David S. Reiner

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 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 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 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.

The major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest.

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16× coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus Giardia can be two different species. Giardia intestinalis is a major contributor to the enormous burden of diarrheal diseases with 250 million symptomatic infections per year, and it is part of the WHO neglected disease initiative. Nonetheless, there is poor insight into how Giardia causes disease; it is not invasive, secretes no known toxin and both the duration and symptoms of giardiasis are highly variable. Currently, there are seven defined variants (assemblages) of G. intestinalis, with only assemblages A and B being known to infect humans. Although assemblage B is the most prevalent worldwide, it is inconclusive whether the various genotypes are associated with different disease outcomes. We have used the 454 sequencing technology to sequence the first assemblage B isolate, and the genome was compared to the earlier sequenced assemblage A isolate. Large genetic differences were detected in genes involved in survival of the parasite during infections. The genomic differences between assemblage A and B can explain some of the observed biological and clinical differences between the two assemblages. Our data suggest that assemblage A and B Giardia can be two different species. The identification of genomic differences between assemblages is indeed very important for further studies of the disease and in the development of new methods for diagnosis and treatment of giardiasis.