; Iinj F10,50=113.8, p<0.01; litter-based analyses, C21 dose × Iinj Interaction F20,80=14.2, p<0.01; C21 dose F2,8=13.6, p<0.01; Iinj F10,40=124.1, p<0.01).

N, Summarized data of the spike number induced by Iinj=500pA showing that C21 at the doses of both 0.1 μM and 1 μM increased the excitability of pDMS MSNs from male rats treated by PAE (cell-based analyses, F2,10=26.3, p<0.01; litter-based analyses, F2,8=22.3, p<0.01). P, Q, Example traces (P) and summarized data (Q) showing that C21 dose-dependently increased the excitability of aDMS MSNs from the male rats treated by PAE (cell-based analyses, C21 dose × Iinj Interaction F20,100=8.3, p<0.01; C21 dose F2,10=28.7, p<0.01; Iinj F10,50=94.3, p<0.01; litter-based analyses, C21 dose × Iinj Interaction F20,80=8.0, p<0.01; C21 dose F2,8=26.5, p<0.01; Iinj F10,40=74.8, p<0.01).

R, Summarized data of the spike number induced by Iinj=500pA showing that C21 at the doses of 1 μM, but not 0.1 μM, increased the excitability of aDMS MSNs from male rats treated by PAE (cell-based analyses, F2,10=16.1, p<0.01; litter-based analyses, F2,8=13.8, p<0.01). The cell number / litter number (i.e., m/n) is shown in parentheses. Data were analyzed by two-way ANOVA (I), two-way ANOVA with repeated measures on Iinj (D, H) or on Iinj and C21 doses (M, Q), and one-way ANOVA with repeated measurements on C21 doses (N, R), followed by Bonferroni post-test. *p<0.05, **p<0.01.

3. Results

3.1. Effects of PAE on acquisition of oral self-administration of sucrose pellets

Sprague-Dawley rats, prenatally treated by PCE or PAE, were trained to acquire oral self-administration of sucrose pellets under the fixed ratio (FR) 1, followed by FR5 schedule as shown in Fig. 1A. The sucrose self-administration training started on P28-P32 and P80-P84, corresponding to the stage of adolescence and adulthood, respectively, in both male and female rats. No effects of PAE were observed on the data collected under the FR1 and FR5 reinforcing schedule in both female and male rats, although there was a significant effect of age on the pellet taking behaviors in FR1 and FR5 training sessions in both sexes (Fig. S1A–D). It was also clear that adolescent rats weighed significantly less than adult rats (Figs. S1E, F). There are at least two possible explanations of age effects on the pellet-taking behaviors. One may be body weight-associated and the other could be learning and memory-related; for example, the capacity of association between lever pressing and pellet delivery during the operant conditioning could be different between adolescent vs. adult rats. Thus, the amount of pellet taking per unit body weight (exemplified by data from FR1 sessions), calculated by dividing the total amount of pellets by the body weight, was used for further analysis. No more age effects were detected in the amount of pellet-taking per unit body weight in both sexes (Fig. S1G,H). Thus, the acquired sucrose self-administration behavior is not affected by PEA, although it is proportionally related to body weight.

3.2. Progressive ratio test of oral self-administration of sucrose pellets: effects of PAE on motivational manipulation by 1-hr lab chow pre-feeding

After acquisition of oral self-administration of sucrose pellets by operant conditioning under FR1 and FR5 schedules, rats were tested for their sucrose taking behavior under progressive ratio (PR). Effects of pre-feeding procedure by 1hr lab chow pre-feeding session were observed in both PCE- and PAE-treated adolescent females. However, there were no significant differences between PCE vs. PAE treatment on break point values in either no pre-feeding or lab chow pre-feeding PR test in these adolescent females (Fig. 1B). Similarly, significant effects of pre-feeding procedure, but no effects of PAE-treatment, were observed in adult females (Fig. 1D) and adolescent males (Fig. 1C). Interestingly, motivational effects of 1-hr pre-feeding session on the break point value was only observed in PCE-, but not PAE-, treated adult males. Specifically, relative to the PCE treatment, PAE treatment decreased the break point value in no pre-feeding PR test, but did not affect the break point value in the lab chow pre-feeding PR test (Fig. 1E). Further data analysis of the resilience index and sensitivity index showed that, although no effects of PAE on the resilience (Fig. 1F) and sensitivity (Fig. 1H) to pre-feeding procedures were observed in female rats, PAE-treated adult males were more resilient (indicated by higher resilience index; Fig. 1G) and less sensitive (indicated by the lower sensitive index; Fig. 1I) to the 1-hr lab chow pre-feeding procedure. A few conclusions are indicated. First, the 1-hr lab chow pre-feeding session is an effective procedure for decreasing the goal-directed component of sucrose taking in laboratory rodents. Second, PAE treatment decreased the goal-directed behavioral output with specificity for developmental stage and sex; i.e., significantly low motivation was detected in adult males treated by PAE. Third, PAE has no effects on the habitual behaviors. Thus, the subsequent studies were designed to explore the effects of PAE on the DMS, the associative striatum contributing to the goal-directed action, but not the DLS, the automatic striatum governing habit expression.

