Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats

Abstract

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

Keywords: CRISPR; caspase 3 lesion; heroin self-administration; knockin; mu opioid receptor; pain.

Figure 1.

CRISPR-mediated knock-in of T2A-iCre downstream of the rat Oprm1 coding sequence. Schematic of the target gene (rat Oprm1) with annotation for the location and sequence of the SpCas9 sgRNA that cleaves within the stop codon. The donor template encoding homologous arms and the T2A-iCre transgene are also shown.

Figure 2.

iCre mRNA and Oprm1 mRNA in NAc, DS, and dHipp. A, Oprm1⁺ cells per mm² for Oprm1 mRNA (wildtype and Oprm1-Cre, n = 5/genotype; males only). B, Oprm1⁺/Cre⁺ double-labeled cells per mm² (Oprm1-Cre rats only). C, Percent Cre⁺/Oprm1⁺ (Oprm1-Cre rats only). D, Representative confocal photomicrographs of Oprm1-Cre rat brains showing colocalization (yellow) between Oprm1⁺ neurons (red) and Cre⁺ neurons (green) in dHipp, DS, and NAc compared with wildtype rats which only showed Oprm1 expression (red). Objective lens magnification: A, C, 10×; B, D, 40×. Scale bars: A, C, 300 µm; B, D, 25 µm. Aca, Anterior commissure; CA1, CA2, CA3, hippocampal subfields; cc, corpus callosum; DG, dentate gyrus; lv, left ventricle.

Figure 3.

Autoradiography in Oprm1-Cre rats and wildtype littermates. A, DAMGO-stimulated [³⁵S]GTPyS in NAc and DS in wildtype and Oprm1-Cre rats (n = 6/genotype & sex). Values are calibrated and expressed as % basal. B, [³H]DAMGO binding in NAc and DS in wildtype and Oprm1-Cre rats (n = 6/genotype & sex). Values are calibrated and expressed as nCi/g. AAV-DIO-Casp3 lesion in NAc: autoradiography and RNAscope. We injected AAV1-EF1a-Flex-taCasp3-TEVP unilaterally into the right hemisphere of NAc shell and PBS into the left hemisphere; injections were 500 nl/side. C, [³H]DAMGO binding (left) and DAMGO-stimulated [³⁵S]GTPyS (right) in NAc of wildtype and Oprm1-Cre rats (n = 5 or 6/sex & genotype). Values are calibrated and expressed as nCi/g and % basal, respectively. D, Mean ± SEM Oprm1⁺ cells expressed as red grains per area (% area covered by red grains) in NAc shell and core in wildtype and Oprm1-Cre rats (n = 5 or 6/genotype & sex). C, D, Individual data points are depicted for males (blue) and females (red). Scale bar, 500 µm. *p < 0.05; different from the control hemisphere.

Figure 4.

Food self-administration, heroin self-administration, and heroin relapse-related behaviors in Oprm1-Cre rats and wildtype littermates. A, Food self-administration. Acquisition (left) and fixed-ratio response (right). Mean ± SEM number of pellets consumed (left) and active lever presses (right). Wildtype (3 males, 6 females), Oprm1-Cre (4 males, 6 females); data were combined for males and females. B, Heroin self-administration. Mean ± SEM number of heroin infusions during heroin self-administration training (days 1-6, 0.1 mg/kg/infusion; days 7-12, 0.05 mg/kg/infusion). Extinction responding. Mean ± SEM number of active lever presses during the seven 6 h extinction sessions. Active lever presses led to contingent presentations of the tone-light cue, but not heroin. Context-induced reinstatement. Mean ± SEM number of active lever presses during the 6 h reinstatement tests in Contexts B and A. Active lever presses led to contingent presentations of the tone-light cue, but not heroin. Individual data points are depicted for males (blue) and females (red). Reacquisition. Mean ± SEM number of heroin infusions (0.05 mg/kg/infusion) per hour during reacquisition. Active lever presses led to the delivery of heroin infusions and the tone-light cue. Wildtype (6 males, 8 females), Oprm1-Cre (5 males, 8 females); data were combined for males and females.

Figure 5.

