Article Figures & Data
Figures
- Figure 1.
Colocalization of Oprk1 and Cre. A–C, In situ hybridization using probes targeting Cre recombinase and mouse Oprk1 reveal 80–90% colocalization between Oprk1 and Cre transcripts across the following three brain regions: the BLA (A), claustrum/dorsal endopiriform nucleus (B), and PVT (C). N = 2 male mice; N = 1 female mouse. Scale bar, 50 µm. See Extended Data Figure 1-1 for generation of Oprk1-Cre transgenic mouse.
- Figure 2.
Brain-wide snapshots of Cre expression in the adult Oprk1-Cre mouse. Tiled images of Cre protein expression at 20× resolution across the brain of an adult Oprk1-Cre mouse are shown. Strongest Cre expression was observed in the claustrum and dorsal endopiriform nucleus. Cre expression was also observed in the PVT, CeA, BLA, PVT, DR, and locus coeruleus (LC). The pattern of Cre expression matched that of Oprk1 expression in the Allen Brain Atlas. Scale bar, 1 mm. Please see Extended Data Figure 2-1 for distribution of BLAKOR cells and Extended Data Figure 2-2 for colocalization between virally delivered Cre-dependent EGFP and Oprk1 in the BLA.
- Figure 3.
KOR function is intact in Oprk1-Cre mice. A, Dyn-stimulated GTPγS binding did not differ significantly between WT and Oprk1-Cre mice. n = 7 males/group. B–D, No differences were observed in phosphorylated (P)-ERK (B), P-JNK (C), and P-p38 (D) in the NAc under basal conditions. N = 4–5/group (WT = 4 females, Cre/+ = 3 females and 2 males). Please see Extended Data Figure 3-1 for Oprk1 mRNA expression in WT and Oprk1-Cre mice; Extended Data Figure 3-2 for [35S]GTPγS binding in the striatum of WT and Oprk1-Cre mice; and Extended Data Figure 3-3 for basal KOR signaling is not altered in the amygdala of Oprk1-Cre mice. Extended Data Table 3-1 lists antibodies used for Western blotting.
- Figure 4.
Baseline anxiety-like and pain behaviors in Oprk1-Cre mice. A–C, Anxiety-like behaviors were tested using the EPM and open field anxiety tests in WT and Oprk1-Cre mice. There were no significant genotypic differences in open arm entries (A) or time (B) in the EPM (WT male = 7, WT female = 12; Cre/+ male = 8, Cre/+ female = 7). C, There were also no genotypic differences in the percentage of time in the center of the open field (WT male = 8, WT female = 11; Cre/+ male = 9, Cre/+ female = 7). D, There were no genotype differences seen in the mean withdrawal threshold in the eVF mechanical nociception test (WT male = 8, WT female = 10; Cre/+ male = 8, Cre/+ female = 7) and response latencies (D) on the hot plate tests (E; WT male = 8, WT female = 10; Cre/+ male = 8, Cre/+ female = 7) and cold plate tests (F) and (WT male = 8, WT female = 9; Cre/+ male = 9, Cre/+ female = 7). See Extended Data Figure 4-1 for closed arm entries were not altered on the EPM in Oprk1-Cre mice.
- Figure 5.
Validation of DREADD-induced activation of BLAKOR neurons. A–C, CNO application increased both spontaneous and electrically evoked excitability in BLAKOR cells expressing hM3DQ. *p = 0.0217, **p = 0.0091, paired t test, n = 7 cells/group. D, Visual representation of c-Fos activation in the BLA of hM3DQ-injected Oprk1-Cre mice treated with vehicle or CNO before killing. E, Quantification of c-Fos shows a significant increase in the activation in CNO-injected mice compared with vehicle-injected mice. **p = 0.0041, unpaired t test, N =3–4 mice/group.
- Figure 6.
Projections of BLAKOR neurons. Image of mCherry injection into the BLA (left). BLAKOR neurons project to the prefrontal cortex (PFC), the claustrum/dorsal endopiriform nucleus, the NAc, the bed nucleus of the stria terminalis (BNST), and the ventral hippocampus (VHipp; right).
- Figure 7.