3.3. Effects of PAE on excitability of MSNs in the pDMS vs. aDMS

As shown in Fig. 2A, the excitability of MSNs in the DMS was evaluated by whole-cell patch clamp on rats treated by PCE vs. PAE only or prenatal treatment followed by operant procedures including both acquisition by FR schedule and test by PR test (n=2–3 litters in each group). No differences were detected in spike number measured in rats without vs. with operant history (p>0.5 by Student’s t test; see individual data in Fig. 2I), thus data were pooled together for statistics. Compared to PCE, PAE significantly decreased the spike number of MSNs in the pDMS (Fig. 2C,D,I) but not in the aDMS (Fig. 2G,H,I).

3.4. Effects of chemogenetic activation on excitability of MSNs in the pDMS vs. aDMS from PAE-treated adult males

To determine whether the decreased excitability of MSNs in the pDMS was involved in low motivation in adult males treated by PAE, designer receptors exclusively activated by designer drugs (DREADDs) system was used. Specifically, an engineered Gq-coupled receptor-expressing AAV5 tagged with a fluorescent protein mCherry (hM3Dq-mCherry-AAV5) was intracranially delivered to the pDMS or aDMS at least 5–7 days before the operant procedures on PAE-treated adult males (Fig. 2J). Whole-cell patch clamp recordings were performed 1–3 days after the last day of operant procedure. The spike number of MSNs was measured under current clamp mode during bath application of ACSF, followed by addition of the DREADD agonist, C21, at 0.1 μM and then 1.0 μM. Our data showed that spike number of MSNs in the pDMS from PAE-treated adult males was significantly increased by C21 at both doses (Fig. 2L–N). The spike number in aDMS was only increased by the high dose of C21 but not by the low dose (Fig. 2P–R). It can be concluded that MSNs in the DMS can be excited by C21 and, more importantly, in PAE-treated adult males, the MSNs in the pDMS, relative to those in the aDMS, are more sensitive to the chemogenetic manipulation. The decreased excitability of MSNs in the pDMS can be reversed by C21 at low dose. Thus, the relatively low dose of C21 (i.e., 1 mg/kg, but not 3 mg/kg used in most publications) was used for the in vivo studies, which was assumed to be more selective at inhibiting the pMDS.

3.5. Effects of chemogenetic activation on motivation for sucrose taking behavior in PAE-treated adult males

As shown in Fig. 3A, at least 5 days after microinjection of hM3Dq-mCherry-AAV5 into the pDMS or aDMS, PAE-treated adult males were trained to acquire oral sucrose self-administration by FR1 and FR5 schedules. Subsequently, 2 blocks of 2–4 daily PR tests were performed 70 min after IP injection of vehicle or C21 (1 mg/kg, according to (Jendryka et al., 2019)) for each block in a pseudo-randomized order (no-pre-feeding sessions). This was followed by 2 more blocks of 2–4 daily PR test in which the 1-hr feeding session was 10 min after the IP injection and right before PR test. The potential effects of C21 on general locomotion were evaluated by running 2 days of open field tests, 5 min per day, 30 min after the IP injection of vehicle and C21 (1mg/kg) on each day.

Our data showed a significant increase of the break point value in the no pre-feeding session by C21, compared to the vehicle treatment, in PAE-treated adult males expressing hM3Dq-mCherry-AAV5 in the pDMS (Fig. 3B) but not aDMS (Fig. 3C). Furthermore, the C21 treatment resulted in a significant decrease of the break point values in pDMS-expressing hM3Dq-mCherry-AAV5, PAE-treated adult males with 1-hr pre-feeding session, compared to the break point value in no pre-feeding session (Fig. 3B). This indicated that in vivo activation of MSNs in the pDMS can rescue goal-directed operant behaviors in PAE-treated adult males. The total distance travelled in the 5-min open field test was not influenced by the C21 treatment, compared to the vehicle treatment in PAE-treated adult males expressing hM3Dq-mCherry-AAV5 in either pDMS or aDMS (Fig. 3D). The average food intake during daily 1-hr-feeding session was not affected in PAE-treated male adults injected with vehicle (gram per 1-hr post-and pre-feeding session: 8.7±0.6 and 8.4±0.8 in pDMS groups; 8.5±0.8 and 8.6±0.5 in aDMS groups) vs. C21 (gram per 1-hr post- and pre-feeding session: 8.1±0.5 and 8.7±0.6 in pDMS groups; 8.1±0.8 and 8.2±0.6 in aDMS groups).