Morphine analgesia, tolerance, and pain-related suppression of operant responding in Oprm1-Cre rats and wildtype littermates. A, von Frey test and timeline of experiment for morphine analgesia and tolerance. Left, Baseline and von Frey thresholds (g) after ascending doses of morphine (0.625, 1.25, and 2.5 mg/kg, i.p.). Middle, von Frey thresholds (g) after vehicle, 2.5 mg/kg morphine, or 2.5 mg/kg morphine + naloxone (1.0 mg/kg, i.p.). Right, von Frey thresholds (g) after vehicle, 2.5 mg/kg morphine, or 2.5 mg/kg morphine after 21 d of chronic morphine (2.5 mg/kg/day, i.p.). Wildtype (7 males, 3 females), Oprm1-Cre (7 males, 3 females). Individual data points are depicted for males (blue) and females (red). B, Tail flick test and timeline of experiment for morphine analgesia and tolerance. Left, Latency (s) measured after ascending doses of vehicle and morphine (1.25, 2.5, 5, and 10 mg/kg, i.p.). Middle, Latency (s) after vehicle, 5 mg/kg morphine, or 5 mg/kg morphine + naloxone (1.0 mg/kg, i.p.). Right, Latency (s) after vehicle, 5 mg/kg morphine, or 5 mg/kg morphine after 21 d of chronic morphine (5 mg/kg/day, i.p.). Wildtype (5 males, 5 females), Oprm1-Cre (5 males, 4 females). Individual data points are depicted for males (blue) and females (red). C, Acute lactic acid-induced suppression of operant responding for food pellets. Left panels, Mean ± SEM pellet intake change score from baseline after injections of lactic acid (0%, 0.9%, 1.35%, and 1.8%, i.p.), morphine (0, 1, 3, and 10 mg/kg, s.c.), and lactic acid (1.8%) plus morphine (0, 1, and 3 mg/kg, s.c). Right panels, Mean ± SEM percent change from baseline of the data presented on the left panels. Wildtype (4 males, 4 females) and Oprm1-Cre (4 males, 3 females) rats.

Figure 6.

Effect of AAV-DIO-Casp3 NAc lesions on acquisition and maintenance of food and heroin self-administration in heterozygote Oprm1-Cre rats and wildtype littermates. A, Food. Left, Acquisition. Mean ± SEM number of pellets consumed during the 3 h sessions. Wildtype (14 males, 12 females), Oprm1-Cre (14 males, 15 females). Right, Within-session fixed-ratio response. Mean ± SEM number of pellets consumed during the 1 h sessions. Wildtype (8 males, 5 females), Oprm1-Cre (8 males, 6 females). B–F, Heroin. B, Acquisition: daily infusions. Mean ± SEM number of heroin infusions during the 3 h heroin self-administration sessions. C, Acquisition: dose–response (3 d mean). Mean ± SEM of heroin infusions at each unit dose. Wildtype (13 males, 12 females), Oprm1-Cre (13 males, 15 females) for B, C. D, Maintenance: within-session dose–response. Mean ± SEM number of heroin infusions at each unit dose. E, Maintenance: within-session fixed-ratio response. Mean ± SEM number of heroin infusions at each hour of heroin self-administration for each fixed-ratio requirement. F, Maintenance: extended access. Mean ± SEM number of heroin infusions during the 9 h heroin self-administration session. Wildtype (13 males, 12 females), Oprm1-Cre (11 males, 14 females) for D–F. G, [³H]DAMGO binding in NAc in wildtype (12 males, 11 females) and Oprm1-Cre (10 males, 12 females) rats. Values are calibrated and expressed as nCi/g. Individual data points are depicted for males (blue) and females (red). *p < 0.05; different from the control hemisphere. *p < 0.05; different from the Oprm1-Cre group.

Figure 7.

Effect of NAc injections of AAV-DIO-EYFP on acquisition and maintenance of food and heroin self-administration in heterozygote Oprm1-Cre rats and wildtype littermates. A, Food. Left, Acquisition. Mean ± SEM number of pellets consumed during the 3 h sessions. Wildtype (7 males, 8 females), Oprm1-Cre (6 males, 7 females). Right, Within-session fixed-ratio response. Mean ± SEM number of pellets consumed during the 1 h sessions. Wildtype (7 males, 8 females), Oprm1-Cre (6 males, 7 females). B–F, Heroin. B, Acquisition: daily infusions. Mean ± SEM number of heroin infusions during the 3 h heroin self-administration sessions. C, Acquisition: dose–response (3 d mean). Mean ± SEM of heroin infusions for each unit dose. Wildtype (7 males, 8 females), Oprm1-Cre (6 males, 7 females) for B, C. D, Maintenance: within-session dose–response. Mean ± SEM number of heroin infusions for each unit dose. E, Maintenance: within-session fixed-ratio response. Mean ± SEM number of heroin infusions during each hour of heroin self-administration for each fixed-ratio requirement. F, Maintenance: extended access. Mean ± SEM number of heroin infusions during the 9 h heroin self-administration session. Wildtype (6 males, 7 females), Oprm1-Cre (6 males, 7 females) for D–F.