Pan-striatal reduction in the expression of the astrocytic dopamine transporter precedes the development of dorsolateral striatum dopamine-dependent incentive heroin seeking habits

The emergence of compulsive drug seeking habits, a hallmark feature of substance use disorder, has been shown to be predicated on the engagement of dorsolateral striatum (DLS) control over behaviour, which is underpinned by a dopamine-dependent functional coupling of the nucleus accumbens and the DLS. However, the mechanisms by which this coupling occurs have not been fully elucidated. The striatum is tiled by a syncytium of astrocytes that express the dopamine transporter (DAT), whose expression is altered in individuals with a heroin use disorder. Thus, astrocytes are uniquely placed functionally to bridge dopamine-dependent mechanisms across the striatum. Thus, here we tested the hypothesis that exposure to heroin, which does not interact with DAT, inﬂuences its expression in astrocytes across the striatum before the development of DLS-dependent incentive heroin seeking habits. Using Western-blot, qPCR and RNAscope we measured DAT protein and mRNA levels in whole tissue, cultured or in situ astrocytes from striatal territories of rats with a well-established cue-controlled heroin seeking habit and rats trained to respond for heroin or food under continuous reinforcement. Incentive heroin seeking habits were associated with a reduction in DAT protein levels in the anterior DLS (aDLS) that was preceded by a heroin-induced reduction in DAT mRNA and protein content in astrocytes across the striatum. aDLS astrocytes were also shown to be uniquely susceptible to direct dopamine-and opioid-induced downregulation of DAT expression. These results suggest that astrocytes may critically regulate the striatal dopaminergic adaptations that lead to the development of incentive habits for heroin

astrocytes may critically regulate the striatal dopaminergic adaptations that lead to the development of incentive heroin seeking habits.
K E Y W O R D S addiction, astrocytes, dopamine, dopamine transporter, heroin, incentive habits

| INTRODUCTION
Individuals with a substance use disorder (SUD) not only take drugs for their reinforcing properties, which depend on dopaminergic and opioidergic mechanisms in the nucleus accumbens (for stimulants and opiates, respectively, Ettenberg et al., 1982;Pettit et al., 1984); they also spend an inordinate amount of time and energy seeking out or foraging for the drug.This drug-seeking behaviour, manifested mostly in a drug-free state, is often under the motivational control of response-produced drug-paired conditioned stimuli (CSs) that, by acting as conditioned reinforcers, bridge delays to the eventual procurement and ensuing consumption of the drug (Belin et al., 2013).
This engagement of aDLS dopamine-dependent control of behaviour after a long history of cue-controlled drug seeking, as experienced by humans (Koob, 2021) and laboratory rats exposed to second-order schedules of reinforcement (SOR) (Belin-Rauscent et al., 2016;Everitt et al., 2018), reflects the emergence of incentive habits (Belin et al., 2013;Fouyssac et al., 2022) that precede and contribute to the development of compulsive drug seeking in vulnerable individuals (Cox et al., 2017;Fouyssac et al., 2022;Giuliano et al., 2019;Marti-Prats et al., 2023).
The development of incentive habits is mediated by the NAcC and the multisynaptic ascending striatomesostriatal spiralling dopaminergic circuitry that functionally connects the ventral and the dorsal striatum (Belin & Everitt, 2008;Haber et al., 2000), the coupling of which is aberrantly increased in former heroin addicts (Xie et al., 2014) and in rats with well-established cuecontrolled drug seeking behaviour (Fouyssac et al., 2018).However, the cellular and molecular nature of the striatal mechanisms involved has not been fully elucidated.
In nonhuman primates, drug-induced decreases in striatal dopamine transporter (DAT) protein expression first occurs in the ventromedial striatum and progressively spreads laterally and dorsally, eventually encompassing the dorsolateral territories of the striatum (Letchworth et al., 2001).This spatiotemporal pattern parallels the spread of alterations in metabolic activity throughout adjacent domains of the corticostriatal circuity over the course of drug exposure (Porrino et al., 2007).In former heroin addicts, similar reductions in striatal DAT expression have been reported to occur alongside that of many proteins expressed in astrocytes (Liu et al., 2013;Reynolds et al., 2006;Xu et al., 2015Xu et al., , 2017)).
Since striatal astrocytes also express the DAT (Asanuma et al., 2014;Inazu et al., 1999;Karakaya et al., 2007;Socan et al., 2024), they too may play a role in the drug-induced systems-level alterations in the DAT previously assumed to occur in neurons.Indeed, astrocytes have increasingly been shown to contribute to functions long considered to be uniquely neuronal, such as the regulation of behaviour (Nagai et al., 2019).Importantly, striatal astrocytes are involved in the control of the balance between goal-directed actions and habits (Kang et al., 2020), and drug-induced adaptations that contribute to SUDs (Fouyssac & Belin, 2019;Spencer & Kalivas, 2017).Striatal astrocytes play a pivotal role in drug-induced alterations in NacC glutamate homeostasis and its influence on cue reactivity (Scofield & Kalivas, 2014;Spencer & Kalivas, 2017), and they contribute to aDLS dopamine-dependent cue-controlled cocaine (Murray et al., 2012) and heroin (Hodebourg et al., 2019) seeking.In addition, striatal astrocytes control the tendency to relapse following self-imposed abstinence in the face of punishment in rats with a history of escalated cocaine intake (Ducret et al., 2016).
Not only do striatal astrocytes express the DAT (Asanuma et al., 2014;Inazu et al., 1999) and the enzymes responsible for the catabolism of dopamine (Hansson & Sellstrom, 1983;Huang et al., 2005), conferring them the ability to control local striatal dopamine transmission, they also form a syncytium that tiles the entire striatum (Adermark & Lovinger, 2008), with each astrocyte interacting with up to 2 million neuronal synapses (Oberheim et al., 2009).Altogether, this suggests that astrocytes are uniquely positioned to spatially tune and coordinate dopaminergic volume transmission across adjacent domains of the striatum, thereby representing a mechanism by which aDLS dopaminedependent mechanisms are progressively recruited by the NAcC over the course of the development of incentive habits.
However, this hypothesis relies on the premise that heroin-induced alterations in striatal DAT expression emerge in the aDLS before its functional engagement in the control over drug seeking, which in the rat occurs only after several weeks of training under a SOR --that is what we sought to establish in the present study.
Since psychostimulant drugs affect regulatory mechanisms that alter the expression of DAT through their direct interaction with the protein (Bu et al., 2021), we focused on opioids, which produce hyperdopaminergic states without directly interacting with the DAT.Thus, we investigated whether incentive heroin seeking habits (which develop after several weeks of training under a SOR) were associated with reductions in DAT expression across the functional domains of the striatum.Furthermore, we tested the extent to which these reductions (1) took place in striatal astrocytes and (2) emerged in the aDLS before its engagement in the control over behaviour (i.e.following exposure to heroin self-administration under continuous reinforcement; Hodebourg et al., 2019;Murray et al., 2012).
In four independent cross-sectional experiments, we assessed alterations in astrocytic DAT expression in ventral and dorsal functional domains of the striatum at two critical stages in the developmental trajectory of incentive heroin seeking habits: following heroin selfadministration under continuous reinforcement, which does not engage the aDLS-dopamine dependent habit system (Hodebourg et al., 2019;Murray et al., 2012), and after a long history of cue-controlled heroin seeking under a FI15(FR10:S) SOR, which promotes the development of aDLS dopamine-dependent incentive heroin seeking habits (Fouyssac et al., 2022;Hodebourg et al., 2019).We quantified (1) the gross expression of DAT protein and (2) mRNA in striatal lysates containing both neurons and astrocytes, (3) the expression of astrocyte-specific DAT protein in striatal astrocyte monocultures, and (4) the in-situ expression of DAT mRNA in striatal astrocytes.
Because opiates do not control DAT expression by direct physical interaction, we reasoned that any alterations observed following a history of heroin selfadministration may be mediated by the hyperdopaminergic states the drug engenders via its μ-OR-mediated GABAergic-interneuron inhibition-induced activation of dopaminergic neurons (Margolis et al., 2014).Thus, to further determine whether dopamine neuron-astrocyte interactions are necessary for opioid-induced alterations of striatal astrocytic DAT, we conducted a fifth experiment in which we exposed cultured striatal astrocytes of drug-naïve rats to either heroin, morphine or dopamine in vitro.We anticipated that dopamine, but not opioids, would decrease the expression of DAT in pure monocultures of striatal astrocytes.

