Filipiak, W. E. | bioRxiv Science

bioRxiv2022

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

The brain {micro}-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 anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the role of nucleus accumbens (NAc) MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and fluorescence in situ hybridization chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum (DS), and dorsal hippocampus, or MOR receptor density (except DS) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly co-expressed 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 and report sex-specific effects: 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 with these rats suggests a sex-specific role of NAc MOR-expressing cells in heroin self-administration. Significance statementThe brain {micro}-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 a potential sex-specific role of nucleus accumbens MOR-expressing cells in heroin self-administration. The new Oprm1-Cre rats can be used to study both the general and sex-specific role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

neuroscience↗

bioRxiv2018

Knock-in rat lines with Cre recombinase at the dopamine D1 and adenosine 2a receptor loci.

Genetically-modified mice have become standard tools in neuroscience research. Our understanding of the basal ganglia in particular has been greatly assisted by BAC mutants with selective transgene expression in striatal neurons forming the direct or indirect pathways. However, for more sophisticated behavioral tasks and larger intracranial implants, rat models are preferred. Furthermore, BAC lines can show variable expression patterns depending upon genomic insertion site. We therefore used CRISPR/Cas9 to generate two novel knock-in rat lines specifically encoding Cre recombinase immediately after the dopamine D1 receptor (Drd1a) or adenosine 2a receptor (Adora2a) loci. Here we validate these lines using in situ hybridization and viral vector mediated transfection to demonstrate selective, functional Cre expression in the striatal direct and indirect pathways respectively. We used whole-genome sequencing to confirm the lack of off-target effects, and established that both rat lines have normal locomotor activity and learning in simple instrumental and Pavlovian tasks. We expect these new D1-Cre and A2a-Cre rat lines will be widely used to study both normal brain functions and neurological and psychiatric pathophysiology.

neuroscience↗