PMC

Dfe Improved Mitochondrial Dysfunction of DRG Neuron in DPN Mice through Kv2.1/CaMKKβ/AMPK/PGC-1α Pathway
(A and C) Oxygen consumption rate (OCR) was measured in DRG neuron from (A) Control, STZ or Dfe (20 mg/kg)-treated STZ mice (STZ + Dfe 20 mg/kg) and (C) db/m, db/db or Dfe (20 mg/kg)-treated db/db mice (db/db + Dfe 20 mg/kg) using XF96 analyzer. N = 4–7. STZ + Dfe (20 mg/kg) versus STZ by Student's t test: #p < 0.05; db/db + Dfe (20 mg/kg) versus db/db by Student's t test: ##p < 0.01.
(B and D) ATP production, basal respiration, spare respiratory capacity, and maximal respiration were measured in DRG neuron from (B) Control, STZ or Dfe (20 mg/kg)-treated STZ mice (STZ + Dfe 20 mg/kg) and (D) db/m, db/db or Dfe (20 mg/kg)-treated db/db mice (db/db + Dfe 20 mg/kg). N = 3. For ATP production, STZ versus Control by Student's t test: ∗∗p < 0.01, STZ + Dfe (20 mg/kg) versus STZ by Student's t test: ∗∗p < 0.01; db/db versus db/m by Student's t test: ∗∗p < 0.01, db/db + Dfe (20 mg/kg) versus db/db by Student's t test: ∗p < 0.05. For basal respiration, STZ versus Control by Student's t test: ∗p < 0.05, STZ + Dfe (20 mg/kg) versus STZ by Student's t test: ∗∗∗p < 0.01; db/db versus db/m by Student's t test: ∗p < 0.05, db/db + Dfe (20 mg/kg) versus db/db by Student's t test: ∗p < 0.05. For spare respiratory capacity, STZ versus Control by Student's t test: ns, STZ + Dfe (20 mg/kg) versus STZ by Student's t test: ∗p < 0.05; db/db versus db/m by Student's t test: ∗p < 0.05, db/db + Dfe (20 mg/kg) versus db/db by Student's t test: ns. For maximal respiration, STZ versus Control by Student's t test: ∗p < 0.05, STZ + Dfe (20 mg/kg) versus STZ by Student's t test: ∗∗∗p < 0.01; db/db versus db/m by Student's t test: ∗p < 0.05, db/db + Dfe (20 mg/kg) versus db/db by Student's t test: ∗p < 0.05.
(E) OCR data in DRG neuron from Control, AAV9-NC or AAV9-Kv2.1-RNAi-injected STZ (STZ + AAV9-NC, STZ + AAV9-Kv2.1) and Dfe (20 mg/kg)-treated AAV9-Kv2.1-RNAi-injected STZ mice (STZ + AAV9-Kv2.1 + 20 mg/kg) were obtained. N = 5–7. STZ + AAV9-Kv2.1 + Dfe versus STZ + AAV9-Kv2.1 by Student's t test: ns.
(F) ATP production, basal respiration, spare respiratory capacity, and maximal respiration were measured in DRG neuron from Control, AAV9-NC or AAV9-Kv2.1-RNAi-injected STZ (STZ + AAV9-NC, STZ + AAV9-Kv2.1) and Dfe (10, 20 mg/kg)-treated AAV9-Kv2.1-RNAi-injected STZ mice (STZ + AAV9-Kv2.1 + Dfe). N = 3. For ATP production, STZ + AAV9-NC versus Control by Student's t test: ∗∗p < 0.01, STZ + AAV9-Kv2.1 versus STZ + AAV9-NC by Student's t test: ∗p < 0.05. Dfe (20 mg/kg) versus STZ + AAV9-Kv2.1 by Student's t test: ns; For basal respiration, STZ + AAV9-NC versus Control by Student's t test: ∗∗p < 0.01, STZ + AAV9-Kv2.1 versus STZ + AAV9-NC by Student's t test: ∗p < 0.05. Dfe (20 mg/kg) versus STZ + AAV9-Kv2.1 by Student's t test: ns; For spare respiratory capacity, STZ + AAV9-NC versus Control by Student's t test: ∗p < 0.05, STZ + AAV9-Kv2.1 versus STZ + AAV9-NC by Student's t test: ∗p < 0.05. Dfe (20 mg/kg) versus STZ + AAV9-Kv2.1 by Student's t test: ns; For maximal respiration, STZ + AAV9-NC versus Control by Student's t test: ∗∗p < 0.01, STZ + AAV9-Kv2.1 versus STZ + AAV9-NC by Student's t test: ∗∗p < 0.01. Dfe (20 mg/kg) versus STZ + AAV9-Kv2.1 by Student's t test: ns.
