Article Figures & Data
Figures
- Figure 1.
Intact MOR function in the Oprm1-Cre line. a, Targeting strategy: a cDNA encoding a functional EGFP/Cre recombinase fusion protein is inserted in frame and upstream the stop codon of the Oprm1 gene. In addition, a T2A cleavable peptide sequence is joining Oprm1 gene to the EGFP/Cre sequence, so that the EGFP/Cre enzyme is released from the receptor on translation of the MOR-T2A-EGFP/Cre fusion protein (Extended Data Fig. 1-1, Knockin (KI) genotyping strategy). b, Oprm1 mRNA expression levels are identical between Oprm1Cre/Cre and Oprm1+/+ mice. Quantification was done by RT-qPCR in samples from DS, NAc, Hb, IPN, VTA/SN, Cer, PAG, and SC and shows comparable Oprm1 transcript levels across genotypes. c, Confocal imaging of RNAscope probes targeting eGFP (green) and Oprm1 (purple) mRNAs in addition of DAPI staining (blue) shows colocalization of the two transcripts in dorsal striatum sections of Oprm1Cre/Cre mice (inset). Dashed lines delimit examples of MOR-EGFP/Cre-positive neurons. Magnification, 60× with immersion oil. Scale bar, 5 μm. d, Left, Immunohistochemistry shows EGFP/Cre and MOR protein expression in the same habenular subdivisions. Coronal brain sections of heterozygote Oprm1Cre/+ mice were stained with EGFP and MOR antibodies (Extended Data Fig. 1-2, MOR antibody validation), and fluorescence microscopy shows the expected staining mainly in basolateral (bl) and apical (a) parts of the MHb. Bm, Basomedial; sm, stria medullaris tract. Magnification:, 10×. Scale bar, 50 μm. Right, Four panels at higher magnification (inset, basolateral part of the MHb), staining reveals nuclear DAPI (blue) and EGFP/Cre (green) staining, whereas MOR staining (purple) is exclusively extranuclear. Magnification, 60× with immersion oil. Scale bar, 5 μm. Dashed lines delimit one example of an MOR-EGFP/Cre-positive neuron. e, MOR signaling is preserved in Oprm1Cre/Cre mice. G-protein activation was evaluated using [35S]-GTPγS binding: DAMGO-induced G-protein activation is similar in striatal membranes to the two genotypes (n = 2 pools × 4 striatum; EC50, 324 nm for Oprm1+/+ and 392 nm for OprmCre/Cre; Emax, 148 ± 7 for Oprm1+/+ and 156 ± 11 for Oprm1Cre/Cre). f, Locomotor sensitization of morphine is intact in Oprm1Cre/Cre mice. Mice were injected at days 1, 4, 8, 11, 14, and 18 with morphine (40 mg/kg, i.p.). Total traveled distances recorded for 2 h are comparable in Oprm1+/+ and Oprm1Cre/Cre mice (n = 7 animals/group). Data are presented as the mean ± SEM. g, Analgesic effects of morphine are intact in Oprm1Cre/Cre mice. Analgesia was assessed by tail immersion test: identical tail withdrawal latencies were measured at 48°C and 52°C, in wild-type Oprm1+/+ and Oprm1Cre/Cre mice 45 min after a single saline or morphine injection (2.5 or 5 mg/kg; n = 10 animals/group). Dashed horizontal lines show cutoff at 15 s for 48°C and 10 s for 52°C. Data are presented as the mean ± SEM. ***p < 0.001 morphine effect compared with saline.
- Figure 2.
Cre-mediated recombination is efficient in the Oprm1-Cre line. a, Cre-mediated expression of the fluorescent reporter tdTomato in the habenula–interpeduncular pathway. Left, Cre recombination on local injection of an AAV2.EF1a.DIO.mCherry viral reporter in the MHb; right, Cre recombination on breeding Cre-dependent tdTomato reporter mice with the Oprm1-Cre line both lead to a strong fluorescent signal apical (a) and basolateral (bl) of the MHb, as well as in the rostral (ipr) and lateral (ipl) areas of the IPN. Scale bar, 100 μm. b, Cre-mediated tdTomato expression in the adult brain of Oprm1-Cre × tdTomato mice. Top, Whole-brain sagittal views show high tdTomato expression in the thalamus, as well as the entire mesolimbic (left) and MHb-IPN (right) pathways. Scale bar, 500 μm. Middle, Coronal sections show strong fluorescence in the basal ganglia and thalamus, as well as in most amygdalar nuclei with the notable exception of basolateral amygdala. Scale bar, 500 μm. Bottom, Higher-magnification highlights both cell bodies and/or fibers patterns of labeled neurons. Scale bar, 200 μm. Slides were scanned on the Olympus VS120 with a 10× objective. BLA, Basolateral amygdala; CeA, central amygdala; EP, endopiriform nucleus; fr, fasciculus retroflexus; GP, globus pallidus; HIP, hippocampus; IA, intercalated amygdala; LHb, lateral Hb; OT, olfactory tubercle; Th, thalamus.
- Figure 3.
The Oprm1-Cre line is amenable to optogenetics. Oprm1-CreVTA-VTA::ChR2 photostimulation causes behavioral avoidance. a, Diagram showing viral delivery of AAV2.EF1a.DIO.ChR2-mCherry (channelorhodopsin) or AAV2.EF1a.DIO.mCherry (control) into the VTA and fiber-optic implantation above the VTA, a well as a timeline for the experimental procedure. b, Representative image of viral expression (red), optic fiber implantation, and VTA dopamine cells immunostained with anti-tyrosine hydroxylase (TH)/Alexa Fluor 647 (blue). Tissues were observed on an inverted epifluorescence microscope. c, Confocal imaging of VTA sections stained with GABA antibody show overlapping with viral expression. Scale bar, 10 μm. d, Occupancy plots for representative individual at 20 Hz. Mice were free to explore the two- chamber RTPT apparatus for 20 min. Mice then received a blue stimulation when entering the light-paired side at 0, 10, 20, and 40 Hz (473 nm, 10 mW, 10 ms pulse width) on 4 consecutive days, as described by Seo et al. (2016). e, Activation of VTA MOR neurons produces place avoidance. Light stimulation in Oprm1-CreVTA-VTA::ChR2 mice induced significant behavioral avoidance to the light-paired side compared with control group (n = 7–8/group). The graph shows frequency responses of mice receiving a blue stimulation when entering the light-paired side (473 nm, 10 mW, 10 ms pulse width at 0, 10, 20, and 40 Hz; n = 9–10, control vs ChR2). Data are expressed as the mean ± SEM. **p < 0.01, ***p < 0.001. f, Further analysis of the 20 Hz simulation condition indicated significant avoidance for the light-paired side without affecting the total distance traveled (p > 0.05).
Movies
- Movie 1.
3D views of the whole brain.
Figure 1-1
KI genotyping strategy. Diagram describing the position of the primers used for genotyping. Download Figure 1-1, TIF file.
Figure 1-2
UMB3 expression and antibody validation. Coronal sections of habenula were stained with UMB3 antibody and show MOR expression in wild-type mice (left), but no signal could be detected in the MOR KO mice (right). Scale bar, 100 μm. Download Figure 1-2, TIF file.
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