Figures and data in Oxytocin neurons mediate the effect of social isolation via the VTA circuits

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7 figures, 1 table and 1 additional file

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Figure 1 with 3 supplements

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Adolescence acute social isolation induces social craving.

(A) Experimental design: WT mice were isolated between P28 and P35 or kept in group. After isolation, mice (from different cohorts) were subjected to different behavioral task. (B) Free direct interaction task paradigm. (C) Time exploring social stimulus (Mann-Whitney U-test=14, p=0.0052, n=10 mice each group). (D) Time exploring object (Mann-Whitney U-test=33, p=0.2176, n=10 mice each group). (E) Experimental design: mice were house four per cage from P21 to P28. At P28, one group of mice were divided in two cages with two mice each, while a second group was divided in one cage with two mice and two cages with one mouse until P35. After isolation, a free-interaction with former cage mate was carried out. (F) Interaction time with former cage mate (Unpaired sample t-test, t₍₁₃₎=6.885, p<0.001). (G) Three-chamber task experimental paradigm. (H) Interaction time with object or social stimulus 1 (Grouped ## Wilcoxon matched-pairs signed rank test, W=118, p=0.001; Isolated ### Paired sample t-test, t₍₁₅₎=5.975, p<0.001. Two-way RM-ANOVA. Target main effect F_{(1, 30)}=51.47, p<0.001; House condition main effect F_{(1, 30)}=3.935, p=0.0565; Target × House condition F_{(2, 60)}=9.487, p>0.001, n=16). (I) Interaction time with stimulus 1 (familiar) and stimulus 2 (unfamiliar) (Grouped ### Paired sample t-test, t₍₁₅₎=5.774, p<0.001; Isolated ### Paired sample t-test, t₍₁₅₎=0.102, p=0.9201. Two-way RM-ANOVA followed by Bonferroni’s multiple comparisons test. Target main effect F_{(1, 30)}=9.251, p=0.0049; House condition main effect F_{(1, 30)}=1.164, p=0.2892; Target × House condition F_{(1, 30)}=8.215, p=0.0075, n=16). (J) Habituation task paradigm. (K) Interaction time on Day 1 (Unpaired-samples t-test, t₍₁₀₎=3.076, p=0.0117, n=6 mice each group). (L) Interaction time across 4 days (Two-way RM ANOVA followed by Tukey’s multiple comparisons test, Days main effect F_{(2.215, 22.15)}=6.775, p=0.0041, House condition main effect F_{(1, 10)}=1.455, p=0.2555, Days × House condition F_{(3, 30)}=4.349, p=0.0117, Grouped Day 1 versus Day 4, p=0.0087, Isolated Day 1 versus Day 4, p=0.9094, n=6 mice each group). (M) Interaction time during Day 1 and Day 4 for Grouped mice (Paired samples t-test, t₍₅₎=5.706, p=0.0023). (N) Interaction time during Day 1 and Day 4 for isolated mice (Paired samples t-test, t₍₅₎=0.6552, p=0.5413). Data are represented as mean ± SEM.

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Effects of social isolation on social behavior.

(A) Experimental design: WT mice were isolated for 24 hr between P34 and P35 or kept in group. After isolation, mice were subjected to free direct interaction task paradigm. (B) Time exploring social stimulus (Unpaired-samples t-test, t₍₁₈₎=0.2817, p=0.7814, n=10 mice each group). (C) Experimental design: WT mice were isolated between P28 and P35 or kept in group. After isolation, mice were subjected to three-chamber task and habituation task (different cohorts). (D) Distance moved during the three-chamber task related to Figure 1G–I (Social preference: unpaired samples t-test t₍₃₀₎=1.383, p=0.1786. Social novelty: unpaired samples t-test t₍₃₀₎=0.8638, p=0.3946, Grouped n=16, Isolated n=16). (E) Time in chamber during social preference phase (Two-way RM-ANOVA followed by Tukey’s multiple comparisons test. Chamber main effect F_{(1.168, 35.03)}=156.7, p<0.001; House condition main effect F_{(1, 30)}=1.155, p=0.2911; Chamber × House condition F_{(2, 60)}=9.487, p>0.001, n=16). (F) Preference index calculated as object interaction time/(object+stimulus 1) or stimulus 1 interaction time/(object+stimulus 1) (# One-sample t-test against chance level 0.5; Grouped t₍₁₅₎=4.443, p<0.001; Isolated t₍₁₅₎=6.766, p<0.001). (G) Time in chamber during social preference phase (Two-way RM-ANOVA followed by Tukey’s multiple comparisons test. Chamber main effect F_{(1.5, 44.99)}=97.47, p<0.001; House condition main effect F_{(1, 30)}=1.1376, p=0.2501; Chamber × House condition F_{(2, 60)}=9.938, p=0.061, n=16).(H) Preference index calculated as stimulus 1 interaction time/(stimulus 2+stimulus 1) or stimulus 2 interaction time/(stimulus 2+stimulus 1) (# One-sample t-test against chance level 0.5; Grouped t₍₁₅₎=7.104, p<0.001; Isolated t₍₁₅₎=0.077, p=0.9). (I) Experimental paradigm: WT mice were isolated between P28 and P29 or kept in group. After isolation, mice were subjected to habituation task consisted in four repeated exposure to a same social stimulus within the same day. (J) Interaction time during trail 1 (Unpaired sample t-test, t₍₁₀₎=17.01, p<0.001, n=6 each group). (K) Intercation time across Trails normalized on Trial 1 (Two-way RM-ANOVA, Trial main affect F_{(2.007, 20.07)}=18.47, p<0.001, House condition main effect F_{(1. 10)}=2.6, p=0.1379, Trial × House condition F_{(3, 30)}=1.050, p=0.3848). (**L–M**) Interaction time within Trial 1 and Trial 4 (Paired samples t-test, Grouped (L) t₍₅₎=6.020, p=0.0018; Isolated (M) t₍₅₎=4.735, p=0.0052). Data are represented as mean ± SEM.