(G and H) Mitochondrial membrane potential levels in DRG neuron from (G) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (H) db/m, db/db and Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe). N = 3. STZ versus Control by Student's t test: ∗p < 0.05. Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 40.94. ∗∗p < 0.01, ∗∗∗p < 0.001; db/db versus db/m by Student's t test: ∗p < 0.05. Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 7.388. ∗p < 0.05.
(I and J) Western blot results of CaMKKβ, AMPK, p-AMPK, PGC-1α and p-ACC in DRG homogenates from (I) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (J) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe). T-ERK was used as an internal control. N = 3.
(K and L) Quantitative analyses for (I) and (J). p-AMPK (STZ versus Control by Student's t test: ∗∗∗p < 0.001, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 91.83. ##p < 0.01, ###p < 0.001; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 56.34. ##p < 0.01, ###p < 0.001), AMPK (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 3.235. ns; db/db versus db/m by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 1.179. ns), CaMKKβ (STZ versus Control by Student's t test: ∗∗∗p < 0.001, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 22.41. ##p < 0.01; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 10.58. #p < 0.05), PGC1-α (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 79.92. ##p < 0.01, ###p < 0.001; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 20.91. ##p < 0.01), p-ACC (STZ versus Control by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 3.615. ##p < 0.01; db/db versus db/m by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.675. #p < 0.05).
(M) Western blot results of CaMKKβ, AMPK, p-AMPK, PGC-1α, and p-ACC in DRG homogenates from Control, AAV9-NC or AAV9-Kv2.1-RNAi-injected STZ (STZ + AAV9-NC, STZ + AAV9-Kv2.1), and Dfe (10, 20 mg/kg)-treated AAV9-Kv2.1-RNAi-injected STZ mice (STZ + AAV9-Kv2.1 + Dfe). T-ERK was used as an internal control. DRG tissue isolated from three mice per group was mixed and homogenized.
(N) Quantitative analysis for (M). p-AMPK (STZ + AAV-NC versus Control by Student's t test: ∗∗∗p < 0.001. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ##p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 3.97. ns), AMPK (STZ + AAV-NC versus Control by Student's t test: ns. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ns. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 1.121. ns), CaMKKβ (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ###p < 0.001. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 3.787. ns), PGC1-α (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: #p < 0.05. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 0.4457. ns), p-ACC (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ##p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 10.19. &p < 0.05). All data were presented as means ± SEM.

Dfe Treatment Reduced Inflammation in DPN Mice by Inhibiting Kv2.1 Channel
(A and B) ELISA analysis of proinflammatory cytokines IL-6, IL-1β, and TNF-α in the serum from (A) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (B) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe). N = 3. IL-6 (STZ versus Control by Student's t test: ∗∗∗p < 0.001, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 55.44. ∗∗∗p < 0.001; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 1.884. ∗∗p < 0.01), IL-1β (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 13.92. ∗p < 0.05, ∗∗p < 0.01; db/db versus db/m by Student's t test: ∗∗∗p < 0.001, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 23.84. ∗∗p < 0.01, ∗∗∗p < 0.001), TNF-α (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 25.92. ∗∗p < 0.01, ∗∗∗p < 0.001; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 45.48. ∗∗p < 0.01, ∗∗∗p < 0.001).
(C) ELISA analysis of proinflammatory cytokines IL-6, IL-1β, and TNF-α in the serum from Control, AAV9-NC or AAV9-Kv2.1-RNAi-injected STZ (STZ + AAV9-NC, STZ + AAV9-Kv2.1) and Dfe (10, 20 mg/kg)-treated AAV9-Kv2.1-RNAi-injected STZ mice (STZ + AAV9-Kv2.1 + Dfe). N = 3. IL-6 (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ∗p < 0.05. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 7.015. ns), IL-1β (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ∗p < 0.05. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 2.671. ns), TNF-α (STZ + AAV-NC versus Control by Student's t test: ∗∗∗p < 0.001. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ∗∗p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 2.023. ns).