| Subjects
As described in Figure 1, one hundred and eight $300 g male Sprague Dawley rats (Charles River, UK) housed under a reversed 12 h light/dark cycle (lights off at 7:00 AM) were used across five independent experiments carried out over 10 years.After a week of habituation to the vivarium, rats were single-housed and food-restricted, gradually reaching 85% of their theoretical free-feeding body weight before starting behavioural training.Water was always available ad libitum.Except for the drug naïve animals of Experiment 5 (n = 24), experiments were performed 6-7 days/week between 8 AM and 5 PM.All experimental protocols were conducted under the project licences 70/8072 and PP0463796 held by David Belin in accordance with the regulatory requirement of the UK Animals (Scientific Procedures) Act 1986, amendment regulations 2012, following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB).

| Drugs
Heroin hydrochloride (Macfarlan-Smith, Edinburgh, UK) used for self-administration was dissolved in sterile 0.9% saline.Heroin hydrochloride, morphine hydrochloride (Macfarlan-Smith, Edinburgh, UK) and dopamine (Sigma Aldrich, UK) used in cell culture experiments were dissolved in sterile water.

| Surgery
All rats that underwent heroin self-administration were implanted with a home-made indwelling catheter into their right jugular vein under isoflurane anaesthesia (5% for induction; 2%-3% for maintenance; O 2 : 2 L/min), as previously described (Jones et al., 2023).Perioperative analgesia was provided with Metacam (1 mg/kg, sc., Boehringer Ingelheim).Following surgery, rats received daily oral treatment with the analgesic for 3 days and an antibiotic (Baytril, 10 mg/kg, Bayer) for a week, which they first received on the day prior to surgery.Catheters were flushed with 0.1 mL of heparinized saline (50 U/ mL, Wockhardt ® in 0.9% sterile NaCl) every other day after surgery and then before and after each daily selfadministration session.

| Apparatus
All behavioural procedures were conducted in operant chambers (31.8 cm Â 25.4 cm Â 26.7 cm, Med associates, St. Albans, USA), which were located within ventilated sound-attenuating cubicles.The front and back panels of the test chambers were aluminium, while the right and left walls and the roof were transparent acrylic plastic with a stainless-steel grid floor.A pellet dispenser was installed behind the front wall of each chamber, F I G U R E 1 Experimental design and timeline of the experiments.Rats were trained instrumentally to respond under a fixed ratio 1 (FR1) schedule of reinforcement either for food pellets (FR1F group; n = 23) or heroin (FR1H group; n = 26) or to seek heroin under the control of the drug-paired cue, as operationalised under a FI15(FR10:S) second order schedule of reinforcement (SOH group; n = 35).In a first experiment (experiment 1), we sought to characterise how a history of heroin self-administration impacted striatal dopamine transporter (DAT) protein levels.The nucleus accumbens core (NacC), the anterior and posterior territories of the dorsomedial (aDMS and pDMS, respectively) and dorsolateral striatum (aDLS and pDLS, respectively) were micro-punched from the frozen brain of each individual of each of these three groups.DAT protein levels were measured using western blot.To delve deeper into the cellular substrate of these adaptations, namely, neurons or astrocytes, in experiment 2, we extracted primary astrocytes from freshly dissected striatal territories of FR1F, FR1H and SOH rats from an independent cohort.DAT protein levels in these cultures were then assessed using western blot.To investigate whether the reduction in striatal astrocytic DAT protein levels following heroin exposure was paralleled with a reduction in DAT mRNA levels, we then used qPCR to quantify total DTA mRNA content of each striatal territory across FR1F, FR1H and SOH rats of another independent cohort (experiment 3).To confirm in situ the results of experiments 2 and 3, that is, the reduction in striatal astrocytic DAT following heroin exposure, in a fourth experiment (experiment 4), we carried out RNAscope TM assays to measure DAT mRNA levels in GFAP+ cells on 12 μm brain sections from FR1F, FR1H and SOH rats from yet another independent cohort.Finally, to tease apart the contribution of between-system effects to the heroin-dependent decrease of DAT expression from the pharmacological effect of heroin on astrocytes per se, in experiment 5, astrocytes cultured from freshly dissected striatal territories of drug naïve rats (n = 24) were treated with heroin, morphine or dopamine for 2 h daily for 10 days.Cells were harvested and DAT protein levels were assessed by western blot.
supplying food pellets to a food magazine located 2 cm above the grid floor.The magazine was flanked by two response levers (4 cm wide; 8 cm above the grid floor), above which were white cue lights.For heroin selfadministration, a spring leash was attached to a swivel that connected to a balanced metal arm secured outside of the chamber.Tygon tubing extended from a 10-mL syringe mounted on a syringe pump (Semat Technical, Herts, UK) located outside each cubical to the swivel and from the swivel, through the leash, to attach to the catheter.Each test chamber was illuminated by one 3-watt light bulb (house light) during the experimental session.
The scheduling and recording of experimental events were controlled by either MED-PC IV software (Med Associates, St. Albans, USA) or the Whisker software suite (Whisker, Cambridge, UK).