3.6. Devaluation by sucrose pellets in adult males

Devaluation by lab chow, which is different from the outcome during the PR test, could be an experimental confound when trying to identify the biological significance of behavioral and neuronal alterations. Thus, in a series of additional experiments sucrose pellets were used in the 1-hr pre-feeding session in adult male rats as illustrated in Fig. 4A. Similar to the lab-chow procedure, the 1-hr sucrose pre-feeding procedure significantly decreased the break point values of PR test in PCE-, but not PAE-treated rats (Fig. 4C). Comparison of the data acquired from lab chow and sucrose pellets pre-feeding sessions showed no effects of the food types (i.e., lab chow vs. sucrose pellets) on the resilience index and sensitivity index in PCE- or PAE-treated male adults (Fig. 4D, E). In addition, the average sucrose pellet-intake during daily 1 hr sucrose-feeding session was not affected by PAE treatment (gram of sucrose pellets per 1-hr pre-feeding session: 1.2±0.2 in PCE, 1.1±0.2 in PAE). Together with no effects of PAE on lab chow-intake (gram per 1-hr pre-feeding session: 8.4±0.7 in PCE, 8.7±0.6 in PAE), we assume that PAE-treated male adults have no deficit in maintaining their basic life needs, but they do have deficits in working for the positive reinforcer. Thus, the PAE-treated adult males are hypothesized to have a significantly low motivation level. Further experiments on PAE-treated adult males with intra-pDMS delivery of hM3Dq-mCherry-AAV5 showed that activation of pDMS-hM3D (Gq) by C21 significantly increased the sucrose pellet-devaluatable component of oral sucrose self-administration (Fig. 4F), indicating that the decreased excitability of MSNs in the pDMS may be the neuronal substrate of low motivation in adult males pre-exposed to PAE. Not surprisingly, the total distance travelled in the 5-min open field test was not influenced by the C21 treatment, compared to the vehicle treatment in PAE-treated adult males expressing hM3Dq-mCherry-AAV5 in pDMS (meters of total distance traveled with no pre-feeding vs. pre-feeding: 19.4±1.3 and 21.3±1.1 in vehicle groups, 19.0±1.6 and 20.4±0.8 in C21 groups). It is worth noting that the devaluated break point values detected in PCE adult males were significantly lower than those in PAE adult males with the sucrose pellet procedure (Fig. 4C), but no differences of the devaluated break point values were observed between PCE vs. PAE adult males (Fig. 1E). It thus appears that the sucrose pellet, the exact reinforcer available during the operant chamber session, may have higher potency in devaluation of sucrose taking behaviors.

3.7. Prolonged training sessions with FR schedule before PR test in adult males

It is well accepted that extensive training sessions, as well as random intervals, facilitate the development of habitual behaviors (Corbit and Janak, 2016; Corbit et al., 2012; Leong et al., 2016; Zapata et al., 2010). The effects of PAE on habituated sucrose-taking were then systematically evaluated in adult female and male rats by PR test after a prolonged FR training period, consisting of 2 weeks of FR1 and 3 weeks of FR5 training sessions (Fig. 5A). We found that, regardless of prenatal treatment (i.e., PCE or PAE), sucrose pellet pre-feeding session did not significantly affect the break point values in adult female (Fig. 5B) or male (Fig. 5C) rats, indicating that sucrose taking is largely habituated after a prolonged training period in rats in spite of their sex or prenatal history. Then, the effects of sucrose pellet pre-feeding on the break point values after the prolonged FR training period were evaluated on PAE-treated adult male rats microinjected with hM3Dq-mCherry-AAV5 to the pDMS 1 week before (Fig. 5A). IP injection of C21 had no influence on the break point values between no pre-feeding vs. sucrose pellet pre-feeding session (Fig. 5D), indicating that once the action is fully habituated, restoration of pDMS excitability in the PAE-treated adult males would not rescue any motivational component. There were no effects of C21 on the general locomotion (meters of total distance traveled with no pre-feeding and pre-feeding: 22.4±1.9 and 21.0±1.5 in vehicle groups; 20.7±1.3 and 20.9±1.4 in C21 groups).

3.8. In vitro and in vivo effects of C21 on non-hM3Dq-expressing PAE-treated adult males

In order to evaluate the potential non-specific effects of C21 independent from DREADDs, the excitability of MSNs in the pDMS, sucrose taking behaviors and general locomotion were measured in non-hM3Dq-expressing PAE-treated adult male rats. Our data showed that the decreased excitability of MSNs was not affected by bath application of C21 at the dose of 1μM for pDMS-containing slices (Fig. S2A, B). Furthermore, by comparing the operant behaviors with 1-hr post-feeding or lab chow pre-feeding procedures, no effects of C21 were detected on goal-directed and habitual sucrose taking in rats with no hM3Dq-mCherry-AAV5 expressed (Fig. S2C). Not surprisingly, C21 did not change the general locomotion if there were no hM3Dq to be targeted (Fig. S2D).

4. Discussion

By evaluating the effects of PAE on goal-directed vs. habitual operant behaviors in both females and males during adolescent and adult stages, we found that PAE-treated adult males display reduced motivation for sucrose self-administration. This anhedonia was the result of decreased MSN excitability in pDMS. Motivational deficits may be involved in a group of overlapping neuropsychiatric symptoms, including apathy, akinetic mutism, abulia, avolition, psychomotor retardation or slowing, and anergia (Epstein and Silbersweig, 2015), most of which have been reported by PAE-subjects in the clinic and in laboratory studies (Symons et al., 2018). Lack of motivation is associated with inflexible, habitual behavioral outputs, resulting in high risk of compulsivity and impulsivity, both highly related to obesity, drug abuse, etc. Our success in rescuing the motivational deficits in adult males may provide insights for preventing PAE-induced/associated neuropsychiatric symptoms. It is noticeable that PAE, particularly that broadly covering the prenatal stage, significantly disrupted efficient habitual actions during the adult stage, which was attributed to the decreased excitability of GABAergic interneurons in the DLS (Cuzon Carlson et al., 2020).