Chemogenetic activation of BLAKOR cells and anxiety-like behavior. A, There was no significant difference between mCherry and hM3DQ-injected male mice in open arm entries or open arm time in males (N = 8/group). B, There was a significant increase in the percentage of open arm entries (**p = 0.0016) and open arm time (***p = 0.0010, Sidak post-test) in hM3DQ-injected female mice compared with mCherry controls (N =9–10 mice/group). Please see Extended Data Figure 7-1 for closed arm entries in mCherry-injected and hM3DQ-injected mice. C, There were no differences in the time spent in the center of an open field in male or female hM3DQ-injected mice compared with mCherry controls (mCherry male = 9, mCherry female = 11; hM3DQ male = 9, hM3DQ female = 9). D, There was no difference in sociability index in male mCherry-injected and hM3DQ-injected mice. Female hM3Dq mice showed increased sociability compared with mCherry controls. This result did not reach statistical significance (p = 0.088, Sidak post-test; mCherry male = 9, mCherry female = 11; hM3DQ male = 9, hM3DQ female = 10).
- Figure 8.
Chemogenetic activation of BLAKOR cells attenuates U-50488 induced CPA. A, CNO administration attenuated U-50488-induced CPA in hM3DQ-injected males compared with mCherry controls. However, this effect did not reach statistical significance (p = 0.0524, Sidak post-test; N = 8–9/group) in males and in females (N = 9–11/group). B, Neither male nor female hM3DQ-injected Oprk1-Cre mice form a preference for or avoid the CNO-paired chamber. N = 5–6/group for males and N = 5–7/group for females. See Extended Data Figure 8-1 for U-50488-induced CPA in C57BL/6J mice.
Tables
Figure Parameter Type of test Sample size Statistical data 3A [35S]GTPγS binding Nonlinear curve fit WT male = 7
Cre/+ male = 7EC50 WT = 1.33 × 10–7 M for WT and 1.268 × 10−7 for Cre/+
R2 = 0.7756 for WT and 0.7597 for Cre/+3B P-ERK levels normalized to T-ERK Unpaired t test
Two tailedWT female = 4, Cre/+ female = 3, Cre/+ male = 2 t = 0.68, df = 10,
p = 0.513C P-JNK levels to T-JNK Unpaired t test, two-tailed WT female = 4, Cre/+ female = 3, Cre/+ male = 2 t = 0.498. df =7,
p = 0.63353D P-p38 levels normalized to total p38 Unpaired t test, two-tailed WT female = 4, Cre/+ female = 3, Cre/+ male = 2 t = 0.043, df =10.
p =0. 974A Elevated plus maze, percentage open arm entries Two-way ANOVA WT male = 7, WT female = 12; Cre/+ male = 8, Cre/+ female = 7 Fgenotype (1,30) = 0.21, p = 0.66
Fsex (1,30) = 4.2, p = 0.049
Fgenotype × sex (1,30) = 0.32, p = 0.574B Elevated plus maze, percentage open arm time Two-way ANOVA WT male = 7, WT female = 12; Cre/+ male = 8, Cre/+ female = 7 Fgenotype (1,30) = 0.74, p = 0.4
Fsex (1,30) = 0.99, p = 0.33
Fgenotype × sex (1,30) = 0.46, p = 0.504C Percentage time in center, open field Two-way ANOVA WT male = 8, WT female = 11; Cre/+ male = 9, Cre/+ female = 7 Fgenotype (1,31) = 1.97, p = 0.17
Fsex (1,31) = 4.4, p = 0.04
Fgenotype × sex (1,31) = 0.00003, p = 0.994D Mean withdrawal threshold, electronic von Frey Two-way ANOVA WT male = 8, WT female = 10; Cre/+ male = 8, Cre/+ female = 7 Fgenotype (1,29) = 0.18, p = 0.68
Fsex (1,29) = 9.82, p = 0.0039
Fgenotype × sex (1,29) = 3.78, p = 0.064E Response latency, hot plate Two-way ANOVA WT male = 8, WT female = 10; Cre/+ male = 8, Cre/+ female = 7 Fgenotype (1,29) = 1.28, p = 0.27
Fsex (1,29) = 3.76, p = 0.06
Fgenotype × sex (1,29) = 2.58, p = 0.124F Response latency,
cold plateTwo-way ANOVA WT male = 8, WT female = 9; Cre/+ male = 9, Cre/+ female = 7 Fgenotype (1,29) = 0.09, p = 0.77
