Paraventricular nucleus-central amygdala oxytocinergic projection modulates pain-related anxiety-like behaviors in mice

Abstract

Aims: Anxiety disorders associated with pain are a common health problem. However, the underlying mechanisms remain poorly understood. We aimed to investigate the role of paraventricular nucleus (PVN)-central nucleus of the amygdala (CeA) oxytocinergic projections in anxiety-like behaviors induced by inflammatory pain.

Methods: After inflammatory pain induction by complete Freund's adjuvant (CFA), mice underwent elevated plus maze, light-dark transition test, and marble burying test to examine the anxiety-like behaviors. Chemogenetic, optogenetic, and fiber photometry recordings were used to modulate and record the activity of the oxytocinergic projections of the PVN-CeA.

Results: The key results are as follows: inflammatory pain-induced anxiety-like behaviors in mice accompanied by decreased activity of PVN oxytocin neurons. Chemogenetic activation of PVN oxytocin neurons prevented pain-related anxiety-like behaviors, whereas inhibition of PVN oxytocin neurons induced anxiety-like behaviors in naïve mice. PVN oxytocin neurons projected directly to the CeA, and microinjection of oxytocin into the CeA blocked anxiety-like behaviors. Inflammatory pain also decreased the activity of CeA neurons, and optogenetic activation of PVN^oxytocin -CeA circuit prevented anxiety-like behavior in response to inflammatory pain.

Conclusion: The results of our study suggest that oxytocin has anti-anxiety effects and provide novel insights into the role of PVN^oxytocin -CeA projections in the regulation of anxiety-like behaviors induced by inflammatory pain.

Keywords: anxiety; central nucleus of the amygdala; oxytocin; pain; paraventricular nucleus.

FIGURE 1

Decreased activity of OTergic neurons in PVN relates to the anxiety behaviors in mice. (A) CFA injection into the left hind paw caused a significant decrease in mechanical paw withdrawal threshold in mice (n = 8–10). (B, C) Elevated plus maze test results 4, and 7 days after CFA injection. (D, E) Light–dark transition test and marble burying test results 4, and 7 days after CFA injection. (F) Locomotor activity 4, and 7 days after CFA injection. (G) Overlap of oxytocin marked by OT‐EGFP (green), c‐Fos immunoreactivity (red) in the saline group (up) and CFA group (down). Scale bar of three pictures on the left: 100 μm. Scale bar of picture on the right: 50 μm. (H) Number of c‐Fos and OT‐EGFP co‐localizations (n = 4–5). (I) OT‐GcaMP6s microinjection schematic diagram. (J, K) The mean shows that calcium ion (Ca2+) signals decreased in CFA‐injected mice compared to saline‐treated mice when subjected to elevated plus maze test. (K) indicates the area under the curve. The colored bars indicate ΔF/F (%) (n = 5). CFA, Complete Freund's adjuvant.

FIGURE 2

Microinjections of oxytocin into the lateral ventricle prevent anxiety‐like behavior. (A) Schematic diagram and timeline of the experiment. (B–E) Elevated plus maze, light–dark transition, and marble burying tests, respectively, of CFA‐injected mice after 20 μg bilateral oxytocin microinjection. Error bars indicate standard error. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with saline‐treated control group, two‐way ANOVA with Bonferroni's post hoc test. CFA, Complete Freund's adjuvant; PVN, paraventricular nucleus; ANOVA, analysis of variance; N.S, not significant.

FIGURE 3

Activation of PVN oxytocin neurons blocked pain‐induced anxiety‐like behaviors. (A) Timeline of the experiment. (B) Schematic diagram of rAAV‐oxytocin‐Cre‐WPREs and rAAV‐EF1α‐DIO‐hM3Dq‐mCherry‐hGHpA microinjections. (C) Co‐localization of immunoreactive DIO‐ hM3Dq ‐mCherry (red) and oxytocin (green). Scale bar of three pictures on the left: 100 μm. Scale bar of picture on the right: 50 μm. (D–G) Results of the elevated plus maze, light–dark transition, and marble burying tests after clozapine‐N‐oxide (CNO) intraperitoneal injection 30 min prior. (H) Representative images of the PVN illustrating c‐Fos in neurons expressing DIO‐mCherry group(up) and DIO‐hM3Dq‐mCherry group(down). Scale bar of three pictures on the left: 100 μm. Scale bar of picture on the right: 50 μm. (I) Number of c‐Fos and mCherry co‐localizations. OE/OE + CE means ratios of entries into open arms to the sum of entries into open arms and entries into closed arms. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with mCherry group in unpaired two‐tailed t‐test. CNO, clozapine‐N‐oxide. Error bars indicate standard error.