Motivation of operant behaviors for sucrose seeking can be attenuated by either satiety (by both lab chow and sucrose feeding) or devaluation of sucrose value (undoubtedly by sucrose pellet pre-feeding). Thus, the decreased excitability of the pDMS in PAE males may be involved in mediating both the satiety- and sucrose value-related sucrose-taking behaviors. However, the amount of lab chow or sucrose pellet taking in the pre-feeding session was not different in the FR training sessions between PCE- vs. PAE-treated male adult rats. This might support the idea that the decreased sucrose taking behaviors in PAE-treated male adults could be more possibly attributed to the devaluated sucrose pellets, although further experiments need to be performed for a conclusive answer.

Disruption of outcome devaluation of operant behaviors can be related to undevaluatable and/or devaluatable components. Undevaluatable components can be quantified by the break point values acquired in the PR test after pre-feeding session, and devaluatable components can be quantified by subtraction of the break point values acquired in the PR test after pre-feeding session from those acquired with no pre-feeding session. Our data support that PAE has no effects on the undevaluatable components but significantly disrupts the devaluatable components of sucrose-taking behaviors. The pre-feeding session with either lab chow or sucrose pellet was efficient at decreasing the motivation of sucrose taking in PAE-treated adolescent female, adult female and adolescent male rats. Furthermore, open field tests (Figs. 4, 5) showed no differences in the total distance traveled between the sessions with or with no pre-feeding procedures. Thus, we conclude that PAE specifically affects the motivational components of sucrose-taking in adult males, although the exact source of the low motivation cannot be identified between satiety vs. devaluation of sucrose pellets.

The DREADD approach, which evolved a decade ago (Armbruster et al., 2007), has been increasingly used to selectively modulate neuronal activity at specific circuits. CNO, broadly applied as a DREADD agonist since the DREADD system was developed, has been challenged due to its back-metabolization to clozapine and low penetration across the blood-brain barrier (BBB) (Gomez et al., 2017; Manvich et al., 2018). C21 represents an alternative agonist for muscarinic-based DREADDs. It has been reported that C21 has excellent bioavailability, high BBB penetrance and no back-metabolization to clozapine (Thompson et al., 2018). Our data showed DREADD-C21 system increased MSN excitability and restored motivation of sucrose taking in PAE-treated male adults. Importantly, systemic application of C21 showed no influence on general locomotion and neuronal excitability if there was no expression of hM3Dq-based DREADDs. Therefore, our data support that the DREADD-C21 approach is effective and selective at modulating behavioral deficits rather than disrupting tonic cellular activity and non-specific behavioral output. It is interesting to note that no increases of intrinsic excitability in aDMS were detected in PAE-treated male adults, and higher dose of C21 was needed to increase significantly the excitability of hM3Dq-expressing MSNs in the aDMS. Together with the fact that no in vivo effects on goal-directed/habitual operant performance or general locomotion were observed in rats in which the aDMS excitability was increased by C21, we assume that the MSNs (e.g., MSNs in the PCE rats) which were not pathologically excited by PAE history, are less sensitive to the DREADD-C21 activation. Further experiments of C21 on MSN excitability in PCE rats could address this assumption directly.

We also demonstrated that sex matters for the expression of some operant behaviors. Three-way ANOVA of the data pool from both males and females showed sex is not a significant factor on adolescent/adult FR1- (Fig. S1A,B) or FR5-(Figs. S1C,D) test, adolescent PR test (Fig. 1B,C), or adult PR test following a prolonged FR training period (Fig. 5B,C). However, significant sex effects were detected in adult PR test after a limited FR training period (Fig. 1D,E, male/female F1,34=15.5, p<0.01, and male/female × PCE/PAE F1,34=4.0, p=0.046). It is noticeable that female PCE controls, either at the adolescent or adult stage, showed higher goal-directed and habitual components in the PR test. Sex differences in learning and memory as well as cognitive capacity have been analyzed broadly in the general population (Hyde, 1981). Better memory and higher cognitive abilities have been reported in women, although more often observed in verbal ability-associated tasks (Andreano and Cahill, 2009). As the primary female sex hormones are not functionally limited to the reproductive system, estrogen can be synthesized in the brain by both neurons and astrocytes. Profound effects of estrogen have been reported on learning, memory, and mood as well as neuroprotection in cognitive deficits such as Alzheimer’s disease associated dementia (Li et al., 2014). Furthermore, significant effects of PAE on operant behaviors were observed in males only. Sex-specific effects of PAE have been reported (Lunde-Young et al., 2019). High placental global DNA methylation was specifically observed in PAE-treated male newborns (Loke et al., 2018). Data from magnetic resonance imaging (MRI) indicated a delayed maturation of white matter in early adult, but not adolescent stage in PAE-treated males (Uban et al., 2017). It is worth noting that greater cognitive deficits in men have been reported among those with neuropathological alterations, including multiple sclerosis and traumatic brain injury (Donaldson et al., 2019; Tucker et al., 2019).