Fsex (1,29) = 1.7, p = 0.2
Fgenotype × sex (1,29) = 1.94, p = 0.175B Number of action potentials Paired t test, two-tailed Baseline = 7, CNO = 7 t = 3.08, df = 9, p = 0.022 5C Resting membrane potential Paired t test, two-tailed Baseline = 7, CNO = 7 t = 3.757, df = 9, p = 0.0094 5E c-Fos counts Unpaired t test, two-tailed Veh = 3, CNO = 4 t = 4.995, df = 5, p = 0.0041 7A Elevated plus maze, percentage open arm entries Two-way ANOVA mCherry male = 8, mCherry female = 10; hM3DQ male = 8, hM3DQ female = 9 Fvirus (1,31) = 6.64, p = 0.015
Fsex (1,31) = 2.51, p = 0.12
Fvirus × sex (1,31) = 7.49, p = 0.017B Elevated plus maze, percentage open arm time Two-way ANOVA mCherry male = 8, mCherry female = 10; hM3DQ male = 8, hM3DQ female = 9 Fvirus (1,31) = 9.4, p = 0.005
Fsex (1,31) = 5.07, p = 0.032
Fvirus × sex (1,31) = 12.22, p = 0.0017C Percentage time in center, open field Two-way ANOVA mCherry male = 9, mCherry female = 11; hM3DQ male = 9, hM3DQ female = 9 Fvirus (1,34) = 1.12, p = 0.29
Fsex (1,34) = 0.03, p = 0.87
Fvirus × sex (1,34) = 0.48, p = 0.497D Sociability index Two-way ANOVA mCherry male = 9, mCherry female = 11; hM3DQ male = 9, hM3DQ female = 10 Fvirus (1,35) = 0.48, p = 0.83
Fsex (1,35) = 0.01, p = 0.92
Fvirus × sex (1,35) = 6.76, p = 0.018A U-50488 CPA, difference score Two-way ANOVA mCherry male = 9, mCherry female = 10; hM3DQ male = 8, hM3DQ female = 11 Fvirus (1,34) = 1.656, p = 0.21
Fsex (1,34) = 0.016, p = 0.89
Fvirus × sex (1,34) = 4.67, p = 0.0378B CNO CPP, difference score Two-way ANOVA mCherry male = 5, mCherry female = 7; hM3DQ male = 6, hM3DQ female = 7 Fvirus (1,21) = 0.0004, p = 0.98
Fsex (1,21) = 0.85 p = 0.37
Fvirus × sex (1,21) = 1.005, p = 0.3276Extended Data 3-1A Relative Oprk1expression, whole brain Unpaired t test, two-tailed WT female = 5
Cre/+ female = 5t = 7.43, df = 8, p < 0.0001 Extended Data 3-1B Relative Oprk1expression, NAc Unpaired t test, two-tailed WT male = 4
WT female = 2
Cre/+ male 3 = Cre/+ female = 5t = 2.934, df = 12, p = 0.013 Extended Data 3-2A Maximal G-protein stimulation Unpaired t test, two-tailed WT male = 7
Cre/+ male = 7t = 0.12, df = 12, p = 0.9 Extended Data 3-2B EC50 Unpaired t test, two-tailed WT male = 7
Cre/+ male = 7t = 0.07, df = 12, p = 0.95 Extended Data 3-2C Basal [35S]GTPγS binding Unpaired t test, two-tailed WT male = 7
Cre/+ male = 7t = 0.22, df = 12, p = 0.83 Extended Data 3-3A Amygdala p-ERK/T-ERK Unpaired t test, two-tailed WT female = 5, Cre/+ male = 2 male, Cre/+ female = 2 t = 0.712, df = 7, p = 0.499 Extended Data 3-3B Amygdala p-JNK/T-JNK Unpaired t test, two-tailed WT female = 5, Cre/+ male = 2 male, Cre/+ female = 2 t = 0.715, df = 7, p = 0.498 Extended Data 3-3C Amygdala P-p38/T-p38 Unpaired t test, two-tailed WT female = 5, Cre/+ male = 2 male, Cre/+ female = 2 t = 1.922, df = 6, p = 0.1030 Extended Data 4-1 Number of closed arm entries, WT vs Oprk1-Cre Two-way ANOVA mCherry male = 7, mCherry female = 12; hM3DQ male = 8, hM3DQ female = 7 Fgenotype (1,30) = 1.335, p = 0.26
Fsex (1,30) = 0.76, p = 0.39
Fgenotype × sex (1,30) = 0.66, p = 0.43Extended Data 7-1 Number of closed arm entries, mCherry vs hM3DQ Two-way ANOVA mCherry male = 8, mCherry female = 10; hM3DQ male = 8, hM3DQ female = 9 Fvirus (1,31) = 0.02, p = 0.88
Fsex (1,31) = 0.43, p = 0.52
Fvirus × sex (1,31) = 0.62, p = 0.43Extended Data 8-1 Difference score percentage (Post-Pre) Unpaired t test WT male = 8–10 t = 2.322, df =18, p = 0.032 P, Phosphorylated; T, total.