(D and E) Paraffin sections of DRG tissue from (D) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (E) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe) for NF-κB immunostaining. Scale bar: 25 μm.
(F and G) Quantitative analyses for (D, E). N = 3. STZ versus Control by Student's t test: ∗∗∗p < 0.001. Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 16.15. ∗p < 0.05, ∗∗p < 0.01; db/db versus db/m by Student's t test: ∗∗p < 0.01. Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 42.06. ∗∗p < 0.01, ∗∗∗p < 0.001).
(H and I) Paraffin sections of DRG tissue from Control, AAV9-NC or AAV9-Kv2.1-RNAi-injected STZ (STZ + AAV9-NC, STZ + AAV9-Kv2.1) and Dfe (10, 20 mg/kg)-treated AAV9-Kv2.1-RNAi-injected STZ mice (STZ + AAV9-Kv2.1 + Dfe) for NF-κB immunostaining and quantitative analysis. N = 3. STZ + AAV-NC versus Control by Student's t test: ∗∗∗p < 0.001. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ∗∗∗p < 0.001. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 0.3936. ns.
(J and K) Western blot results indicating the expressions of iNOS, TNF-α, phosphorylated IκBα and IκBα in DRG tissue from (J) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (K) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe). N = 3.
(L and M) Quantitative analyses for (J, K). iNOS (STZ versus Control by Student's t test: ∗∗∗p < 0.001, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 9.918. ∗p < 0.05; db/db versus db/m by Student's t test: ns, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 12.43. ns. ∗∗p < 0.01), p-IκBα (STZ versus Control by Student's t test: ns, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 3.739. ∗p < 0.05; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 15.79. ∗∗p < 0.01), TNF-α (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 11.13. ns, ∗∗p < 0.01; db/db versus db/m by Student's t test: ns, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 25.83. ns, ∗∗∗p < 0.001).
(N and O) Western blot assays in DRG tissue from Control, AAV9-NC or AAV9-Kv2.1-RNAi-injected STZ (STZ + AAV9-NC, STZ + AAV9-Kv2.1) and Dfe (10, 20 mg/kg)-treated AAV9-Kv2.1-RNAi-injected STZ mice (STZ + AAV9-Kv2.1 + Dfe). DRG tissue from three mice per group were mixed and homogenized. iNOS (STZ + AAV-NC versus Control by Student's t test: ∗∗∗p < 0.001. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ∗p < 0.05. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 1.224. ns), p-IκBα (STZ + AAV-NC versus Control by Student's t test: ∗∗∗p < 0.001. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ∗∗∗p < 0.001. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 3.089. ns), TNF-α (STZ + AAV-NC versus Control by Student's t test: ∗∗∗p < 0.001. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ∗∗p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 0.2739. ns). All data were presented as means ± SEM.

Dfe Treatment Alleviated Apoptosis of DRG Neuron in DPN Mice Involving Regulation of Bcl-2 Family Proteins and Caspase-3 by Inhibiting Kv2.1 Channel
(A and B) Representative images of double immune-fluorescent staining showing Annexin V-FITC and PI labeled early and late stage of apoptosis of DRG neuron from (A) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (B) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe). Hoechst was used to mark nuclei. Scale bar: 250 μm.
(C) Quantitative analyses for (A, B). N = 3. STZ versus Control by Student's t test: ∗∗p < 0.01. Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 27.24. ∗p < 0.05, ∗∗∗p < 0.001; db/db versus db/m by Student's t test: ∗∗p < 0.01. Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 14.77. ∗∗p < 0.01.
(D and E) The paraffin sections of DRG tissue from (D) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (E) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe) were fluorescently stained for IHC assay. Neurons were located by NeuN and apoptotic cells were labeled by tunnel. Scale bar: 25 μm.
(F) Quantitative analyses for (D, E). N = 3. STZ versus Control by Student's t test: ∗∗∗p < 0.001. Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 54.09. ∗∗∗p < 0.001; db/db versus db/m by Student's t test: ∗∗∗p < 0.001. Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 41.48. ∗∗p < 0.01, ∗∗∗p < 0.001.