It also was interesting to see the effects of PAE in adult but not adolescent stage, in partial agreement with human observations. Adolescence is a stage during which the brain, including the DMS, undergoes a rapid period of development. Functional alterations may lead to a high vulnerability of brain output. For example, alcohol-related neurodevelopmental disorders include neurobehavioral deficits in attention, learning and memory and so on, although generally more subtle than those seen with fetal alcohol syndrome (Jacobson and Jacobson, 2002). However, rapid alterations may also provide the young brain with a better ability for compensation during pathological progression. With the passage of time, less adaptation in the adult brain may lead to an unmasking of behavioral consequences triggered by prenatal insults of the brain. Another possibility is that the onset of the behavioral phenotype is not detectable until enough accumulation of pathological processes occur in the brain.

Compared to the well-accepted ventromedial-to-dorsolateral gradient in the striatum (Voorn et al., 2004), the striatal gradient along the anterior-to-posterior axis has been less investigated. Despite limited evidence, the heterogeneity within the DMS along the anterior-posterior axis has been demonstrated by both human neuroimaging studies with high-resolution functional MRI (Mattfeld et al., 2011; Mestres-Misse et al., 2012) and laboratory rodent studies with neuroanatomical tracing (Kelley et al., 1982; Nauta, 1989) and region-specific lesions (Yin and Knowlton, 2004; Yin et al., 2005). Laboratory rodent studies showed that, relative to the aDMS, the pDMS is innervated by more dense inputs from the basolateral amygdala (Mcgeorge and Faull, 1989), a critical brain region for encoding the value of expected output. By either excitotoxic lesion or local activation of GABAA receptors, inhibition of pDMS, but not aDMS, prevents the goal-directed sucrose taking behaviors (Yin et al., 2005). Together with our data showing that activation of the pDMS MSNs restored the goal-directed behaviors, the pDMS output is, at least within a certain range, proportionally related to the expression of the goal-directed performance. In human studies, higher levels of cognitive processing were detected in the aDMS (Mattfeld et al., 2011; Mestres-Misse et al., 2012). Thus, the dissociation between aDMS vs. pDMS is confirmed and each sub-region may contribute to the specific behavioral output.

Anatomical and electrophysiological evidence demonstrated significant differences in morphology as well as membrane and synaptic properties of D1 and D2 MSNs (Cepeda et al., 2008; Day et al., 2006; Gertler et al., 2008; Kravitz et al., 2010). Although one could have expected divergent responses based on previous literature, particularly responses to the motivation for drugs (Lobo et al., 2010), a more recent publication from Tanaka’s group, in which the motivation for natural rewards (i.e., food pellets) was evaluated by PR schedule, demonstrated optogenetically that inhibition of either D1- or D2-MSNs in the ventrolateral striatum can inhibit the goal-directed behaviors (Natsubori et al., 2017). Our data also indicated the independence of D1 and D2 cell types in controlling goal-directed operant actions. However, we acknowledge that use of animals expressing fluorescent reporters in D1 and D2 neurons would be required to confirm our findings.

The functional output of a brain region relies on the integration of synaptic transmission and intrinsic excitability. PAE-increased excitatory synaptic transmission in D1-MSNs in the DMS (Cheng et al., 2018), led to a higher probability of depolarization to the threshold of action potential generation. This enhanced excitatory synaptic transmission, together with the decreased intrinsic excitability, supports the hypothesis of synapse-membrane homeostatic crosstalk (SMHC) (Ishikawa et al., 2009; Wang et al., 2018), which postulates that an increase or decrease in excitatory synaptic strength induces a homeostatic decrease or increase in the intrinsic membrane excitability, respectively. Cocaine exposure was reported to disrupt SMHC regulation (Wang et al., 2018). Recognition of SMHC mechanisms will guide effective manipulations on the brain pre-exposed to PAE in future studies.

5. Conclusions

In summary, our data highlight the age- and sex-dependent deficits of PAE and their rescue by chemogenetic activation of the pDMS. The biological mechanisms of decreased excitability in pDMS in adult male rats with a PAE history need to be further explored in future studies.

Supplementary Material

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2
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Highlights:

  • PAE decreased goal-directed behavioral output in adult males.

  • PAE decreased excitability of MSNs in the pDMS.

  • Chemogenetic activation of pDMS reverses low motivation in adult male rats by increasing MSN excitability.

  • The pDMS excitability may be targeted to rescue the prolonged, deleterious effects of PAE.

Acknowledgements

This work was supported by NIH grants (P50AA017823, R01AA025784, R21NS108128) and Brain & Behavior Research Foundation grant #24989.

Footnotes

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Conflict of interest The authors report no conflicts of interest.