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Effects of social isolation on social behavior.

(A) Experimental design: WT mice were isolated between P28 and P35 or kept in group. After isolation, mice were subjected to Novel object recognition (NORT) and Elevated plus maze (same cohorts). (B) NORT task experimental paradigm. (**C–D**) Preference index during familiarization phase calculated as object 1 interaction time/(object 1+object 2) or object 2 interaction time/(object 1+object 2) (Grouped: Paired-samples t-test t₍₉₎=2.181, p=0.0571, n=10, Isolated: Paired-samples t-test t₍₉₎=0.5301, p=0.6088, n=10). (**E–F**) Preference index during novelty phase calculated as object 1 interaction time/(object 1 +object 3) or object 3 interaction time/(object 1+object 3) (Grouped: Paired-samples t-test t₍₉₎=2.276, p=0.0489, n=10, Isolated: Paired-samples t-test t₍₉₎=4.487, p=0.0015, n=10). (G) Distance moved (Unpaired sample t-test t₍₁₈₎=0.4078, p=0.6434, Grouped n=10, Isolated n=10). (H) Elevated Plus Maze experimental paradigm. (I) Distance moved (Unpaired sample t-test t₍₁₈₎=3.419, p=0.0031, Grouped n=10, Isolated n=10). (J) Time spent in compartment (One-way RM-ANOVA followed by Tukey’s multiple comparisons test. Grouped; compartment main effect F_{(1.306,11.76)}=50.54, p<0.001, n=10; Isolated: compartment main effect F_{(1.182, 10.64)}=56.21, p<0.001, n=10). (**K–L**) Preference index calculated as time in open arm/(open+close) or time in close arm/(open+close) (Grouped: Paired-samples t-test t₍₉₎=8.101, p<0.001, n=10, Isolated: Paired-samples t-test t₍₉₎=7.025, p<0.001, n=10). (M) social conditioned place preference (sCPP) task experimental paradigm. (N) Time in chamber during Pre-test (Grouped: Paired-samples t-test t₍₉₎=1.448, p=0.1815, Isolated: Paired-samples t-test t₍₉₎=1.157, p=0.2772; Two-way RM-ANOVA, Chamber main effect F_{(1, 18)}=3.372, p=0.0829, House condition main effect F_{(1, 18)}=1.027, p=0.3243, Chamber × House condition F_{(1, 18)}=0.02581, p=0.8742, n=10). (O) Time in chamber during Post-test (Grouped: # Paired-samples t-test t₍₉₎=2.297, p=0.0472, Isolated: # Paired-samples t-test t₍₉₎=3.075, p=0.0132; Two-way RM-ANOVA, Chamber main effect F_{(1, 18)}=14.73, p=0.0012, House condition main effect F_{(1, 18)}=0.1818, p=0.6749, Chamber × House condition F_{(1, 18)}=1.279, p=0.273, n=10). (P) Time in social chamber during Pre/Post-test (Grouped: # Paired-samples t-test t₍₉₎=3.183, p=0.0111, Isolated: ### Paired-samples t-test t₍₉₎=6.638, p<0.001; Two-way RM-ANOVA, House condition main effect F_{(1, 18)}=0.9767, p=0.3361, Time main effect F_{(1, 18)}=33.81, p<0.001, Time × House condition F_{(1, 18)}=0.01413, p=0.9067, n=10). (**Q–R**) Preference score for social chamber (Grouped: Paired-samples t-test t₍₉₎=2.323, p=0.0452, Isolated: Paired-samples t-test t₍₉₎=4.118, p=0.0026, n=10). Data are represented as mean ± SEM.

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Effects of social isolation during adulthood.