| Fixed ratio 1 (FR1) schedule of reinforcement for heroin (FR1H) or food (FR1F)
Rats were trained instrumentally to respond for heroin (40 μg/infusion; 100 μL/5 s) or food (one 45 mg food pellet) under a fixed ratio 1 (FR1) schedule of reinforcement.Under this schedule, one active lever (AL) press resulted in the delivery of the outcome associated with the presentation of a conditioned stimulus (CS, the cue light above the active lever).For heroin self-administration, each active lever press resulted in drug infusion, initiated concurrently with a 20-s time out that included onset of the CS, offset of the house light and retraction of both levers.Inactive lever pressing was recorded but had no scheduled consequence.Active and inactive lever assignment was counterbalanced, and a maximum of 30 infusions of heroin or 100 food pellets were available for this stage.Rats in the FR1H group were trained for approximately 20 consecutive days, such that the number of heroin infusions they received matched that of the individuals of the "second-order heroin seeking" group (SOH).Rats in the FR1F group were trained for 10 daily sessions.

| Cue-controlled seeking under a second-order schedule of reinforcement (SOH)
Rats trained to develop aDLS dopamine-dependent incentive heroin seeking habits (Hodebourg et al., 2019) after extended exposure to a SOR (Belin et al., 2013) initially acquired heroin self-administration under the same FR1 schedule of reinforcement as described above for 7 days.Then, the schedule of reinforcement was changed to fixed intervals, the duration of which progressively increased across daily training sessions from 1 min (fixed interval 1 min, FI1) to FI2, FI4, FI8, FI10 and eventually FI15 min (Everitt et al., 2008).Under these schedules of reinforcement, responding on the AL after each interval has elapsed results in an infusion of heroin and concurrently initiates the presentation of the CS, retraction of the levers, and a 20s timeout.From the first FI15 session onwards, heroin infusions were limited to five per 2-h session, as previously described (Belin & Everitt, 2008;Murray et al., 2015).Thus, under a FI15 min schedule of reinforcement, each day instrumental responding is maintained over the 15 min interval, in the absence of the drug, but in anticipation of the eventual, contingently delivered i.v.infusion of heroin (Everitt et al., 2018).After 3 days of training to seek heroin under a FI15 schedule of reinforcement, rats were trained for an additional 21 sessions to seek heroin under the control of the conditioned reinforcing property of the drugpaired cue, as operationalised as a FI15(FR10:S) schedule of reinforcement under which rats continue receiving the drug after 15 min periods of drug seeking but during which they receive a presentation of the heroin-paired CS every 10 lever presses.Under these conditions, which closely resemble drug foraging in humans (Koob, 2021), heroin seeking is invigorated and maintained over long periods of time by the response-contingent presentation of the drugpaired CS, which thereby acts as a conditioned reinforcer (Everitt & Robbins, 2000).It is important to note that the well-established drug-seeking behaviour measured here differs in psychological terms from what is measured in cued reinstatement of drug-seeking paradigms, in which rats with a brief history of drug self-administration (which operationalizes drug taking), usually under low ratio schedules, are required once to respond under extinction conditions (i.e., no drug is ever delivered).Because instrumental responding never leads to a drug infusion under these conditions, it decreases rapidly even during a single session, thereby reflecting the learning of a new responseno unconditioned stimulus association conflated with the introduction of a new response-CS contingency but not a seeking behaviour, that is, one that persists over time, until the attainment of the outcome (Belin-Rauscent et al., 2016).

| Astrocytes primary cell culture
For experiments 2 and 5, rats were briefly anaesthetized (5% isofluorane, O 2 : 2 L/min) and decapitated.Sacrifice took place 22 to 24 h after the last heroin/food selfadministration session for experiment 2. The anterior (aDLS) and posterior dorsolateral striatum (pDLS), the anterior (aDMS) and pDMS, as well as the NacC were quickly dissected from fresh brains and mechanically dissociated with a pellet mixer (Argos Technologies) in 300 μL of DMEM (Gibco™, Fisher Scientific, UK) and subsequently vortexed then centrifuged at 1300 rpm during 5 min at room temperature.Supernatants were removed, and the pellets were suspended in 300 μL of fresh DMEM.Samples were vortexed and centrifuged once again, supernatants were removed, and cells were resuspended in 1 mL DMEM.Following 10 min of decantation, supernatants were plated directly into 12-well plates and stored in an incubator (Binder, Germany) with a controlled environment set up at 37 C, 5% CO2.Four days later, 500 μL of medium was removed and replaced by 500 μL of fresh medium supplemented with basic Fibroblast Growth Factor (bFGF, Gibco, Fisher Scientific, UK) at a final concentration of 10 ng/mL, which promotes astrocytes proliferation.Subsequently, over a period of 4 to 5 weeks, the media was changed, and the cells were treated with bFGF (10 ng/mL) weekly.Once the cells had reached confluence, those from experiment 2 were immediately processed for Western blot.Those for experiment 4 subsequently underwent in vitro drug exposure.

