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Increased anxiety and decreased sociability induced by paternal deprivation involve the PVN-PrL OTergic pathway

  1. Zhixiong He
  2. Larry Young
  3. Xin-Ming Ma
  4. Qianqian Guo
  5. Limin Wang
  6. Yang Yang
  7. Luo Luo
  8. Wei Yuan
  9. Laifu Li
  10. Jing Zhang
  11. Wenjuan Hou
  12. Hui Qiao
  13. Rui Jia
  14. Fadao Tai  Is a corresponding author
  1. Shaanxi Normal University, China
  2. Emory University, United States
  3. University of Tsukuba, Japan
  4. University of Connecticut Health Center, United States
Research Article
Cite this article as: eLife 2019;8:e44026 doi: 10.7554/eLife.44026
9 figures, 1 table and 1 additional file

Figures

Effect of paternal deprivation on anxiety-like behavior and social preference in adult mandarin voles (n = 7).

(A, B) Percentage of time in the central area, (C, D) representative path and (E, F) total distance of mandarin voles in the open field test. *p<0.05; **p<0.01. Independent sample t-tests. (G, H) Percentage of time in the light area and (I, J) animal traces of mandarin voles in the light and dark box. *p<0.05; **p<0.01. Independent sample t-tests. Effect of PD on social preference in (K) males and (L) females. Error bars indicate SEM. **p<0.025 vs. object stimulus. ##p<0.025 vs. PC. Two-way ANOVA (factors: treatment × stimulus). PC, biparental care; PD, paternal deprivation.

https://doi.org/10.7554/eLife.44026.003
Figure 1—source data 1

Statistical results of the percentage of time spent in the central area and total distance in the open field, percentage of time in the light area in the light-dark box, and the percentage of time in investigating the social stimulus or object stimulus in the social preference test.

https://doi.org/10.7554/eLife.44026.004
Figure 2 with 1 supplement
Effect of paternal deprivation on the percentage of c-fos/OT double-labeled neurons in the PVN in adult mandarin voles (n = 5).

Voles were subjected to interaction with either (A) a juvenile voles or (B) a magic cube. (C, D) Double-immunohistochemical staining of c-fos (red) and OT (blue). Effect of PD on the percentage of neurons double-labeled for OT and c-fos in (E) males and (F) females. Error bars indicate SEM. **p<0.01 vs. object stimulus. ##p<0.01 vs. PC. Two-way ANOVA (factors: treatment × stimulus). PC, biparental care; PD, paternal deprivation.

https://doi.org/10.7554/eLife.44026.005
Figure 2—source data 1

Numbers of c-fos/OT double-labeled positive cells in the PVN.

https://doi.org/10.7554/eLife.44026.007
Figure 2—figure supplement 1
Paternal deprivation diminishes social approach in (A) male and (B) female mandarin voles (n = 5).

Error bars indicate SEM. **p<0.025 vs. Toy. ## p<0.025 vs. PC. PC, biparental care; PD, paternal deprivation.

https://doi.org/10.7554/eLife.44026.006
Effect of paternal deprivation on c-fos expression in the PrL and the NAc of adult mandarin voles (n = 5).

(A) Voles were subjected to interaction with either a juvenile vole or a magic cube. (B) Immunohistochemical staining of c-fos (red) and 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) (blue). The effect of PD on c-fos expression in the PrL of (D) males and (E) females. (C) Images show c-fos immunoreactivity in the NAc shell and the NAc core. Effect of PD on c-fos expression in (F) males and (G) females of the NAc shell. Effect of PD on c-fos expression in the NAc core of (H) males and (I) females. Error bars indicate SEM. **p<0.01 vs. object stimulus. ##p<0.01 vs. PC. Two-way ANOVA (factors: treatment × stimulus). PC, biparental care; PD, paternal deprivation.

https://doi.org/10.7554/eLife.44026.008
Figure 3—source data 1

Numbers of c-fos-positive cells in the PrL, NAc shell and NAc core.

https://doi.org/10.7554/eLife.44026.009
Effect of paternal deprivation on PVN OT-IR neurons.