References

  1. Alati R, Al Mamun A, Williams GM, O’Callaghan M, Najman JM, Bor W, 2006. In utero alcohol exposure and prediction of alcohol disorders in early adulthood: a birth cohort study. Arch Gen Psychiatry 63, 1009–1016. [DOI] [PubMed] [Google Scholar]
  2. Andreano JM, Cahill L, 2009. Sex influences on the neurobiology of learning and memory. Learn Mem 16, 248–266. [DOI] [PubMed] [Google Scholar]
  3. Armbruster BN, Li X, Pausch MH, Herlitze S, Roth BL, 2007. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci U S A 104, 5163–5168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burton AC, Nakamura K, Roesch MR, 2015. From ventral-medial to dorsal-lateral striatum: neural correlates of reward-guided decision-making. Neurobiol Learn Mem 117, 51–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cepeda C, Andre VM, Yamazaki I, Wu N, Kleiman-Weiner M, Levine MS, 2008. Differential electrophysiological properties of dopamine D1 and D2 receptor-containing striatal medium-sized spiny neurons. Eur J Neurosci 27, 671–682. [DOI] [PubMed] [Google Scholar]
  6. Chappell TD, Margret CP, Li CX, Waters RS, 2007. Long-term effects of prenatal alcohol exposure on the size of the whisker representation in juvenile and adult rat barrel cortex. Alcohol 41, 239–251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cheng Y, Wang X, Wei X, Xie X, Melo S, Miranda RC, Wang J, 2018. Prenatal Exposure to Alcohol Induces Functional and Structural Plasticity in Dopamine D1 Receptor-Expressing Neurons of the Dorsomedial Striatum. Alcohol Clin Exp Res. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Corbit LH, Janak PH, 2016. Habitual Alcohol Seeking: Neural Bases and Possible Relations to Alcohol Use Disorders. Alcohol Clin Exp Res 40, 1380–1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Corbit LH, Nie H, Janak PH, 2012. Habitual alcohol seeking: time course and the contribution of subregions of the dorsal striatum. Biol Psychiatry 72, 389–395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cullere ME, Spear NE, Molina JC, 2014. Prenatal ethanol increases sucrose reinforcement, an effect strengthened by postnatal association of ethanol and sucrose. Alcohol 48, 25–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cuzon Carlson VC, Gremel CM, Lovinger DM, 2020. Gestational alcohol exposure disrupts cognitive function and striatal circuits in adult offspring. Nat Commun 11, 2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Day M, Wang Z, Ding J, An X, Ingham CA, Shering AF, Wokosin D, Ilijic E, Sun Z, Sampson AR, Mugnaini E, Deutch AY, Sesack SR, Arbuthnott GW, Surmeier DJ, 2006. Selective elimination of glutamatergic synapses on striatopallidal neurons in Parkinson disease models. Nat Neurosci 9, 251–259. [DOI] [PubMed] [Google Scholar]
  13. Donaldson E, Patel VP, Shammi P, Feinstein A, 2019. Why Sex Matters: A Cognitive Study of People With Multiple Sclerosis. Cogn Behav Neurol 32, 39–45. [DOI] [PubMed] [Google Scholar]
  14. Epstein J, Silbersweig D, 2015. The neuropsychiatric spectrum of motivational disorders. J Neuropsychiatry Clin Neurosci 27, 7–18. [DOI] [PubMed] [Google Scholar]
  15. Gertler TS, Chan CS, Surmeier DJ, 2008. Dichotomous anatomical properties of adult striatal medium spiny neurons. J Neurosci 28, 10814–10824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gomez JL, Bonaventura J, Lesniak W, Mathews WB, Sysa-Shah P, Rodriguez LA, Ellis RJ, Richie CT, Harvey BK, Dannals RF, Pomper MG, Bonci A, Michaelides M, 2017. Chemogenetics revealed: DREADD occupancy and activation via converted clozapine. Science 357, 503–507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gore-Langton JK, Spear LP, 2019. Prenatal ethanol exposure attenuates sensitivity to the aversive effects of ethanol in adolescence and increases adult preference for a 5% ethanol solution in males, but not females. Alcohol 79, 59–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hausknecht K, Shen YL, Wang RX, Haj-Dahmane S, Shen RY, 2017. Prenatal Ethanol Exposure Persistently Alters Endocannabinoid Signaling and Endocannabinoid-Mediated Excitatory Synaptic Plasticity in Ventral Tegmental Area Dopamine Neurons. J Neurosci 37, 5798–5808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hausknecht KA, Acheson A, Farrar AM, Kieres AK, Shen RY, Richards JB, Sabol KE, 2005. Prenatal alcohol exposure causes attention deficits in male rats. Behav Neurosci 119, 302–310. [DOI] [PubMed] [Google Scholar]
  20. Hille B, 2001. Ion channels of excitable membranes 507. [Google Scholar]
  21. Hyde JS, 1981. How large are cognitive gender differences? American Psychologist 36, 10. [Google Scholar]
  22. Ishikawa M, Mu P, Moyer JT, Wolf JA, Quock RM, Davies NM, Hu XT, Schluter OM, Dong Y, 2009. Homeostatic synapse-driven membrane plasticity in nucleus accumbens neurons. J Neurosci 29, 5820–5831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jacobson JL, Jacobson SW, 2002. Effects of prenatal alcohol exposure on child development. Alcohol Res Health 26, 282–286. [PMC free article] [PubMed] [Google Scholar]