| In vitro application of dopamine, heroin and morphine to primary cell cultures
For experiment 5, once the cultures reached confluence, drugs were applied daily for 2 h for 10 consecutive days.Each day, the DMEM+ was aspirated from the culture wells and replaced with 1 mL DMEM containing the sterilised drug (Dopamine: 1.5 ng/mL; Heroin: 1.75 μM; Morphine: 1.0 μM) or DMEM vehicle.This drug application regimen was designed such that astrocyte populations from each striatal territory from each source rat were exposed daily to the various drugs according to a counterbalanced Latin square design for the same duration as they were in situ in vivo in the animals that had selfadministered heroin.The concentration of each drug was selected based on reports of rat brain extracellular fluid concentrations of heroin, morphine and dopamine following intravenous delivery of heroin (Gottas et al., 2012(Gottas et al., , 2014)).After 2 h of incubation with the drug or vehicle at 37 C and 5% CO 2 , the cultures were rinsed once with 1 mL DMEM, and then 1 mL of fresh DMEM was replaced in each well.The culture plates were then incubated at 37 C, 5% CO 2 , until the next application.After the 10th daily drug application, the cultures were processed for Western blot.

| Lysis and protein extraction
For culture samples, DMEM was aspirated from the wells and cells were treated with 100 μl lysis buffer (Complete Lysis-M kit, Roche).For brain samples, rats were decapitated 22 to 24 h after the last heroin/food selfadministration session, and their brains were harvested and snap-frozen in À40 C isopentane (Sigma-Aldrich, UK).Bilateral punches of each striatal territory were then taken with a 1 mm micro-puncher from 300 μm coronal sections cut at À18 C with a cryostat (Leica, UK).Punched samples were weighed and mixed with lysis buffer (10 μL/mg; Complete Lysis-M kit, Roche).For both culture and brain samples, following pipette homogenisation and centrifugation (15 min, 15,000 g, 4 C), supernatants were collected, and protein levels were quantified via spectrophotometry (Nanodrop, ND-1000, Thermo Fisher Scientific).

| Electrophoresis
Proteins were diluted to a final concentration of 1 g/L in LDS NuPAGE buffer (Invitrogen-Life Technologies, UK) and 0.05 M DTT.The samples were heated for 5 min at 90 C, and 10 μg of protein was loaded onto 4%-20% NuPAGE Tris-Glycine gel (Invitrogen-Life Technologies, UK).One well contained the ladder for determining molecular weights (Chameleon ® Vue Pre-stained Protein Ladder, LI-COR Biosciences, UK in experiment 5 or EZ-Run, Fisher BioReagents in experiments 1 and 2).

| Antibody incubation and visualisation
Following the transfer of proteins on nitrocellulose membranes (Invitrogen-Life Technologies, UK) with an iBlot device (Invitrogen) for 7 min at 20 V, the membranes were washed in Tris-buffer saline containing Tween 0.05% (TBST) for 10 min and blocked for 1 h (Intercept ® Blocking Buffer, LI-COR Biosciences in experiment 5 or BSA 5% in TBST in experiments 1 and 2) before being incubated for 24 h at 4 C and then for 1 h at room temperature with a custom-made anti-DAT primary antibody (Giros et al., 1996) (1:750) in experiments 1 and 2 or the ZRB1525 anti-DAT primary antibody (1:250) (Russo et al., 2023) previously tested against a Recombinant DAT protein (Abnova, H00006531-P01) in experiment 5.The membranes were then washed in TBST and incubated with an anti-Actin primary antibody (1:10,000; biotechne, MAB8929-SSP in experiment 5 or 1:75,000; Abcam, ab8226 in experiments 1 and 2) for 1 h at room temperature.The membranes were then washed again in TBST and incubated with the secondary antibodies (1:20,000; IRDye ® 800 Goat anti-Mouse; IRDye ® 680 Goat anti-Rabbit; LI-COR Biosciences, UK in experiment 5 or HRP-linked secondary antibodies: 1:100; antirabbit, cell signalling, 7074S and antimouse, ImmunoReagents Inc., gtxmu-003-dhrpx in experiments 1 and 2) for 1 h at room temperature.The membranes then underwent a final wash and were imaged using the Odyssey ® CLx Infrared Imaging System (LI-COR Biosciences, UK) (experiment 5).For experiment 1 and 2, the membranes were imaged using an electrochemoluminescence (ECL) detection system (ChemiDoc-It, Ultra-violet products) after receiving 1 mL of the HRP substrate (Luminata, Millipore).
Infrared signal intensities were quantified in Image Studio™ (LI-COR Biosciences, UK) in experiment 5 or with ImageJ software (imagej.net) in experiments 1 and 2. The percentage of DAT relative to Actin (i.e.(DAT/Actin)*100) was calculated for each sample to control for variations in protein loading.All samples were run in duplicate, and the average %DAT/Actin (log) value was used as the dependent variable for statistical analyses.

| Tissue collection and RNA extraction
Brains were harvested 22 to 24 h after the last heroin/ food self-administration session, snap-frozen by immersion in À35/40 C isopentane for $3 min and stored at À80 C. Bilateral samples from each striatal territory were collected using the same procedure as that described for western blots.RNAs were extracted using the Quick-RNA™ Microprep kit (Zymo Research) following the manufacturer guidelines and quantified using the NanoDrop ® ND-1000 spectrophotometer (Thermo Fisher Scientific).