Figure 1-1
Schematic illustrating insertion of Cre recombinase into the 3′UTR of the KOR locus. Download Figure 1-1, TIF file.
Figure 2-1
Distribution BLAKOR cells. GFP expression was examined in Oprk1-Cre:: L10A-EGFP mice. GFP expression was distributed throughout the BLA in anterior sections (left). This expression pattern shifted medially and ventrally in more posterior sections. This expression pattern matched the pattern found on the Allen Brain Atlas (right). Scale bar, 200 µm. The numbers shown in panels on the right correspond to image numbers in the Allen Brain Atlas for Oprk1 mRNA expression (coronal sections). Download Figure 2-1, TIF file.
Figure 2-2
Colocalization between EGFP-expressing and OPRK1-expressing cells in the BLA. We examined colocalization between Oprk1 and virally delivered Cre-dependent EGFP expression in the BLA of Oprk1-Cre mice. A, Representative images showing EGFP and Oprk1 expression in the BLA. B, Quantification of the results are shown and revealed that 93% of EGFP-expressing cells also expressed Oprk1. N = 3 female mice. Scale bar, 100 µm. Download Figure 2-2, TIF file.
Figure 3-1
Oprk1 mRNA expression in WT and Oprk1-Cre mice. We measured Oprk1 mRNA levels in WT and Cre/+ mice by quantitative PCR. Oprk1 mRNA levels (relative to Gapdh and Tfrc) were significantly increased in (A) whole brain mRNA from Cre/+ mice (*, p < 0.001, N = 5 female mice/group). B) Oprk1 expression was also increased in the NAc of Cre/+ mice (*, p = 0.0125, WT male = 4, WT female = 2, Cre/+ male = 3, Cre/+ female = 5). Download Figure 3-1, TIF file.
Figure 3-2
[35S]GTPγS binding in the striatum of WT and Oprk1-Cre mice. A–C, WT and heterozygous Oprk1-Cre mice did not differ in maximal stimulation in response to agonist over basal activity (A), EC50 values of Dyn to activate G-protein (B), and basal GTPγS binding (C). N =7 males/group. Download Figure 3-2, TIF file.
Figure 3-3
Basal KOR signaling is not altered in the amygdala of Oprk1-Cre mice. A–C, Basal phosphorylation levels of ERK (A), JNK (B), and p38 (C) were not different between WT and Oprk1-Cre mice in the amygdala (WT female = 5; Cre/+ male = 2, Cre/+ female = 2). Download Figure 3-3, TIF file.
Table 3-1
List of antibodies used with catalog numbers and dilutions. Download Table 3-1, DOCX file.
Figure 4-1
Closed arm entries were not altered on the EPM in Oprk1-Cre mice. No genotype or sex differences were found in the number of closed arm entries between male and female WT and Oprk1-Cre mice. N = 7–10/group for males and N = 7–12/group for females. Download Figure 4-1, TIF file.
Figure 7-1
Closed arm entries were not altered on the EPM in mCherry-injected and hM3DQ-injected mice. The number of closed arm entries was not significantly different between mCherry-injected and hM3DQ-injected male and female mice. N = 8/group for males and N = 9–12/group for females. Download Figure 7-1, TIF file.
Figure 8-1
U-50488-induced CPA in C57BL/6J mice. Systemic injection of 2.5 mg/kg U-50488 produced CPA in WT mice. Difference score obtained by subtracting the percentage of time spent in drug-paired compartment before and after conditioning is shown for saline-injected and U-50488-injected mice. U-50488-injected mice showed CPA compared with mice that received saline injections on both sides of the chamber. *p = 0.032, t test, N =8–10 male mice/group. Download Figure 8-1, TIF file.
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