(G and I) Western blot results demonstrated that Kv2.1 inhibitor Dfe reduced apoptosis in DRG neuron from (G) Control, STZ or Dfe (10, 20 mg/kg)-treated STZ mice (STZ + Dfe) and (I) db/m, db/db or Dfe (10, 20 mg/kg)-treated db/db mice (db/db + Dfe) by regulating apoptosis-related proteins. T-ERK was used as loading control. N = 3.
(H and J) Quantitative analyses for (G, I). Bcl-xl (STZ versus Control by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 14.33. ##p < 0.01; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.535. #p < 0.05), Bcl-2 (STZ versus Control by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.205. #p < 0.05; db/db versus db/m by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 15.61. #p < 0.05, ##p < 0.01), Bax (STZ versus Control by Student's t test: ∗∗p < 0.01, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 11.06. #p < 0.05. ##p < 0.01; db/db versus db/m by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 8.733. #p < 0.05), p-Bad (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 5.134. #p < 0.05; db/db versus db/m by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 5.357. #p < 0.05), Cleaved-caspase-3 (STZ versus Control by Student's t test: ∗p < 0.05, Dfe (10, 20 mg/kg) versus STZ by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.543. #p < 0.05; db/db versus db/m by Student's t test: ∗∗∗p < 0.001, Dfe (10, 20 mg/kg) versus db/db by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 20.9. ##p < 0.01).
(K and L) Western blot assays in DRG tissue from Control, AAV9-NC or AAV9-Kv2.1-RNAi-injected STZ (STZ + AAV9-NC, STZ + AAV9-Kv2.1) and Dfe (10, 20 mg/kg)-treated AAV9-Kv2.1-RNAi-injected STZ mice (STZ + AAV9-Kv2.1 + Dfe). DRG tissue from three mice per group was mixed and homogenized. Bcl-xl (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ###p < 0.001. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 5.186. ns), Bcl-2 (STZ + AAV-NC versus Control by Student's t test: ∗∗∗p < 0.001. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ##p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 6.773. ns. #p < 0.05), Bax (STZ + AAV-NC versus Control by Student's t test: ∗p < 0.05. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ##p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 2.492. ns), p-Bad (STZ + AAV-NC versus Control by Student's t test: ∗p < 0.05. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ns. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 5.101. &p < 0.05, ns), Cleaved caspase-3 (STZ + AAV-NC versus Control by Student's t test: ∗∗p < 0.01. STZ + AAV-Kv2.1 versus STZ + AAV-NC by Student's t test: ##p < 0.01. Dfe (10, 20 mg/kg) versus STZ + AAV-Kv2.1 by one-way ANOVA with Dunnett's post hoc test: F (2, 6) = 4.272. &p < 0.05, ns). All data were presented as means ± SEM.

Chemogenetic inhibition of excitatory neurons in the mouse vHPC. A, Schematic of bilateral targeting of vCA1. B, Representative image of the vHPC from a GAD67GFP(+) mouse receiving AAV8-CaMKIIα-mCherry in vCA1. Dashed lines highlight vCA1, vCA3, and vDG subregions. The white rectangle shows the area enlarged in panel C. C, Image of the vCA1 subregion showing GFP-positive (GABA) neurons within the area of viral infusion (left), CaMKIIα-driven mCherry expression (middle), and minimal overlap of GFP and mCherry fluorescence (right). D, Change in rheobase induced by CNO (10 μm) in control or hM4Di-expressing vCA1 pyramidal neurons (t₍₅₎ = 5.456, **p = 0.0025; unpaired Student's t test with Welch's correction; N = 2 mice and n = 6–8 total recordings/group). E, F, left, Percentage of time spent in open (t₍₁₇₎ = 0.8167, p = 0.4254; unpaired Student's t test) and closed (t₍₁₇₎ = 1.557, p = 0.1378; unpaired Student's t test) arms of the EPM by male C57BL/6J mice treated with intra-vCA1 hM4Di or control vector (N = 8–11 mice/group), following