| qPCR reaction and analysis
RNA was reverse-transcribed into cDNA with the RT2 First Strand Kit (Qiagen, UK) according to the manufacturer instructions.The following primers were used to assess the relative level of DAT mRNA (Quiagen Ref. PPR44664C; Slc6a3; Rn.10093) in comparison with that of Cyclophilin A (Quiagen Ref. PPR06504A; Ppia; Rn.1463), used as housekeeping gene (Qiagen, UK).Realtime-PCR was performed on the CFX96 Real-Time PCR Detection System (Bio-Rad, UK) using the RT2 SYBR Green Mastermix (Qiagen, UK) under the following conditions: reaction volume of 25 μL (1 μL cDNA, 24 μL PCR reaction mixture), 1 initial step at 95 C (10 min) followed by 40 temperature cycles (95 C for 15 s, 60 C for 60 s).The relative mRNA level of the target gene was calculated using CFX Manager Software (Bio-Rad, UK) and expressed as 2 ÀΔCT (Bernal et al., 2005;Sugden et al., 2009) relative to Cyclophilin A.

| RNAscope™ in situ hybridisation assay
RNAscope TM was performed according to the manufacturer's instructions for fresh frozen tissue using the RNAscope TM Multiplex Fluorescent Reagent Kit (Advanced Cell Diagnostics) as previously described (Velazquez-Sanchez et al., 2023).Brain sections were first fixed in chilled 10% NBF for 30 min on ice, rinsed three times in PBS and dehydrated in increasing concentrations of ethanol (50%, 70%, 100% and 100%).Slides were then kept in fresh 100% ethanol at À20 C overnight.Next, a hydrophobic barrier was drawn around each section to avoid the loss of the reagents during the assay.Sections were treated with Protease IV for 20 min at room temperature and then rinsed 3 Â 5 min with PBS prior to being incubated with the target probes in a HybEZ oven for 2 h at 40 C.
Each probe consists of a unique oligonucleotide mixture designed to bind to a specific target RNA, which, for this study, was Rattus norvegicus glial fibrillary acidic protein (Rn-Gfap-C2; 407,881-C2, Bio-Techne, UK), R. norvegicus solute carrier family 6 member 3 (i.e., DAT) (Rn-Slc6a3; 319,621, Bio-Techne, UK).Following the 2 h incubation with the target probes, sections were incubated with the preamplifier and amplifier probes (AMP1, 40 C for 30 min; AMP2, 40 C for 15 min) and washed with washing buffer in between each incubation step for 3 Â 5 min.Then sections were incubated with the fluorescence-labelled probe to detect GFAP in orange (Opal 570) and DAT in green (Opal 520).The sections were rinsed in washing buffer and incubated with DAPI for 20 s before being coverslipped under Fluoroshield mounting medium (Abcam, ab104135).

| Imaging and quantification
Images for quantitative RNAScope™ analysis were acquired at 40Â magnification using a Leica SP7 confocal microscope.Three to five 2.75 mm Â 3.68 mm images were acquired bilaterally from each striatal territory, which required two brain slices per animal.For each condition (i.e.FR1F, FR1H and SOH), images were acquired from five animals.Our in-house MATLAB-based pipeline was used to quantitatively detect clusters of RNA molecules (i.e.'puncta') around the nuclei of identified cells (see Velazquez-Sanchez et al., 2023 for a detailed description of the technical and theoretical elements of our analysis pipeline).The software package is available online (https://gitlab.com/lemur01/rnascopeanalysis).
Images that could not be accurately quantified by the pipeline due to the presence of fluorescence artefacts or inadequate tissue staining were not included in the final analysis, resulting in at least three and, at most, five representative animals per condition per region.To reduce false positives, we classified GFAP+ nuclei as those with >5 GFAP mRNA puncta and DAT mRNA puncta were counted only when the number of perinuclear DAT puncta exceeded 4.

Blinding and randomisation
The experimenters performed the western blots, the qPCR and RNAscope assays were not the same as those who sliced, dissected or micropunched the brains and who ascribed a specific code to each sample or microscope slide.
Rats trained to self-administer heroin under continuous reinforcement (FR1F) or under a second order schedule of reinforcement (SOH) were pseudo-randomly allocated to the different experiments so that their overall level of heroin intake was similar.This thereby controlled for any potential cellular and molecular differences merely resulting from differential levels of heroin exposure.

| Data and statistical analyses
Group sizes were determined a priori by power analyses simulations (G*Power 3) (Faul et al., 2007) for ttests, analyses of variance (ANOVA) and correlations based on effect sizes obtained from pilot studies.
Data presented as means ± SEM and/or individual data points were analysed with STATISTICA-10 Software (Statsoft, Inc., Tulsa, OK, USA) or GraphPad Prism version 10.0.0 (GraphPad Software, Boston, MS, USA).Assumptions for parametric analyses, namely, homogeneity of variance and normality of distribution were verified prior to each analysis with the Cochran and Shapiro-Wilk's tests, respectively.Where normality was substantially violated, data were Log transformed.
For experiments 1, 3, 4 and 5, all data sets were first subject to a one-way ANOVA to examine the effect of treatment on DAT mRNA or protein expression in each striatal territory separately.Significant omnibus effects were followed up by Holm-Šid ak's multiple comparison tests.For experiment 2, a one-sample t-test was used to test if the control group was significantly greater than zero.P-values displayed on the figures are the results of post-hoc comparisons wherever significant differences were observed.For all analyses, significance was set at α = 0.05.Effect sizes are reported as partial eta squared (η p 2 ).

