Fig. 4: mMORp-GCaMP6f in vivo photometry recording of MOR+ neural population calcium activity in response to noxious stimulation, opioid administration, and opioid withdrawal.

a In vivo fiber photometry recording set up and overall experimental design, as well as AAV1/5-mMORp-GCaMP6f injection schema in C57BL/6J mice. b mMORp-GCaMP6f viral targeting and transduction efficiency within CeA neurons (NeuN+) and fiber optic implant placement; scale bars = 500 μm (left), 100 m (right). c Photometry experimental timeline for mMORp-GCaMP6f (N = 12) and mMORp-eYFP (N = 3) injected mice. d Normalized calcium-mediated activity responses observed in the CeA of mMORp-GCaMP6f and mMORp-eYFP mice in response to 10 air puff applications (1-min inter-stimulation interval. Thick lines = averaged responses, thin lines = individual mice average responses to all 10 stimulations). Inset graphs depict differences in the area under the curve (AUC) quantified between averaged GCaMP6f and eYFP control response (two tailed unpaired t-test, P = 0.035) and peak z-score between groups (two tailed unpaired t-test, P = 0.011). e Plots for normalized, calcium-mediated responses to the application of 10 ~ 55 °C hot water droplets to the left hind paw (1-min inter-stimulation interval). Thick lines = average group response, thin lines = individual mice averaged response. Insets show AUC (two tailed unpaired t-test, P = 0.051) and peak z-score (two tailed unpaired t-test, P = 0.009) comparisons for eYFP (N = 3) and GCaMP6f (N = 12) expressing mice. f Normalized, calcium-mediated responses to an additional round of hot water hind paw stimulations in the mMORp-GCaMP6f mice (N = 12) before and 30 min after morphine (i.p., 10 mg/kg). Opioid-naïve responses (green) and post-morphine responses (blue) are shown as grouped averages (thick lines) and individual averages (thin lines) following 10 stimulations. Insets show AUC (two tailed unpaired t-test, P = 0.016) and peak z-scores (two tailed unpaired t-test, P = 0.005) comparisons between the pre- and post-morphine responses in mMORp-GCaMP6f mice. Gray lines track changes in AUC and peak z-score for individual mice in pre- and post-morphine treatment conditions. g Normalized, calcium-mediated responses from mMORp-GCaMP6f mice undergoing an escalating forced morphine drinking paradigm (Fig. 4c) for morphine drinking (red, N = 6 mice) or saccharin drinking (gray, N = 5 mice) subjects following naloxone induced precipitated withdrawal (i.p., 3 mg/kg). Insets show the global withdrawal behavior score (left) and total calcium-mediated events (right) post-naloxone between morphine and saccharin mice (W/D score: two tailed unpaired t-test, P = 0.0001; Ca²⁺ events: two tailed unpaired t-test, P = 0.0007). All data are presented as means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001.