Fig. 3: Activity of µOR-expressing neurons during acute and chronic fentanyl exposure.
From: Distinct µ-opioid ensembles trigger positive and negative fentanyl reinforcement
a, Top, schematic of mouse preparation for recording Ca2+ activity of VTA neurons expressing dopamine and GABA. Bottom, schedule of the recording experiment in fentanyl-dependent mice. Withdrawal is precipitated by naloxone on day 7 (intraperitoneal injection, 5 mg kg−1). b, Ca2+ signal (ΔF/F0) of dopamine (n = 8 mice) and GABA (n = 8 mice) neurons after intraperitoneal injection of naloxone (5 mg kg−1), fentanyl (0.3 mg kg−1) and fentanyl plus naloxone. c, Top, schematic representation of NAc dLight recordings after µOR deletion in VTA. Bottom, schedule of intraperitoneal injections for dLight recordings after saline, fentanyl (0.3 mg kg−1) and apomorphine (10 mg kg−1) treatments. d, Accumbal dLight signal (ΔF/F0) in mice with deletion of µORs in VTA (n = 8 mice) versus control mice (n = 7 mice). e, Quantification of the area under the curve (AUC) after intraperitoneal injection of saline or fentanyl in mice with deletion of µORs in VTA (n = 8 mice) versus control mice (n = 7 mice). Two-way repeated measures ANOVA: group effect, F(1,26) = 4.371, P < 0.05; injection effect, F(1,26) = 23.95, P < 0.0001; group × injection, F(1,26) = 5.777, P < 0.05; Bonferroni post hoc analysis, **P = 0.0076. Data are mean ± s.e.m. f, Top, schematic of mice preparation to label µOR-expressing neurons in CeA. Bottom, schedule of the experiment to induce fentanyl dependence and precipitation of withdrawal on the challenge day (day 6). g, Box plot representation of jumps and immobility time withdrawal symptoms quantified in Oprm1-cre (n = 8 mice) and Sst-cre (n = 8) mice. The centre line is the median, box edges delineate first and third quartiles, and whiskers extend to maximum and minimum values. h, Left, representative example of CeA µOR-expressing neurons co-localizing with cFOS (white arrows) after precipitated withdrawal. Right, fraction of cFOS-expressing neurons among CeA µOR or SST-expressing neurons (n = 8 mice for SST and n = 8 mice for µOR; two-sided unpaired t-test, ***P < 0.0001). Scale bar, 20 μm. i, Schematic of mouse preparation for Ca2+recording of CeA µOR-expressing neurons during precipitation of withdrawal. j, Average Ca2+ signal (ΔF/F0) of CeA µOR-expressing neurons in naive mice after intraperitoneal injection of naloxone (left) and independent mice after intraperitoneal injection of fentanyl (0.3 mg kg−1) plus saline or fentanyl (0.3 mg kg−1) plus naloxone (5 mg kg−1) (n = 7 mice). k, Quantification of AUC after intraperitoneal injection of saline or naloxone in naive and dependent mice (n = 7 mice; Kruskal–Wallis test for AUC H(3) = 7.577, P < 0.05; Dunn’s multiple comparisons test, *P = 0.0258). Data are mean ± s.e.m. l, Bottom, average Ca2+ traces align to different behavioural events during precipitation of withdrawal. Top, trials activity map of each behavioural parameter during precipitation of withdrawal.