| RESULTS
3.1 | A prolonged history of cuecontrolled heroin seeking is associated with a decrease in aDLS DAT protein levels SOH rats in experiment 1 (Figure 1) readily acquired and maintained heroin seeking under the control of the conditioned reinforcing properties of the drug-paired cue over a period of 3 weeks (main effect of session: F 7,63 = 5.57, p < 0.0001, η p 2 = 0.38) (Figure 2a), in line with previous reports (Hodebourg et al., 2019).SOH rats did not differ in their overall level of heroin intake from FR1H rats (t 14 = 1.490, p = 0.15), which had been trained to self-administer, but not seek, heroin under continuous reinforcement (Figure 2b).Analysis of total tissue content of DAT protein across striatal territories of heroin-experienced and control rats that had been trained instrumentally to respond for food (FR1F) revealed an effect of heroin exposure on DAT protein content of the aDLS and the pDLS (main effect of group: F 2,18 = 13.610,p < 0.001, η p 2 = 0.60 and F 2,17 = 9.017, p = 0.002, η p 2 = 0.52, respectively).Thus, in both the anterior and posterior dorsolateral striata, FR1H rats displayed higher DAT protein levels than FR1F controls.In contrast, SOH rats displayed lower DAT protein levels than controls in the aDLS and the NacC, the latter being overall less responsive to heroin exposure than the DLS (main effect of group: F 2,18 = 3.035, p = 0.07, η p 2 = 0.25; FR1F vs. SOH: F 1,14 = 6.109, p = 0.0269, η p 2 = 0.30; FR1F vs. FR1H: F 1,9 < 1, p = 0.80, η p 2 = 0.01).These differences were not attributable to differential exposure to heroin in SOH and FRIH individuals (Figure 2b).No other striatal territories were affected (all F < 2.080, all p > 0.150), despite a clear trend of a pan-striatal decrease in DAT protein levels in SOH rats (Figure 2c-g and Figure S1).monocultures (Figure 3a; for higher resolution, see Figure S2a).These cultures were confirmed to be free of microglial or neuronal contamination, as evidenced by the absence of Iba-1-positive cells (Figure 3b) and NeuNpositive cells (Figure 3c).Like those in experiment 1, SOH rats in experiment 2 readily acquired and maintained heroin seeking under the control of the conditioned reinforcing properties of the drug-paired cue over a period of 3 weeks (main effect of session: F 7,63 = 4.71, p = 0.0003, η p 2 = 0.34) (Figure 4a) and did not differ from in overall level of heroin intake compared with FR1H rats (t 13 = 1.846, p = 0.088) (Figure 4b).