  24. Jendryka M, Palchaudhuri M, Ursu D, van der Veen B, Liss B, Katzel D, Nissen W, Pekcec A, 2019. Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics in mice. Sci Rep 9, 4522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kelley AE, Domesick VB, Nauta WJ, 1982. The amygdalostriatal projection in the rat--an anatomical study by anterograde and retrograde tracing methods. Neuroscience 7, 615–630. [DOI] [PubMed] [Google Scholar]
  26. Kravitz AV, Freeze BS, Parker PR, Kay K, Thwin MT, Deisseroth K, Kreitzer AC, 2010. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature 466, 622–626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lebel C, Mattson SN, Riley EP, Jones KL, Adnams CM, May PA, Bookheimer SY, O’Connor MJ, Narr KL, Kan E, Abaryan Z, Sowell ER, 2012. A longitudinal study of the long-term consequences of drinking during pregnancy: heavy in utero alcohol exposure disrupts the normal processes of brain development. J Neurosci 32, 15243–15251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Leong KC, Berini CR, Ghee SM, Reichel CM, 2016. Extended cocaine-seeking produces a shift from goal-directed to habitual responding in rats. Physiol Behav 164, 330–335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Li R, Cui J, Shen Y, 2014. Brain sex matters: estrogen in cognition and Alzheimer’s disease. Mol Cell Endocrinol 389, 13–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lobo MK, Covington HE 3rd, Chaudhury D, Friedman AK, Sun H, Damez-Werno D, Dietz DM, Zaman S, Koo JW, Kennedy PJ, Mouzon E, Mogri M, Neve RL, Deisseroth K, Han MH, Nestler EJ, 2010. Cell type-specific loss of BDNF signaling mimics optogenetic control of cocaine reward. Science 330, 385–390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Loke YJ, Muggli E, Nguyen L, Ryan J, Saffery R, Elliott EJ, Halliday J, Craig JM, 2018. Time-and sex-dependent associations between prenatal alcohol exposure and placental global DNA methylation. Epigenomics 10, 981–991. [DOI] [PubMed] [Google Scholar]
  32. Lunde-Young R, Ramirez J, Naik V, Orzabal M, Lee J, Konganti K, Hillhouse A, Threadgill D, Ramadoss J, 2019. Hippocampal transcriptome reveals novel targets of FASD pathogenesis. Brain Behav 9, e01334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ma YY, Henley SM, Toll J, Jentsch JD, Evans CJ, Levine MS, Cepeda C, 2013. Drug-primed reinstatement of cocaine seeking in mice: increased excitability of medium-sized spiny neurons in the nucleus accumbens. ASN Neuro 5, 257–271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ma YY, Lee BR, Wang X, Guo C, Liu L, Cui R, Lan Y, Balcita-Pedicino JJ, Wolf ME, Sesack SR, Shaham Y, Schluter OM, Huang YH, Dong Y, 2014. Bidirectional modulation of incubation of cocaine craving by silent synapse-based remodeling of prefrontal cortex to accumbens projections. Neuron 83, 1453–1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Manvich DF, Webster KA, Foster SL, Farrell MS, Ritchie JC, Porter JH, Weinshenker D, 2018. The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice. Sci Rep 8, 3840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mattfeld AT, Gluck MA, Stark CE, 2011. Functional specialization within the striatum along both the dorsal/ventral and anterior/posterior axes during associative learning via reward and punishment. Learn Mem 18, 703–711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mcgeorge AJ, Faull RLM, 1989. The Organization of the Projection from the Cerebral-Cortex to the Striatum in the Rat. Neuroscience 29, 503–537. [DOI] [PubMed] [Google Scholar]
  38. Merrick J, Merrick E, Morad M, Kandel I, 2006. Fetal alcohol syndrome and its long-term effects. Minerva Pediatr 58, 211–218. [PubMed] [Google Scholar]
  39. Mestres-Misse A, Turner R, Friederici AD, 2012. An anterior-posterior gradient of cognitive control within the dorsomedial striatum. Neuroimage 62, 41–47. [DOI] [PubMed] [Google Scholar]
  40. Natsubori A, Tsutsui-Kimura I, Nishida H, Bouchekioua Y, Sekiya H, Uchigashima M, Watanabe M, de Kerchove d’Exaerde A, Mimura M, Takata N, Tanaka KF, 2017. Ventrolateral Striatal Medium Spiny Neurons Positively Regulate Food-Incentive, Goal-Directed Behavior Independently of D1 and D2 Selectivity. J Neurosci 37, 2723–2733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nauta WJ, 1989. Reciprocal Links of the Corpus striatum with the Cerebral Cortex and Limbic System: A Common Substrate for Movement and Thought? Neurology and Psychiatry, 21. [Google Scholar]
  42. Rescorla RA, 1992a. Depression of an instrumental response by a single devaluation of its outcome. Q J Exp Psychol B 44, 123–136. [DOI] [PubMed] [Google Scholar]
  43. Rescorla RA, 1992b. Response-outcome versus outcome-response associations in instrumental learning Animal Learning & Behavior 20, 10. [Google Scholar]