FIGURE 4

Inhibition of the PVN oxytocin neurons increased pain‐induced anxiety‐like behaviors. (A) Timeline of the experiment. (B–E) Elevated plus maze, light–dark transition, and marble burying tests after clozapine‐N‐oxide (CNO) intraperitoneal injection for 30 min. (F) Representative images of the PVN illustrating c‐Fos in neurons expressing DIO‐mCherry and DIO‐hM3D(Gi)‐mCherry. Scale bar of three pictures on the left: 100 μm. Scale bar of picture on the right: 50 μm. (G) Number of c‐Fos. OE/OE + CE means ratios of entries into open arms to the sum of entries into open arms and entries into closed arms. *p < 0.05, ***p < 0.001 compared with mCherry group in unpaired two‐tailed t‐test. Error bars indicate standard error.

FIGURE 5

Oxytocin bilateral microinjection in the CeA blocks inflammatory pain‐induced anxiety‐like behaviors. (A) OT‐EGFP microinjection schematic diagram. (B) Immunoreactive OT‐EGFP (green). Left: OT‐EGFP in the paraventricular nucleus (PVN); right: projection fiber in the central nucleus of the amygdala (CeA). Scale bar: 100 μm. (C) Immunoreactive c‐Fos (green) in the complete Freund's adjuvant (CFA) group compared with the saline group. Scale bar: 100 μm. (D) Number of c‐Fos in the CeA. (E) Timeline of the experiment and oxytocin microinjection schematic diagram. (F–H) Results of elevated plus maze and marble burying tests of CFA‐injected mice after 20 μg oxytocin microinjection into the CeA. (I) Timeline of the experiment. (J) Schematic diagram of rAAV‐oxytocin‐Cre‐WPREs and rAAV‐EF1α‐DIO‐hM3Dq‐mCherry‐hGHpA microinjections with cannula implantation. (K–N) Elevated plus maze, light‐dark transition, and marble burying tests after clozapine‐N‐oxide (CNO) intraperitoneal injection and OTA (40 ug for each mouse) microinjection for 30 min. *p < 0.05, **p < 0.01 compared with saline group in unpaired two‐tailed t‐test. Error bars indicate standard error.

FIGURE 6

Activation of PVN‐CeA oxytocin neuron projection blocked inflammatory pain‐induced anxiety‐like behaviors. (A) Left: timeline of the experiment; Right: schematic representing the OT‐Cre and DIO‐ChR2‐mCherry microinjection sites, and fiber implant location. (B) Schematic diagram of rAAV‐oxytocin‐Cre‐WPREs, rAAV‐EF1α‐DIO‐ hChR2 ‐mCherry‐hGHpA microinjections and fiber implant. (C–F) Results of elevated plus maze, light–dark transition (stimulation parameters: 20 Hz, 10–20 mW, 10 ms, 3 s on 2 s off, 5 min), marble burying (20 Hz, 5 mW, 5 ms, 3 min on, 2 min off, 30 min) with 473 nm blue light. (G) Representative images of the CeA illustrating c‐Fos in neurons expressing DIO‐hChR2‐mCherry projection fibers. Scale bar: 100 μm. (H) Number of c‐Fos. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with mCherry group in unpaired two‐tailed t‐test. Error bars indicate standard error.

FIGURE 7

Inhibition of the PVN‐CeA oxytocin projection increased pain‐induced anxiety‐like behaviors and this projection performed anti‐anxiety effect by oxytocin receptor. (A) Timeline of the experiment. (B) Schematic diagram of Retro‐oxytocin‐Cre‐WPREs and rAAV‐EF1α‐DIO‐hM4Di‐mCherry‐hGHpA microinjections. (C) Co‐localization of immunoreactive DIO‐hM4Di‐mCherry (red) and oxytocin (green). Scale bar of three pictures on the left: 100 μm. Scale bar of picture on the right: 50 μm. (D–G) Elevated plus maze, light–dark transition, and marble burying tests after clozapine‐N‐oxide (CNO) intraperitoneal injection for 30 min. (H) Co‐localization of immunoreactive DIO‐ hM4Di‐mCherry (red) and c‐Fos (green) in the paraventricular nucleus (PVN). Scale bar of three pictures on the left: 100 μm. Scale bar of picture on the right: 50 μm. (I) Number of c‐Fos. OE/OE + CE means ratios of entries into open arms to the sum of entries into open arms and entries into closed arms. *p < 0.05, **p < 0.01, ***p < 0.001 compared with mCherry group in unpaired two‐tailed t‐test. Error bars indicate standard error.