| A prolonged history of heroin seeking or taking is associated with panstriatal reductions in astrocytic DAT mRNA
Considering the low baseline level of expression of astrocytic DAT, as illustrated in the FR1F control group, the observation of an apparent complete ablation of DAT protein expression in astrocytes cultured from striatal samples collected from the brains of animals with a history of heroin exposure may reflect a decrease in expression to levels undetectable by Western blot.We therefore sought further to investigate in an independent cohort of rats the effect of heroin taking and heroin seeking behaviour on the levels of DAT mRNA using qPCR (experiment 3).This approach, which relied on a much more sensitive technique, informed us on the specific nature of these alterations (i.e.posttranslational or/and posttranscriptional).
As in the previous experiments, rats in experiment 3 acquired and maintained cue-controlled heroin seeking (main effect of session: F 19,171 = 4.04, p = 0.0001, η p 2 = 0.31) (Figure 5a) and did not differ from FR1H rats in their overall level of heroin intake (t 19 = 0.151, p = 0.088) (Figure 5b).The results obtained from qPCR analysis of striatal DAT mRNA recapitulated the alterations observed in DAT protein from cultured astrocytes: DAT mRNA levels were profoundly decreased in all striatal territories investigated in FR1H and SOH rats as compared to FR1F controls (Figure 5c-g) (NacC: F 2,23 = 9.37, p = 0.0011, η p 2 = 0.45; aDLS: F 2,23 = 71.64,p < 0.0001, η p 2 = 0.86; pDLS: F 2,23 = 15.62,p < 0.0001, η p 2 = 0.71; aDMS: F 2,23 = 24.06,p < 0.0001, η p 2 = 0.68; pDMS: In order further to confirm that the decrease in striatal DAT mRNA levels occurred in astrocytes, on another cohort of rats (experiment 4), we used RNAscope™ to establish the localisation of DAT mRNA in nuclei expressing GFAP mRNA across the striatum and quantitatively assess in situ the influence of a history of heroin taking and seeking on DAT mRNA levels specifically in astrocytes.
F I G U R E 4 Pan-striatal reductions in astrocytic dopamine transporter (DAT) protein are observed following a prolonged history of heroin taking and cue-controlled heroin seeking.(a) In a second experiment, rats that were trained to seek heroin under a second-order schedule of reinforcement (SOH) readily acquired and maintained heroin seeking under the control of the conditioned reinforcing properties of the drug-paired cue over a period of 3 weeks.(b) They did not differ in their overall heroin intake from rats that had been trained to selfadminister (e.g.take) heroin under continuous reinforcement for 3 weeks (FR1H group).(c-g) Striatal astrocytes cultured from the striatal territories of individuals trained instrumentally to respond for food under continuous reinforcement for 3 weeks (FR1F group) expressed detectable levels of DAT protein, whereas no DAT protein was detected in astrocytes cultured from the FR1H or SOH groups.Inserts show illustrative western blots.
Once again, in this fourth, independent experiment, rats readily acquired and maintained heroin seeking under the control of the conditioned reinforcing properties of the drug-paired cue over 3 weeks (main effect of session: F 7,28 = 3.43, p = 0.009, η p 2 = 0.46) (Figure 6a) and did not differ from FR1H rats in their overall level of heroin intake (t 13 = 1.846, p = 0.088) (Figure 6b).The RNAScope™ signal obtained on 12 μm thick sections (Figure 6c) was of high quality and revealed unambiguously colocalisation of DAT and GFAP mRNAs within the same cells (Figure 6d).As anticipated, a history of heroin exposure resulted in a lower level of DAT mRNA in GFAP+ cells across the striatum as compared to FR1F individuals (main effect of group in the aDLS: main effect of group: F 2,794 = 5.682, p = 0.004, η p 2 = 0.012, the aDMS: F 2,943 = 5.454, p = 0.004, η p 2 = 0.011, pDLS: F 2,1284 = 18.81, p < 0.001, η p 2 = 0.029, pDMS: F 2,727 = 9.013, p < 0.001, η p 2 = 0.024, and NAcC: F 2,1194 = 11.82,p < 0.001; η p 2 = 0.019) (Figure 6e-j).This effect was not due to a change only in GFAP+ cells with the highest level of expression of DAT mRNA across striatal astrocytes, as evidenced by the analysis of the distribution of DAT mRNA puncta per cell per animal (Figure 6f-j), nor was it attributable to a decrease in the overall number of GFAP+ cells (Figure S3).Instead, as shown by the analyses reported in the pie charts in Figure 6f-j, heroin exposure resulted in an overall reduction in the number of astrocytes that expressed a detectable level of DAT mRNA, an effect more pronounced in the SOH than the FR1H group in the NacC (see Table S1 for exact percentages and statistics).
Having demonstrated that heroin exposure results in a profound decrease in DAT mRNA and protein content in astrocytes across the striatum even before the functional engagement of the aDLS dopamine-dependent mechanisms that mediate incentive heroin-seeking habits (i.e. in FR1H rats, where responding is goal-directed), we sought to determine whether these adaptations were due to a direct effect of opiates or dopamine on astrocytes or whether they depend on interactions between the different cell type component of striatal circuits.F I G U R E 5 Pan-striatal reductions in astrocytic dopamine transporter (DAT) mRNA are observed following a prolonged history of either heroin taking or cue-controlled heroin seeking.(a) Rats that were trained to seek heroin under a second-order schedule of reinforcement (SOH) acquired and maintained heroin seeking under the control of the conditioned reinforcing properties of the drug-paired cue and did not differ in their overall heroin intake from rats that had been trained to self-administer (e.g.take) heroin under continuous reinforcement (FR1H group) (b).(c-g) Compared with FR1F rats, FR1H and SOH rats showed a profound decrease in DAT mRNA across all striatal territories.No difference was observed between FR1H and SOH groups.
protein in the aDLS and the pDLS as compared with FR1F controls, while they shared a profound decrease in DAT protein and mRNA levels in astrocytes throughout the striatum with SOH rats.Indeed, qPCR measurements of DAT mRNA content alongside RNAscope™ quantification of astrocytic DAT mRNA in situ revealed a decrease in astrocytic DAT across the entire striatum in heroin-exposed rats.
Together with the evidence of decreased DAT mRNA levels in striatal astrocytes, the observation that a history of heroin self-administration results in the profound downregulation of DAT protein expression in striatal astrocytes (as assessed in primary monocultures of astrocytes harvested from striatal territories of rats with a history of heroin taking, heroin seeking or instrumental responding for food) lends compelling evidence to the hypothesis that exposure to heroin results in a downregulation of astrocytic DAT expression across the striatum that occurs prior to the functional recruitment of the aDLS, which subserves the development of incentive habits.
The overall decrease in NacC and aDLS DAT protein levels observed in SOH rats is in agreement with the previously reported reduction in striatal DAT protein or midbrain DAT mRNA levels observed postmortem in people meeting the diagnostic criteria for heroin use disorder or who have died from heroin overdose (Horvath et al., 2007;Yuan et al., 2017).In the latter, DAT expression was decreased in all striatal territories but predominantly in the nucleus accumbens and only marginally in the dorsal striatum, an observation at odds with the relatively smaller effect size observed in the present study in the NacC as compared with the aDLS.This apparent discrepancy may be due to differences in the overall heroin exposure between the human population and the present one, the former having certainly had years of heroin use, or still, to differences in the precise striatal territories examined.That is, we specifically investigated the NAcC in the present study, since it had previously been shown to be involved in the acquisition of cue-controlled drug seeking (Ito et al., 2004;Puaud et al., 2021) and the subsequent development of aDLS dopamine-dependent incentive habits (Belin & Everitt, 2008).
The increase in total DAT protein in the dorsolateral striatum following 3 weeks of FR1 heroin selfadministration (FR1H; conditions that operationalise recreational, controlled heroin taking) is consistent with that of George et al. (2021), who demonstrated that a similar history of short daily access to heroin selfadministration under continuous reinforcement in rats resulted in an increase in the rate of striatal DA reuptake, suggesting an upregulation, or increased efficacy, of total striatal DAT.However, this increase in total DLS DAT protein content in FR1H rats is at odds both with the profound downregulation of astrocytic DAT mRNA observed in both these individuals, whose habit system has not been engaged and those having developed an incentive heroin seeking habit (SOH), who also display a decrease in total DAT protein content in the aDLS.
This observation suggests that a functional balance may exist in the striatum between presynaptic DAT expressed by dopamine neurons and DAT expressed by post-synaptic striatal cells.Indeed, because local translation of DAT has been shown not to occur in midbrain dopamine neuron axons or in their terminal varicosities (Hobson et al., 2022), the DAT mRNA presently detected in the aDLS is necessarily of a striatal origin, which we confirmed here using primary cultures of striatal astrocytes and in situ, using RNAscope™, to be astrocytic (Asanuma et al., 2014;Karakaya et al., 2007).Therefore, the decrease in striatal astrocytic DAT caused by heroin self-administration or heroin seeking throughout the striatum may be functionally compensated by an upregulation of DAT in dopamine neurons early on (e.g. after 3 weeks of heroin taking) that progressively wears off or even reverses to a downregulation in the aDLS and perhaps to some degree in the NacC when heroin seeking has become habitual.
Future research will be necessary to test this hypothesis, which has far-reaching implications: A shift in presynaptic and postsynaptic/perisynaptic dopamine reuptake may contribute to an alteration of both the temporal and spatial determinants of striatal dopamine transmission, in that dopamine would be able to spread further beyond the synapse from which it is released, hence contributing to a functional coupling of adjacent striatal microcircuits.In addition, because each astrocyte interacts with thousands of synapses and is a component of a syncytium that encompasses the entire striatum, such drug-induced alterations in their glutamatergic (Ducret et al., 2016;Spencer & Kalivas, 2017) and dopaminergic regulatory function could result in the functional coupling of adjacent functional domains of the striatum, as observed in humans (Spencer & Kalivas, 2017), which is associated with the development of incentive drug seeking habits in rats (Belin & Everitt, 2008;Fouyssac et al., 2018;Murray et al., 2015).These two processes may, therefore, contribute to the engagement of the striato-nigro-striatal spiralling circuitry (Ikeda et al., 2013) on which relies the transition from a ventral to dorsal dopaminergic locus of control over behaviour that underlies the development of incentive habits (Belin & Everitt, 2008).Such functional coupling between the NacC and dopamine-dependent mechanisms in the aDLS may be facilitated by astrocytic dopamine receptor-mediated alterations in astrocyte calcium signalling throughout the striatal syncytium (Jennings et al., 2017) and altered terminal dopamine release through perturbed astrocyte-dopamine neuron interactions (for a review of potential molecular mechanisms, see Fouyssac & Belin, 2019).
Having established that heroin exposure decreases striatal astrocytic DAT expression, we sought to determine the neurochemical underpinnings.The cellular and molecular mechanisms underlying the dopamineand psychostimulant-induced regulation of DAT in dopamine neurons are well-characterised and most classically involve their direct binding to DAT (for review, see Bu et al., 2021).As such, we originally hypothesised that opioids may indirectly modulate astrocytic DAT through the hyperdopaminergic states they induce by their μ-ORmediated disinhibition of dopamine neurons (Margolis et al., 2014).The observation that direct in vitro application of dopamine to astrocytes cultured from the aDLS of drug-naive rats results in a decrease in their DAT protein expression suggests instead that the regulation of astrocytic DAT may occur via dopamine-dependent mechanisms, as it does in neurons.Dopamine may not have affected DAT expression in posterior striatal astrocytes because the anterior and posterior striata are molecularly and genetically distinct (Shi et al., 2023), the latter expressing fewer dopamine receptors than the former (Ren et al., 2017).The midbrain dopaminergic innervation of the posterior striatum is also less dense than, and anatomically distinct from, that of the anterior striatum (Menegas et al., 2015(Menegas et al., , 2018)).Since astrocytes tend to assume the molecular portfolio of their surrounding neurons, anterior striatal astrocytes may be uniquely equipped to undergo dopamine receptor-mediated DAT downregulation (Lohani et al., 2018) or downstream processes that are consequent of astrocytic activation resulting from increased cytoplasmic dopamine (Vaarmann et al., 2010).
Astrocytes of the anterior striatum along with those of the pDLS were susceptible to opiate-induced downregulation of DAT protein.While these observations warrant further research, it can be speculated that the underlying mechanism involves opioid receptordependent ERK1/2 signalling.Indeed, striatal astrocytes express mu-and kappa-opioid (MOR and KOR) receptors, which are both powerful effectors of intracellular ERK1/2 signally pathways in astrocytes and neurons (Belcheva et al., 2005;Ikeda et al., 2010;Miyatake et al., 2009).Chronic agonism of the astrocytic MOR receptor causes a decrease in ERK1/2 signalling (Ikeda et al., 2010), and chronic agonism of the KOR receptor reduces dopamine uptake (Thompson et al., 2000).Taken together with the evidence that KOR-mediated regulation of neuronal DAT is ERK1/2-dependent (Kivell et al., 2014), it is plausible that direct chronic agonism of striatal astrocytic MOR and/or KOR by morphine or heroin causes the downregulation of DAT.
DAT protein expression in astrocytes cultured from pDMS of drug naïve rats was unaffected by direct dopamine or opiate application, suggesting that DAT regulation in the pDMS is predicated on an intact striatal microenvironment and not just direct exposure to high levels of dopamine or opiates.Such indirect effects could be mediated by postsynaptic striatal neurons, which influence astrocytes by locally releasing neuromodulators such as endocannabinoids, adenosine and nitrous oxide in response to hyperdopaminergic states (Boison et al., 2010;Martin et al., 2015).
Taken together with these in vitro findings, our in vivo observations suggest that the heroin-induced pan-striatal reductions in astrocytic DAT we observed in vivo may have occurred via several independent and territory-specific mechanisms.More comprehensive transcriptomic profiling would be necessary to uncover the regional differences in striatal astrocytes that may contribute to their differential sensitivity to dopamineand opiate-induced alterations in DAT expression, though we predict regional differences in baseline expression of DAT and dopamine/opioid receptors may be contributing factors.
In summary, the results of the present study demonstrate that exposure to heroin self-administration results in a decrease in astrocytic DAT expression throughout the striatum which precedes the development of aDLS dopamine-dependent incentive heroin seeking habits.These results suggest a striatum-wide role of astrocytes and their syncytium in dopamine-dependent striatal adaptations that were previously considered exclusively to involve neurons.