  44. Richardson NR, Roberts DC, 1996. Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy. J Neurosci Methods 66, 1–11. [DOI] [PubMed] [Google Scholar]
  45. Saalfield J, Spear L, 2015. Consequences of repeated ethanol exposure during early or late adolescence on conditioned taste aversions in rats. Dev Cogn Neurosci 16, 174–182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shan L, Galaj E, Ma YY, 2019. Nucleus accumbens shell small conductance potassium channels underlie adolescent ethanol exposure-induced anxiety. Neuropsychopharmacology 44, 1886–1895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sharma S, Hryhorczuk C, Fulton S, 2012. Progressive-ratio responding for palatable high-fat and high-sugar food in mice. J Vis Exp, e3754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Subramoney S, Eastman E, Adnams C, Stein DJ, Donald KA, 2018. The Early Developmental Outcomes of Prenatal Alcohol Exposure: A Review. Front Neurol 9, 1108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Symons M, Pedruzzi RA, Bruce K, Milne E, 2018. A systematic review of prevention interventions to reduce prenatal alcohol exposure and fetal alcohol spectrum disorder in indigenous communities. BMC Public Health 18, 1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thompson KJ, Khajehali E, Bradley SJ, Navarrete JS, Huang XP, Slocum S, Jin J, Liu J, Xiong Y, Olsen RHJ, Diberto JF, Boyt KM, Pina MM, Pati D, Molloy C, Bundgaard C, Sexton PM, Kash TL, Krashes MJ, Christopoulos A, Roth BL, Tobin AB, 2018. DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs in Vitro and in Vivo. ACS Pharmacol Transl Sci 1, 61–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tucker LB, Velosky AG, Fu AH, McCabe JT, 2019. Chronic Neurobehavioral Sex Differences in a Murine Model of Repetitive Concussive Brain Injury. Front Neurol 10, 509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Uban KA, Herting MM, Wozniak JR, Sowell ER, Cifasd, 2017. Sex differences in associations between white matter microstructure and gonadal hormones in children and adolescents with prenatal alcohol exposure. Psychoneuroendocrinology 83, 111–121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Voorn P, Vanderschuren LJ, Groenewegen HJ, Robbins TW, Pennartz CM, 2004. Putting a spin on the dorsal-ventral divide of the striatum. Trends Neurosci 27, 468–474. [DOI] [PubMed] [Google Scholar]
  54. Wang J, Ishikawa M, Yang Y, Otaka M, Kim JY, Gardner GR, Stefanik MT, Milovanovic M, Huang YH, Hell JW, Wolf ME, Schluter OM, Dong Y, 2018. Cascades of Homeostatic Dysregulation Promote Incubation of Cocaine Craving. J Neurosci 38, 4316–4328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Watts N, Amann M, Ayeb-Karlsson S, Belesova K, Bouley T, Boykoff M, Byass P, Cai W, Campbell-Lendrum D, Chambers J, Cox PM, Daly M, Dasandi N, Davies M, Depledge M, Depoux A, Dominguez-Salas P, Drummond P, Ekins P, Flahault A, Frumkin H, Georgeson L, Ghanei M, Grace D, Graham H, Grojsman R, Haines A, Hamilton I, Hartinger S, Johnson A, Kelman I, Kiesewetter G, Kniveton D, Liang L, Lott M, Lowe R, Mace G, Odhiambo Sewe M, Maslin M, Mikhaylov S, Milner J, Latifi AM, Moradi-Lakeh M, Morrissey K, Murray K, Neville T, Nilsson M, Oreszczyn T, Owfi F, Pencheon D, Pye S, Rabbaniha M, Robinson E, Rocklov J, Schutte S, Shumake-Guillemot J, Steinbach R, Tabatabaei M, Wheeler N, Wilkinson P, Gong P, Montgomery H, Costello A, 2018. The Lancet Countdown on health and climate change: from 25 years of inaction to a global transformation for public health. Lancet 391, 581–630. [DOI] [PubMed] [Google Scholar]
  56. Willey AR, Anderson RI, Morales M, Ramirez RL, Spear LP, 2012. Effects of ethanol administration on corticosterone levels in adolescent and adult rats. Alcohol 46, 29–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yin HH, Knowlton BJ, 2004. Contributions of striatal subregions to place and response learning. Learn Mem 11, 459–463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yin HH, Knowlton BJ, Balleine BW, 2004. Lesions of dorsolateral striatum preserve outcome expectancy but disrupt habit formation in instrumental learning. Eur J Neurosci 19, 181–189. [DOI] [PubMed] [Google Scholar]
  59. Yin HH, Ostlund SB, Knowlton BJ, Balleine BW, 2005. The role of the dorsomedial striatum in instrumental conditioning. Eur J Neurosci 22, 513–523. [DOI] [PubMed] [Google Scholar]
  60. Zapata A, Minney VL, Shippenberg TS, 2010. Shift from goal-directed to habitual cocaine seeking after prolonged experience in rats. J Neurosci 30, 15457–15463. [DOI] [PMC free article] [PubMed] [Google Scholar]

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NIHMS1631973-supplement-1.pdf (197.7KB, pdf)
2
NIHMS1631973-supplement-2.pdf (180.2KB, pdf)
3
NIHMS1631973-supplement-3.docx (14.1KB, docx)

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