(A) PC males, (B) PD males, (C) PC females and (D) PD females. 3V, 3rd ventricle. (E) Schematic drawing illustrating tissue in the PVN. (F) Quantification of OT-IR neurons in the PVN. Error bars indicate SEM. n = 4. **p<0.01 vs. PC. ##p<0.01 vs. male. Two-way ANOVA (factors: treatment × sex). PC, biparental care; PD, paternal deprivation; PVN, paraventricular nucleus.

https://doi.org/10.7554/eLife.44026.010
Figure 4—source data 1

Numbers of OT-positive cells in the PVN.

https://doi.org/10.7554/eLife.44026.011
Effects of paternal deprivation on mesocorticolimbic.

(A–B) OTR, (C–D) V1aR and (E–F) AVP immunoreactivity in male (n = 6) and female (n = 6) mandarin voles. Error bars indicate SEM. *p<0.05; **p<0.01. Independent sample t-tests. AVP, arginine vasopressin; mPFC, medial prefrontal cortex; NAc, nucleus accumbens; OTR,oxytocin receptor; PC, biparental care; PD, paternal deprivation; PVN, paraventricular nucleus; V1aR, vasopressin 1a receptor.

https://doi.org/10.7554/eLife.44026.012
Figure 5—source data 1

Levels of OTR, V1aR or AVP in the mPFC, NAc and PVN measured by Western Blot assay.

https://doi.org/10.7554/eLife.44026.013
Effects of PrL OT administration on anxiety-like behavior and social preference in paternal deprivation mandarin voles.

(A) Experimental schematics. (B) Histological representations of the microinjection site and (C, D) schematic diagrams showing the location of injector tips in the PrL. ×: missed. OT in the PrL is anxiolytic in both of sexes. (E, H) Percentage of time in the central area and (F, I) total distance in the open field test. One-way ANOVA. *p<0.05. OT in the PrL promotes a social preference in (G) males and (J) females. #p<0.0083 vs object stimulus. Two-way ANOVA (factors: treatment × sex). (Male: CSF – n = 6; 1 ng OT – n = 6; 10 ng OT – n = 6; 10 ng OT/10 ng OTA – n = 6; 10 ng OT/100 ng OTA – n = 5; 10 ng OT/10 ng V1aRA – n = 6. Female: CSF – n = 6; 1 ng OT – n = 6; 10 ng OT – n = 6; 1 ng OT/10 ng OTA – n = 5; 1 ng OT/100 ng OTA – n = 5; 10 ng OT/10 ng V1aRA – n = 6).

https://doi.org/10.7554/eLife.44026.014
Figure 6—source data 1

The percentage of time spent in the central area, the total distance in the open field, and the percentage of time spent investigating the social stimulus and the object stimulus after administration of CSF, OT, OT/OTA, or OT/V1aRA to the PrL.

https://doi.org/10.7554/eLife.44026.015
Figure 7 with 4 supplements
Optogenetic stimulation of OT terminals rescued changes in emotion and social preference behavior that were induced by paternal deprivation.

(A) Schematic drawing of the locations of rAAV-Ef1α-DIO-ChR2-mCherry plus rAAV-Oxytocin-Cre or rAAV-Ef1α-DIO-mCherry plus rAAV-Oxytocin-Cre injection into the right PVN and optic fiber implants. (B) Immunohistological image showing the targetting of fiber implants in the right PrL. Colocalization of ChR2-mCherry (red), OT neurons (green) and DAPI (blue) in the PVN of (C) males and (D) females. Confocal images of axonal mCherry signal in the PrL of (E) males and (F) females. (G) Images show expression of c-fos in the PrL after photostimulation. Quantification of c-fos in the PrL of (H) males (Ctrl: n = 4; ChR2: n = 5) and (I) females (Ctrl: n = 4; ChR2: n = 5) after photostimulation. Optogenetic activation of oxytocinergic fibers in the PrL increases social preference of (J) males (n = 6) and (K) females (n = 6). **p<0.01 vs. object stimulus. ## p<0.01 vs. Ctrl. Two-way ANOVA (factors: photostimulation treatment × stimulus type). Activation of PVN-PrL oxytocinergic projection significantly increased the percentage of time in the central area for both (L) males (n = 6) and (M) females (n = 6), but did not influence total distance traveled for (N) males (n = 6) or (O) females (n = 6). *p<0.05; **p<0.01. Independent sample t-tests. Error bars indicate SEM.

https://doi.org/10.7554/eLife.44026.016
Figure 7—source data 1

Statistical result of levels of c-fos in the PrL after optogenetic activation of PrL-projecting PVN OTergic neurons during the open field, the social preference test.

https://doi.org/10.7554/eLife.44026.021
Figure 7—figure supplement 1
Histology and immunostaining.