administration of CNO (2 mg/kg, i.p.). Right, Percentage of time spent in open (t₍₁₅₎ = 0.3685, p = 0.7177; unpaired Student's t test) and closed (t₍₁₅₎ = 0.3007, p = 0.7678; unpaired Student's t test) arms of the EPM by male CaMKIICre(+) mice treated with intra-vCA1 AAV8-hSyn-DIO-hM4Di(mCherry) or AAV8-hSyn-DIO-mCherry vector (N = 8–9 mice/group), following administration of CNO (2 mg/kg, i.p.). G, Total distance traveled during the EPM test by male C57BL/6J mice treated with hM4Di or control vector (left, t₍₁₇₎ = 0.09228, p = 0.9276; unpaired Student's t test), and in CaMKIICre(+) mice treated with intra-vCA1 DIO-hM4Di or control vector (right, t₍₁₅₎ = 0.3286, p = 0.7470; unpaired Student's t test). H, Schematic of bilateral targeting of vCA3/DG. I, Representative image of the vHPC from a GAD67GFP(+) mouse receiving AAV8-CaMKIIα-mCherry in the vCA3/DG subregion. The white open box shows the area enlarged in panel J. J, Image of the vCA3/DG subregion showing GFP-positive (GABA) neurons within the area of viral infusion (left), CaMKIIα-driven mCherry expression (middle), and minimal overlap of GFP and mCherry fluorescence (right). K, left, Change in rheobase induced by CNO (10 μm) in control or hM4Di-expressing vCA3 pyramidal neurons (t₍₃₎ = 3.749, *p = 0.0305; unpaired Student's t test with Welch's correction; N = 1–2 mice and n = 4–5 total recordings/group). Right, Change in rheobase induced by CNO (10 μm) in control or hM4Di-expressing vDG granule cells (t_(6.5) = 7.298, ***p = 0.0002; unpaired Student's t test with Welch's correction; N = 2 mice and n = 7 total recordings/group). L, M, left, Percentage of time spent in open (t_(14.84) = 3.074, **p = 0.0078; unpaired Student's t test with Welch's correction) and closed (t₍₁₇₎ = 3.228, **p = 0.0049; unpaired Student's t test) arms of the EPM by male C57BL/6J mice treated with intra-vCA3/DG hM4Di or control vector (N = 8–11 mice/group), following administration of CNO (2 mg/kg, i.p.). Right, Percentage of time spent in open (t₍₁₃₎ = 3.153, **p = 0.0076; unpaired Student's t test) and closed (t₍₁₃₎ = 2.795, *p = 0.0152; unpaired Student's t test) arms of the EPM by male CaMKIICre(+) mice treated with intra-vCA3/DG DIO-hM4Di or control vector (N = 7–8 mice/group), following administration of CNO (2 mg/kg, i.p.). N, Total distance traveled during the EPM test by male C57BL/6J mice treated with hM4Di or control vector (left, t₍₁₇₎ = 1.185, p = 0.2523; unpaired Student's t test) and in male CaMKIICre(+) mice treated with intra-vCA1 DIO-hM4Di or control vector (right, t₍₁₃₎ = 0.8588, p = 0.4060; unpaired Student's t test). O, left, Percentage of time spent in open-field center by male C57BL/6J mice treated with hM4Di or control vector (t₍₁₈₎ = 2.327, *p = 0.0318; unpaired Student's t test), following administration of CNO (2 mg/kg, i.p.). Right, Percentage of time spent in open-field center by male C57BL/6J mice treated with systemic ML297 (30 mg/kg, i.p.) or vehicle (t₍₂₂₎ = 0.1919, p = 0.8496; unpaired Student's t test) 30 min before testing. P, left, Percentage of time spent in light chamber of light-dark box by male C57BL/6J mice treated with hM4Di or control vector (t₍₁₈₎ = 2.767, *p = 0.0127; unpaired Student's t test), following administration of CNO (2 mg/kg, i.p.). Right, Percentage of time spent in light chamber by male C57BL/6J mice treated with systemic ML297 (30 mg/kg, i.p.) or vehicle (t₍₂₂₎ = 2.198, *p = 0.0387; unpaired Student's t test) 30 min before testing. Q, left, Percentage of marbles buried by male C57BL/6J mice treated with hM4Di or control vector (t₍₁₈₎ = 1.790, p = 0.0903; unpaired Student's t test), following administration of CNO (2 mg/kg, i.p.). Right, Percentage of marbles buried by male C57BL/6J mice treated with systemic ML297 (30 mg/kg, i.p.) or vehicle (t₍₂₂₎ = 0.4238, p = 0.6758; unpaired Student's t test) 30 min before testing.