Fig 2: Comparison of GABAergic neurons subtypes in WT vs. TLE. (A) Representative images of immunostaining of PV+ and SOM+ neurons in dDG of WT and TLE mice. Scale bar, 200 µm. (B) The densities of SOM+ and PV+ neurons in dDG/CA3 (SOM+: WT, n = 6 mice, TLE, n = 6 mice; PV+: WT, n = 6 mice, TLE, n = 5 mice). (C) Representative images of immunostaining of NTSR+, CCK+, and VIP+ neurons in dLS of WT and TLE mice. Scale bar, 200 µm. (D) The densities of NTSR+, CCK+, and VIP+ neurons in dLS (NTSR+: WT, n = 11 mice, TLE, n = 10 mice; CCK+: WT, n = 6 mice, TLE, n = 9 mice, VIP+: WT, n = 11 mice, TLE, n = 6 mice) (Error bars represent s.d; *p < 0.05; **p < 0.01, ***p < 0.001, t-test).

Fig 3: Identification of specific subtypes of CCK receptors and opioid receptors in BLA involved in AOAT.(A) Schematic of BLA infusion of opioid receptor antagonists. (B to E) Quantitative PWL of mice before (pre-M) and after (post-M) morphine (5 mg/kg, intraperitoneally) injection with intra-BLA infusion of naloxone (B), CTOP (C), naltrindole (D), or nor-BNI (E) at various doses. (F) Left: Schematic of AAV-Cre injection in Oprm1fl/fl mice. Right: Quantitative PWL of Oprm1fl/fl mice with (BLA-MOR-/-) or without (BLA-MOR+/+) knockout of BLA MORs. (G) Schematic of 4-day AOAT development and intra-BLA infusion of DAMGO on day 5. (H) Morphine analgesic tolerance in HC-mice and CC-mice. (I) Quantitative PWL of mice before and after intra-BLA infusion of DAMGO (100 ng per mice) on day 5. (J) Schematic of BLA infusion of CCK receptor agonists or antagonists. (K to M) Dose-dependent effects of intra-BLA injection of CCK8s (K), L365260 (L), and MK329 (M) on the development of morphine analgesic tolerance. (N and O) Representative images (N) and percentage analysis (O) of MOR+ neurons and CCK2R+ neurons in BLA. The experiment was repeated with similar results in five mice. Scale bars, 10 µm. (P to U) Schematic of electrophysiological recording paradigm (P and S). AP spiking patterns (Q and T) and spike numbers and thresholds (R and U) for AP firing of the BLA neurons of C57BL/6J mice (P to R) and CCK-CreER mice (S to U). (V) Left: Schematic of chemogenetics. Right: Morphine analgesic tolerance in HC-mice with or without chemogenetic activation of the CCKdmPFC?BLA terminals and the effect of L365260 (30 ng per mice). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by one- and two-way ANOVA with Bonferroni post hoc test; ns, not significant (P > 0.05).

Fig 4: Identification of the GluvHPC?CCKdmPFC?GluBLA pathway.(A) Schematic showing the procedure for retrograde tracing of the GluvHPC?CCKdmPFC neurons using the Cre-dependent retrograde trans-monosynaptic RV. (B) Left: Representative image showing starter cells (yellow) coexpressing AAV-DIO-TVA-GFP (green), AAV-DIO-RVG, and RV-EnvA-?G-DsRed (red) in dmPFC of CCK-CreER mice. Scale bar, 200 µm. Right: Representative image showing DsRed+ neurons in the vHPC retrograde traced from CCKdmPFC neurons. Scale bar, 500 µm. (C) Left: Representative images showing DsRed+ neurons that colocalized with CaMKII immunoreactivity in vHPC (white arrowheads). Scale bars, 10 µm. Right: Percentage of DsRed+ neurons that coexpressed CaMKII in vHPC (n = 3704 neurons from five mice). (D) Schematic of the procedure for verification of the GluvHPC?CCKdmPFC?GluBLA neuronal pathway using optogenetic methods combined with virus-mediated retrograde monosynaptic tracing. Whole-cell patch clamp recordings of the mCherry+ neurons in BLA slices were conducted with laser stimulation. (E) Representative images showing EYFP+ axon terminals of vHPC neurons that intermingled with mCherry+ CCKdmPFC?BLA neurons in dmPFC. Scale bars, 20 and 5 µm (inset). (F) Representative traces (left) and average amplitudes (right) of oEPSCs in the mCherry+ CCKdmPFC?BLA neurons. (G) Working model of the hierarchical GluvHPC?CCKdmPFC?GluBLA neuronal pathway that mediates AOAT. Morphological analyses (B, C, and E) were independently repeated with similar results in three mice. Data are presented as means ± SEM. ***P < 0.001 by paired t test.

Fig 5: Identification of the CCKdmPFC?GluBLA pathway mediating AOAT.(A) Heatmap of RNA-seq data for 16 opioid-related genes; data are reported in FPKM (fragments per kilobase million). (B) Representative image showing EYFP+ neurons in dmPFC of CCK-CreER::Ai3-EYFP reporter mice. Scale bar, 100 µm. (C) Schematic of AAV2-retro virus injection in CCK-CreER mice (upper left) and representative images of viral expression in the indicated nuclei (lower left and middle). Scale bars, 500 and 100 µm (inset). EGFP+ neurons colocalized with CCK8s immunoreactivity in dmPFC (right). Scale bars, 20 µm. (D) Schematic of AAV-Ef1a-DIO-EGFP injection in CCK-CreER mice. (E and F) Representative images showing AAV-Ef1a-DIO-EGFP infection in dmPFC (E, left) and the EGFP+ axon terminals that intermingled with CaMKII+ neurons in BLA (E, right, and F). Scale bars, 100 µm (E) and 10 µm (F). (G) Schematic of the procedure for tracing of Cre-dependent retrograde trans-monosynaptic RV. (H) Representative images showing starter cells in the BLA and DsRed+ neurons in dmPFC. Scale bars, 100 µm. (I) Representative images (left) and percentage analysis (right) of DsRed+ neurons that expressed CCK8s in dmPFC (n = 1372 neurons from three mice). (J) Schematic of electrophysiological recording of EGFP+ CCKdmPFC?BLA neurons. (K and L) AP spiking patterns (K) and spike numbers and thresholds (L) for AP firing of the CCKdmPFC?BLA neurons of HC-mice and CC-mice. (M) Schematic of chemogenetics. (N and O) Morphine analgesic tolerance in CC-mice (N) and HC-mice (O) with or without chemogenetic activation or inactivation of the CCKdmPFC?BLA terminals. Morphological analyses (B, C, E, H, and I) were independently repeated with similar results in four mice. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired t test (L, right) and two-way ANOVA with Bonferroni post hoc test [L (left), N, and O].