The histology of CTB injecting into the right PrL of (A) male and (C) female voles. Immunostaining showing colocalization of OT neurons (green) and CTB (red) in the PVN of (B) male and (D) female voles.

https://doi.org/10.7554/eLife.44026.017
Figure 7—figure supplement 2
Immunostaining showing colocalization of mCherry (red) with OT (green) but not AVP (blue) in PVN neurons of (A) male and (B) female voles.
https://doi.org/10.7554/eLife.44026.018
Figure 7—figure supplement 3
Representative immunostaining allowing the vizualization of (A) OT expression (green), (B) mCherry (red) and (C) DAPI (blue) in PVN neurons of PD voles.

(D) Colocalization image including bands (A), (B) and (C). Arrowheads indicate the OT, mCherry and DAPI. Quantification of OT–meCherry colocalization in (E) males (147/234 = 62.8%, from two voles) and (F) females (203/283 = 71.7%, from two voles).

https://doi.org/10.7554/eLife.44026.019
Figure 7—figure supplement 4
After activation of PVN-to-PrL OT terminals does not elicit long-lasting effects on anxiety-like behavior and social preference in PD voles (n = 6).

The behavioral changes disappeared 8 hr after blue light photostimulation. Quantification of the percentage of time spent in the central area ((A): male; (B): female) and total distance traveled ((C): male; (D): female). **p<0.01. One-way ANOVA. Quantification of social preference ((E): male; F): female)). **p<0.01 vs. object stimulus. ## p<0.01 vs. Ctrl. Two-way ANOVA. Error bars indicate SEM.

https://doi.org/10.7554/eLife.44026.020
Figure 8 with 2 supplements
Optogenetic inhibition of OT terminals elicits anxiety-like behavior and attenuated social preference in naive voles.

(A) Schematic drawing of the locations of rAAV-Ef1α-DIO-eNpHR3.0-mCherry plus rAAV-Oxytocin-Cre or rAAV-Ef1α-DIO-mCherry plus rAAV-Oxytocin-Cre injection bilaterally into the PVN and optic fiber implants. (B) Immunohistological image showing target of fiber implants in the bilateral PrL. Colocalization of eNpHR3.0-mCherry (red), OT neurons (green) and DAPI (blue) in the PVN of (C) males and (D) females. Confocal images of axonal mCherry signal in the PrL of (E) males and (F) females. (G) Images showing the expression of c-fos in the PrL after yellow light photostimulation. Quantification of c-fos in the PrL of (H) males (n = 4) and (I) females (n = 4) after photostimulation. **p<0.01. Independent sample t-tests. Optogenetic inhibition of oxytocinergic fibers in the PrL decreases the social preference of (J) males (n = 6) and (K) females (n = 6); p<0.025. Two-way ANOVA (factors: optogenetic inhibition treatment × stimulus type). Inhibition of PVN-PrL oxytocinergic projection significantly decreases the percentage of time spent in the central area for both (L) males (n = 6) and (M) females (n = 6), but did not influence total distance traveled by (N) males (n = 6) or (O) females (n = 6). **p<0.01. Independent sample t-tests. Error bars indicate SEM.

https://doi.org/10.7554/eLife.44026.022
Figure 8—source data 1

Results of statistical analysis of the levels of c-fos in the PrL after optogenetic inhibition of PrL-projecting PVN OTergic neurons during the open field and the social preference test.

https://doi.org/10.7554/eLife.44026.025
Figure 8—figure supplement 1
Representative immunostaining of (A) OT expression (green), (B) mCherry (red) and (C) DAPI (blue) in PVN neurons of naïve voles.

(D) Colocalization image for bands (A), (B) and (C). Arrowheads indicate the OT, mCherry and DAPI. Quantification of OT/meCherry colocalization in (E) males (427/602 = 70.9%, from two voles) and (F) females (672/803 = 83.6%, from two voles).

https://doi.org/10.7554/eLife.44026.023
Figure 8—figure supplement 2
After inhibition of PVN-to-PrL OT terminals does not elicit long-lasting effects on anxiety-like behavior and social preference in naïve voles (n = 6).

