307 - Oxytocin Neuron Dysfunction in Paraventricular Nucleus Mediates Adverse Outcomes of Maternal Separation

Publication Number: 4301.307

Mingyan Hei, Beijing Chidlren's Hospital, Capital Medical University, Beijing, Beijing, China (People's Republic); Yuan Zhang, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing, Beijing, China (People's Republic)

Background: Maternal separation (MS) for newborns in Neonatal Intensive Care Unit (NICU), as an early life stress for newborns, is a significant risk factor for the infants' neuropsychiatric disorders in adolescent and adult stage. Clinical trials proved that decreasing MS by family integrated care during the infants' stay in NICU mitigate these negative effects, however the underlying mechanisms remain unclear.

Objective: To explore the possible mechanisms of how oxytocin (OXT) neuron dysfunction in the cerebral paraventricular nucleus (PVN) mediates the adverse outcomes of newborns with MS.

Design/Methods: C57BL/6J mice and Oxt-Cre: Ai9 double-transgenic mice pups were randomly assigned to Control (Ctrl) or MS group (mice pups were separated from the dam daily for 4 h from P4 to P20). behavioral testing, histo-immunochemistry staining, single-cell RNA sequencingof PVN, ELISA, Western Blotting and patch-clamp recording in OXT neurons were used. SPSS 26.0 and GraphPad Prism 9.0 were used for statistical analysis.

Results: In this study, we demonstrated that MS in neonatal mice induced anxiety- and depression-like behaviors (Fig 1) in adolescence and disrupted OXT neuron function in the PVN of the hypothalamus. By using single-cell RNA sequencing, we identified a significant reduction in neuronal density and widespread transcriptomic alterations specifically within OXT neurons (Fig 2). Electrophysiological recordings by using whole0cell patch clamp method revealed that MS-induced hyperexcitability of OXT neurons alongside impaired synaptic transmission (Fig 3). Subcutaneous administration of oxytocin during MS period rescued the above anxiety- and depression-like behaviors in adolescence (on P21 for the mice pups), and restored central OXT expression, and normalized corticosterone levels in saliva. The serum corticosterone levels in the MS + OXT group (7.24 ± 0.64 ng/mL) were significantly lower than in the MS + PBS group (11.76 ± 2.42 ng/mL) (P = 0.007). OXT levels in serum and PVN tissue were significantly higher in the MS + OXT group compared to the MS + PBS group (P = 0.011, 0.042). Exogenous OXT administration also significantly ameliorated anxiety- and depression-like behaviors in adolescent MS mice.

Conclusion(s): Dysfunction of OXT neurons in cerebral PVN zone could be a key mediator of MS-induced neurodevelopmental impairments. This could be mechanism basis for OXT-based interventions to improve brain development in sick newborn infants who undergo MS in NICU.

Fig 1. Maternal separation (MS) increases susceptibility to neuropsychiatric behaviors
(A) Experimental timeline and behavioral paradigm. Mice in MS (P4-20) or controls (Ctrl) groups underwent a battery of behavioral tests during adolescence (P28-34). Brain tissues were collected at P21 for comprehensive analyses. (B) There was no significant difference in weight gain between the two groups at different time points. (C) The exploration trajectory map of mice during the open field test (OFT). (D) Entries into central zone in the OFT. (E) Duration in central zone during the OFT. (F) Percentage of entries into the open arms in elevated plus maze (EPM). (G) Duration in central zone during the EPM. (H) Immobility duration in the forced swim test (FST). (I) Immobility duration in the tail suspension test (TST). (J) Sucrose preference ratio in the sucrose preference test (SPT).
*P < 0.05, **P < 0.01. Unpaired Student's t-test (parametric) or Mann Whitney U test (non-parametric). Bar graphs show the mean ± SD

Fig 2. Single-cell RNA sequencing (scRNA-seq) of cells in the paraventricular nucleus (PVN)
(A) Schematic illustration of the experimental workflow for scRNA-seq. (B) UMAP plot showing clusters of major 9 cells isolated from the PVN. (C) Identification of neuronal cell types by canonical marker genes expression. (D) Bar chart depicting the proportional abundance of major cell types identified by scRNA-seq between Control (Ctrl) and maternal separation (MS) groups. (E) Nissl staining on mice PVN sections.
***P < 0.001 (unpaired Student's t-test). Bar graphs show the mean ± SD

Fig 3. Maternal separation (MS) disrupts OXT neuron electrophysiological function in the PVN
(A) Representative spontaneous firing of Ctrl and MS. (B) There was an effect of MS on spontaneous firing frequency compared to Ctrl group. (C) Representative eAP-ramp of Ctrl and MS. (D-F) There was an effect of MS on first spike latency (FSL) and threshold current. MS had no effect on threshold potential. (G) Representative eAP-step of Ctrl and MS. (H-J) There was an effect of MS on half width and amplitude. MS had no effect on afterhyperpolarization (AHP). (K) MS increased the number of action potentials evoked by current injections (150, 175, and 200 pA). (L) Representative mEPSCs of Ctrl and MS. (M-P) There was an effect of MS on amplitude and frequency. (Q) Representative mIPSCs of Ctrl and MS. (R-U) There was an effect of MS on amplitude. MS had no effect on frequency.
*P < 0.05, **P < 0.01, ***P < 0.001. Unpaired Student's t-test (parametric) or Mann-Whitney U test (non-parametric). Bar graphs show the mean ± SD