Journal: bioRxiv
Article Title: (2R,6R)-Hydroxynorketamine elicits rapid antidepressant effects by promoting astrocytic µ-δ opioid receptor heterodimerization
doi: 10.64898/2026.05.31.729044
Figure Lengend Snippet: a, Schematic of hippocampal slice recording: Stimulating electrode in Schaffer collaterals, recording electrode in CA1 stratum radiatum. b, Ketamine increased the rising slope of SC-CA1 fEPSPs elicited at CA1 stratum radiatum in acutely prepared hippocampal slices. When ketamine-induced potentiation reached a plateau, subsequent bath application of HNK further enhanced SC-CA1 fEPSPs. Left: time course of SC-CA1 fEPSPs changes; Right, representative fEPSP traces before, after ketamine treatment, and following subsequent HNK application. c, Increasing ketamine concentration to 30 μM did not further potentiate SC-CA1 fEPSPs after 10 μM ketamine-induced effects plateaued. d, MK-801, a selective and non-competitive NMDA receptor antagonist, mimicked ketamine’s potentiation of SC-CA1 fEPSPs and did not occlude the effect of HNK. e, DAMGO, a selective MOR agonist, increased the rising slope of SC-CA1 fEPSPs and occluded the effect of HNK on SC-CA1 fEPSPs, whereas ketamine further potentiated SC-CA1 fEPSPs on top of DAMGO. f, The selective MOR antagonist CTOP reduced SC-CA1 fEPSP slope and blocked the effect of HNK but not ketamine. g , DPDPE, a selective DOR agonist, increased the rising slope of SC-CA1 fEPSPs and occluded the effect of HNK on SC-CA1 fEPSPs, whereas ketamine further potentiated SC-CA1 fEPSPs on top of DPDPE. h , The selective DOR antagonist ICI blocked the effect of HNK but not ketamine. i , Experimental timeline for assessing the effect of MOR or DOR activation, blockade, or deletion on the antidepressant effect of HNK. j–l, DAMGO (10 mg·kg -1 , i.p.) mimicked and CTOP (3 μg·kg -1 , i.c.v.) blocked the antidepressant actions of HNK (10 mg·kg -1 , i.p.) in FST (j), SPT (k), and three-chamber test (l). m–p, MOR knockout mice exhibited depression-like behaviors in FST (m) and SPT (n) and abolished the antidepressant-like effects of HNK in the two assays (FST, o; SPT, p). q–r, DPDPE (0.5 μg·kg -1 , i.c.v.) mimicked and ICI (3 mg·kg -1 , i.v.) blocked the antidepressant actions of HNK in FST (q) and SPT (r). s–v, Western blot analysis revealed that DAMGO and DPDPE mimicked and occluded the HNK-induced increase in GluA1 levels (s, u), whereas CTOP and ICI blocked this effect (t, v). Scale bar: 5 ms, 0.2 mV in b–d. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, not significant (statistical analyses and n values are provided in Supplementary Table 1).
Article Snippet: (R,S)-ketamine provided by Dr. Sungchil Yang of City University of Hong Kong, (2R,6R)-HNK (Tocris), DAMGO (Tocris), CTOP (Tocris), MK-801 (MCE), D-APV (MCE), DPDPE (MCE), ICI174864 (MCE), Picrotoxin (MCE), CYM51010 (MCE), CGP52432 (Millipore), and MOR-DOR heterodimer allosteric inhibitor peptide (PEP; amino acid sequence: VTACTPSDGPGGGAAAYGRKKRRQRRR) (QYAOBIO, Shanghai, China) were dissolved in 0.9% saline to make a stock solution.
Techniques: Concentration Assay, Activation Assay, Knock-Out, Western Blot
