Fig 1.
Genetic construct generation and sequencing of the Oprm1-Cre mouse line.
(A) Cartoon representation of the genetic targeting strategy to create the desired mouse line. CRISPR/Cas9 mediated insertion encoding a functional Cre-recombinase (Cre) enzyme was inserted upstream of the Oprm1 stop codon, in Exon 4. A T2A-cleavable peptide was included in this genetic construct to allow for Cre release from the mu-opioid receptor (MOR) once the entire Oprm1-T2Acre gene is translated to avoid unwanted protein interactions. (B) Confirmational DNA sequencing of the T2Acre genetic insert. (C) Genotyping PCR products for homozygote (Oprm1Cre/Cre) heterozygote (Oprm1Cre/+), and wild-type (Oprm1+/+) mice. Wild-type allele is 273bp and T2Acre allele is 350bp.
Fig 2.
Oprm1 mRNA and 3HDAMGO binding is not altered in the Oprm1-Cre mouse line compared to wild type C57Bl/6 mice.
Animals expressing the T2A-Cre allele showed no significant molecular differences of Oprm1-expression when compared to wild-type littermates. (A) Quantification of the qPCR analysis of Oprm1 between Oprm1Cre/+ and Oprm1+/+ animals shows that there is comparable Oprm1 mRNA transcript levels in the hippocampus, hypothalamus, PFC, cerebellum, striatum, and thalamus, regardless of genotype (Males, 10-15wks old, n = 6). Two-way ANOVA revealed no main effect of genotype between groups (F (1,60) = 0.5438, p = 0.4637). (B) 3H-DAMGO binding is not different between groups. (Males, 10-15wks old, n = 6–7). Two-way ANOVA revealed a main interaction effect (F(2, 32) = 3.306, p = 0.0495. Post hoc test revealed no difference between Oprm1Cre/+ and Oprm1+/+ mouse lines in any brain region.
Fig 3.
Fluorescence in situ hybridization of Oprm1Cre; RosaLSL-GFP-L10a mice confirms selective reporter expression in Oprm1+ cells in mouse habenula and cortex.
Overlap of Oprm1 (red) and GFP (Cyan) mRNA in (A) the medial and lateral habenula; and (B) Primary Somatosensory Cortex. Left images: Oprm1 signal (red) and DAPI (grey); Middle: GFP signal (cyan) + DAPI (grey); Right images: Merge of all signals. Top images: Entire ROI with cortical layers and/or subnuclei indicated; bottom images: large image of area of densest Oprm1 signal. L1-6b: indicates cortical layer; HbX: HbX subdivision of the habenula; MHb, medial habenula; LHb, lateral habenula; S1; primary somatosensory cortex. Signal was detected using the RNAScope Multiplex Fluorescence V2 kit. 16μm slices were imaged using the BZ-X800 Viewer software in conjunction with a BZ-X Series automated Keyence microscope. Images were taken at 40x magnification. Mice (n = 2) were homozygous for both transgenes. All scale bars: 200μm.
Fig 4.
Imaging brain tissue from GFP-reporter mice crossed with the Oprm1-Cre line confirms functional Cre-mediated recombination.
Recombinant GFP fluorescence reporting in Nucleus Accumbens (NAcc), Cortex (CTX), Hippocampus, Thalamus (Th), Ventral Tegmental Area (VTA) and ventrolateral periaqueductal-gray (VL-PAG). Oprm1-Cre x RosaLSL-GFP-L10a tissue (TRAP mice) and Oprm1-Cre x RosaLSLSun1-sfGFP (Sun1 mice) were imaged at 2x, 10x (z-stack/stitched image) and 40x magnification. Note that the ribosomal-GFP expression pattern is more dispersed and less defined in TRAP mice compared to the nuclear membrane staining in Sun1 mice. Slides were imaged using the BZ-X800 Viewer software in conjunction with a BZ-X Series automated Keyence microscope.
Fig 5.
SUN1-GFP reporter signal in the Oprm1Cre; RosaLSL-Sun1-sfGFP mouse line is observed in brain structures that highly express MOR.
Expression of SUN1-GFP reporter signal within the Oprm1Cre; RosaLSL-Sun1-sfGFP mouse observed across several structures known to highly express the mu opioid receptor (MOR), including the (A) dorsomedial striatum, (B) habenula, and (C) parabrachial nucleus. High magnification images (20X and 40X) of sample areas within section are denoted by boxed (yellow) regions within lower magnification images. Individual channels showing anti-MOR staining (pink) and SUN1-GFP (green) signal overlaying DAPI stained nuclei (gray) are displayed next to merged signal images. White arrows in (A) denoted striosomal regions within the dorsomedial striatum. DMS = dorsomedial striatum; NAcc = nucleus accumbens core; MHb = medial habenula; LHb = lateral habenula; PBN = parabrachial nucleus; c.c. = corpus callosum.
