Figure 7. Antinociceptive interactions between LH^PV neuronal activation and morphine.

(a) Dose-response curves of clozapine-N-oxide (CNO) and morphine alone or in combinations of different fixed proportions in LH^PV:hM3D and (b) LH^PV:Ctrl mice in the hot plate test (n = 8 mice per group). (c, d) Isobolograms constructed from the data shown in panels (a) and (b). Each point represents the ED₅₀ ± 95% CI of each drug alone or in a mixture; ordinates represent the ED₅₀ value of morphine and abscissae represent the ED₅₀ value of CNO. In LH^PV:hM3D mice, the 3:1 morphine:CNO mixture was significantly more potent than predicted by the hypothesis of additivity (paired Student’s t-test, t(7) = 2.92, p=0.022). (e) Both groups of mice developed significant antinociceptive tolerance to 32 mg/kg morphine when administered twice per day for 3 days. Three-way mixed-model ANOVA revealed a significant morphine × test interaction (n = 8 mice per group; F(1, 14) = 134.7, p<0.0001), and Bonferroni multiple comparisons post-tests showed the antinociceptive effects of 32 mg/kg morphine were significantly lower on day 4 than day 1 (both p<0.0001). (f) Activation of LH^PV neurons restored morphine potency, and further tolerance did not develop to combination treatment. Three-way mixed-model ANOVA revealed a significant treatment × group interaction (n = 8 mice per group; F(2, 28) = 42.10, p<0.0001). Bonferroni multiple comparisons post-tests revealed that there were between-group differences in PWL_HP evoked on day 5 by CNO (p=0.0006) and morphine (p<0.0001) and on day 9 by CNO (p=0.016) and morphine (p<0.0001). However, no within-group differences were observed between day 5 and 9 in LH^PV:hM3D mice during CNO (p>0.99) or morphine treatment (p>0.99).