3. 2 |
Striatal astrocytic DAT protein content is profoundly reduced both following heroin self-administration and a prolonged history of cue-controlled heroin seeking Immunohistochemical analysis of the cultures revealed the presence of DAT proteins in pure astrocytic U R E 2 A prolonged history of cue-controlled heroin seeking, but not heroin taking, is associated with a reduction of dopamine transporter (DAT) protein content in the anterior dorsolateral striatum and the nucleus accumbens core.(a) In a first experiment, rats that were trained to seek heroin under a second-order schedule of reinforcement (SOH) readily acquired and maintained heroin seeking under the control of the conditioned reinforcing properties of the drug-paired cue over a period of 3 weeks.(b) They did not differ in their overall heroin intake from rats that had been trained to self-administer (e.g.take) heroin under continuous reinforcement for 3 weeks (FR1H group).(c) Compared with individuals trained instrumentally to respond for food under continuous reinforcement for 3 weeks (FR1F group), SOH rats showed a decrease in the DAT protein content of the nucleus accumbens core (NAcC).In both the anterior dorsolateral striatum (aDLS) (d) and posterior dorsolateral striatum (pDLS) (e), FR1H rats showed an increase in DAT protein levels as compared to FR1F controls.In contrast, SOH rats showed a lower level of DAT proteins in the aDLS than both FR1F and FR1H rats.No heroin-induced alterations in DAT protein levels were observed in the anterior (aDMS) (f) or the posterior (pDMS) dorsomerial striatum (g).Inserts show illustrative Western blots.F I G U R E 3 Primary striatal astrocyte monocultures express dopamine transporter (DAT) proteins.Immunohistochemical analysis of the cultures revealed pure striatal astrocyte monocultures expressing DAT proteins (a) without microglial or neuronal contamination, as evidenced by the presence of GFAP-positive cells and the absence of Iba-1-positive cells (b) and NeuN-positive cells (c).