Fig 6.
Overlap of SUN1-GFP expression from Oprm1Cre; RosaLSL-Sun1-sfGFP mice with mu opioid receptor (MOR) staining across multiple brain regions.
Low (4X) and higher (20X) magnification images of SUN1-GFP signal observed within the (A) somatosensory cortex, (B) dorsal hippocampus, (C) periventricular thalamus, (D) nucleus accumbens, and (E) periaqueductal gray in brain tissue sections taken from the Oprm1Cre; RosaLSL-Sun1-sfGFP mouse. High magnification images of sample areas within each section are denoted by boxed (yellow) regions within the low magnification images. Individual channels showing anti-MOR staining (pink) and SUN1-GFP (green) signal overlaying DAPI stained nuclei (gray) are displayed next to merged signal images. S1 = somatosensory cortex; L1-6b = cortical layers; CA1 = CA1 division of the hippocampus; CA3 = CA3 division of the hippocampus; DG = dentate gyrus of the hippocampus; MHb = medial habenula; LHb = lateral habenula; PVT = periventricular thalamus; IMD = interomediodorsal thalamus; CM = central medial thalamus; MD = mediodorsal thalamus; NAcc = nucleus accumbens core; NAcs = nucleus accumbens shell; dmPAG = dorsomedial periaqueductal gray; dlPAG = dorsolateral; l = lateral; vlPAG = ventrolateral; c.c. = corpus callosum; a.c. = anterior commissure.
Fig 7.
Confirmation of MOR function and acute and chronic opiate-mediated behaviors/phenotypes in the Oprm1-Cre mouse line.
Animals expressing the T2A-Cre allele showed no significant behavioral differences of morphine-induced phenotypes when compared to wild-type littermates. (A) No significant differences of acute morphine-induced locomotion responses occurred between Oprm1Cre/+ and Oprm1+/+ animals. Following 20mg/kg morphine injections activity was recorded for 2hrs (males, 10-15wks old, n = 8/group) (2-way ANOVA: significant effect of time post-injection on locomotor activity (F(9, 120) = 2.53, p < 0.05), no effect of genotype (F(1, 120) = 0.60, p = 0.44). (B) Morphine-mediated antinociception, as measured by hind-paw lick latency on a 55°C hot-plate assay using a cumulative-dosing paradigm, was not different (males, 10-15wks old, n = 8/group) (2-way ANOVA analysis: significant effect of morphine dose (F(1.43, 12.89) = 6.36, p = 0.0178) and no genotype effect (F(1,9) = 0.016, p = 0.901). Results are presented as percentage of maximal possible effect (MPE) [(morph jump latency − saline jump latency)/(total time − saline jump latency) × 100] (mean ± SEM, n = 18). (C) Naloxone induced precipitated withdrawal after a chronic morphine exposure paradigm is intact between Oprm1Cre/+ and Oprm1+/+ animals. Cumulative withdrawal score per animal was calculated by tallying all instances of somatic signs in the 30-minute test interval (males, 10-15wks old, n = 5–6) (unpaired two tailed t-test: p = 0.523).
Fig 8.
TRAP-sequencing in Oprm1-T2A-Cre striatum enriches for neuronal cell types.
(A) GO analysis for enrichment in top 10,000 genes from TRAP-seq. The top 10,000 expressed genes (by average normalized gene count across all samples) were analyzed for GO term enrichment using goProfiler. All significant (p<0.05) GO terms were then reduced using Revigo. Top GO term categories are shown here. (B) Brain cell type enrichment. Enrichment for genes characteristic of brain cell types [21] was calculated for using a hypergeometric test. Data sets from each sequencing experiment included the top 10,000 expressed genes (by average normalized gene count across control samples for bulk sequencing experiments, and across all samples for TRAP-sequencing experiment). (C and D). Striatum cell type enrichment. Enrichment for genes characteristic of striatum cell types was calculated in an identical manner for cell-specific gene lists from [22] (C) as well as gene lists from Merienne et. al. 2019 (D) [23].
Fig 9.
Chronic morphine withdrawal increases expression of membrane and synapse related genes in MOR+ cells in the striatum.
(A) Differentially expressed genes in MOR+ cells in the striatum following chronic morphine withdrawal. (B) GO analysis for enrichment in DEGs (p<0.05) upregulated in MOR+ cells in the striatum following chronic morphine withdrawal. All significantly upregulated DEGs were analyzed for GO term enrichment using goProfiler. All significant (p<0.05) GO terms were then reduced using Revigo. Top GO term categories are shown here.