The behavioral changes (except social preference of male: within 8 hr) occur within 8–24 hr following yellow light photostimulation. Quantification of the percentage of time in the central area ((A): male; (B): female) and total distance traveled ((C): male; (D): female). **p<0.01. One-way ANOVA. Quantification of social preference ((E): male; (F): female). *p<0.05; **p<0.01 vs. object stimulus. Two-way ANOVA. Error bars indicate SEM.

https://doi.org/10.7554/eLife.44026.024
Author response image 1
Lane 2 and Lane 3: Anti-Oxytocin Receptor antibody [EPR12789] (ab181077, Abcam) at 1/2000 dilution.

Lane 1: Protein marker. Lane 2: Voles mPFC tissue lysate. Lane 3: Mouse spleen tissue lysate.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Antibodyanti-oxytocin mouse monoclonal antibodyMilliporeCat#MAB5296
RRID:AB_2157626
concentration: 1:7500
Antibodyanti-vasopressin rabbit polyclonal antibodyMilliporeCat#AB1565
RRID:AB_90782
concentration:1:4000
Antibodyanti-c-fos rabbit polyclonal antibodyAbcamCat#ab190289
RRID:AB_2737414
concentration:1:1500
Antibodyanti-oxytocin receptor rabbit monoclonal
antibody
AbcamCat#ab181077concentration:1:2000
Antibodyanti-vasopressin receptor 1A (V1aR) goat polyclonal antibodyGeneTexCat#GTX89114
RRID:AB_10724608
concentration:1:7000
Antibodyanti-β-tubulin mouse monoclonal antibodyComWin BiotechnologyCat#CW0098Mconcentration:1:5000
Antibodyanti-rabbit goat conjugated with TRITCJackson ImmunoResearchCat#111-025-003
RRID:AB_2337926
concentration:1:200
Antibodyanti-rabbit goat conjugated with DyLight 405Jackson ImmunoresearchCat#111-475-003
RRID:AB_2338035
concentration:1:200
Antibodyanti-rabbit goat conjugated with DyLight 488BosterCat#BA1127concentration:1:200
Antibodyanti-mouse goat antibody conjugated with DyLight 488BosterCat#BA1126concentration:1:200
Antibodyanti-rabbit goat conjugated with horseradish peroxidaseZhongShan GoldenbridgeZB-2301
RRID:AB_2747412
concentration:1:10,000
Antibodyanti-mouse goat conjugated with horseradish peroxidaseZhongShan GoldenbridgeZB-2305
RRID:AB_2747415
concentration:1:10,000
Antibodyanti-goat rabbit conjugated with horseradish peroxidaseZhongShan GoldenbridgeZB-2306concentration:1:10,000
Reagentnormal goat serumBosterCat#AR0009
VirusAAV-Ef1α-DIO-ChR2-mCherryBrainVTACat#PT-0002
VirusAAV-Ef1α-DIO-eNpHR3.0-mCherryBrainVTACat#PT-0007
VirusAAV-Ef1α-DIO-mCherryBrainVTACat#PT-0013
VirusAAV-Oxytocin-CreBrainVTACat#PT-0263
Chemical compound, drugradioimmunoprecipitation assay buffer (RIPA)SolarbioCat#R0010
Chemical compound, drugenhanced chemiluminescence (ECL) reagentMilliporeCat#WBKLS0500
Chemical compound, drugantifade solutionBosterCat#AR1109
Chemical compound, drug4',6-diamidine-2'-phenylindole dihydrochloride (DAPI)BosterCat#AR1176
Chemical compound, drugCholera Toxin Subunit B (CTB)−594Thermo Fisher ScientificCat#C34777
Software, algorithmOBSERVER v5.0Noldushttps://www.noldus.com/knowledge-base/observer-50
Software, algorithmSPSSIBMRRID:SCR_002865
Software, algorithmImageJNIHRRID:SCR_003070
Software, algorithmGraphPad Prism 5GraphPadRRID:SCR_002798
Software, algorithmSuperMazeShanghai XinRuanXR-XJ117
Software, algorithmSocialScanClever Syshttp://cleversysinc.com/CleverSysInc/home/software/socialscan/
OtherPVDF membranesMilliporeC3117

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all Figures.

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