(A) Experimental design: WT mice were isolated between P53 and P60 or kept in group. After isolation, mice were subjected to different behavioral task. (B) Free direct interaction task paradigm. (C) Time exploring social stimulus (Unpaired-samples t-test, t₍₂₀₎=2.237, p=0.0369, Grouped n=12, Isolated n=10). (D) Time exploring object (Unpaired-samples t-test, t₍₁₉₎=1.876, p=0.0761, Grouped n=12, Isolated n=9). (E) Three-chamber task experimental paradigm. (F) Interaction time with object or social stimulus 1 (Paired sample t-test. ### Grouped: t₍₉₎=4.941, p<0.01, n=10. ## Isolated: t₍₉₎=4.082, p=0.0027. Two-way RM-ANOVA. Target main effect F_{(1, 18)}=40.32, p<0.001; House condition main effect F_{(1, 18)}=0.02823, p=0.8684; Target × House condition F_{(1, 18)}=0.1188, p=0.7343, n=10). (G) Time in chamber during social preference phase (One-way RM-ANOVA followed by Tukey’s multiple comparisons test. Grouped; chamber main effect F_{(1.270, 11.43)}=71.56, p<0.001, n=10; Isolated: chamber main effect F_{(1.594, 14.35)}=75.35, p<0.001, n=10). (H) Preference index calculated as object interaction time/(object+stimulus 1) or stimulus 1 interaction time/(object+stimulus 1) (One-sample t-test against chance level 0.5; ### Grouped t₍₉₎=5.329, p<0.001; ## Isolated t₍₉₎=4.026, p=0.003). (I) Interaction time with stimulus 1 (familiar) and stimulus 2 (unfamiliar) (Paired sample t-test. ###Grouped: t₍₉₎=4.736, p=0.0011; ### Isolated: t₍₉₎=4.939, p<0.001. Two-way RM-ANOVA. Target main effect F_{(1, 18)}=46.45, p<0.001; House condition main effect F_{(1, 18)}=0.4045, p=0.5328; Target × House condition F_{(1, 18)}=0.1068, p=0.7476, n=10). (J) Time in chamber during social preference phase (One-way RM-ANOVA followed by Tukey’s multiple comparisons test. Grouped; chamber main effect F_{(1.871 16.84)} = 36.35 p < 0.001, n = 10; Isolated: chamber main effect F_{(1.993, 17.93)} = 46.27 p < 0.001 n = 10). (K) Preference index calculated as stimulus1 interaction time/(stimulus1 +stimulus2) or stimulus2 interaction time/(stimulus1 +stimulus2) (One-sample t-test against chance level 0.5; ## Grouped t₍₉₎=4.494 P = 0.0015; ### Isolated t₍₉₎=5.502 p < 0.001). (L) Distance moved during the 3-chamber task (Social preference: Mann Whitney test U = 13, P = 0.0039. Social novelty: Unpaired samples t-test t₍₁₈₎=2.26 P = 0.0364, n = 10 mice each group). Data are represented as mean ± SEM.

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Figure 2

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Adolescence acute social isolation induces putative VTA DA neurons hyperexcitability.

(A) Experimental design: WT mice were isolated between P28 and P35 or kept in group. After isolation, mice were subjected whole-cell patch-clamp recordings. (B) Left: experimental paradigm, putative VTA DA neurons were subjected at multiple depolarizing current steps. Right: example traces from 250 pA depolarizing current injection. (C) Number of action potentials (APs) across increasing depolarizing current steps (Two-way RM ANOVA followed by uncorrected Fisher’s LSD post doc analysis, House condition × Current step F_{(6, 132)}=2.230, p=0.044, current step main effect F_{(2.310, 50.82)}=36.65, p<0.001, House condition main effect F_{(1, 22)}=8.016, p=0.0097). (D) Resting membrane potential of recorded cells (Mann-Whitney U-test=66, p=0.7762. Grouped n=13, Isolated n=11 from three mice each group). Data are represented as mean ± SEM.

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PVN OXT neurons as main orchestrator of social isolation induced social craving.

(A) Representative confocal images of PVN stained with cFOS, OXT, and AVP antibody (scale bar: 20 µm). (B) cFos⁺ cells (as % of DAPI⁺ cells) (Unpaired samples t-test, t₍₂₂₎=2.510, p=0.0199, n=12 slices from three mouse each group). (C) OXT⁺ cells (as % of DAPI⁺ cells) (Mann-Whitney U-test=33, p=0.0439, n=11–12 slices from three mouse each group). (D) OXT⁺/ cFos⁺ cells (as % of total OXT⁺ cells) (Unpaired samples t-test, t₍₂₂₎=3.491, p=0.021, n=12 slices from three mouse each group). (E) Left: experimental paradigm, PVN neurons projecting to VTA (CTB-488 was injected in the VTA at P21) were subjected at multiple depolarizing current steps. Right: example traces from 250 pA depolarizing current injection. (F) Number of action potentials (APs) across increasing depolarizing current steps (Two-way RM ANOVA followed by uncorrected Fisher’s LSD post doc analysis, House condition main effect F_{(1, 30)}=7.306, p=0.0112, Current step main effect F_{(1.857, 55.71)}=30.47, p<0.001, House condition × Current step F_{(6, 180)}=2.828, p=0.018). (G) Resting membrane potential of recorded cells (Mann-Whitney U-test=85.5, p=0.1483. Grouped n=13, Isolated n=19 from three mice each group). (H) Experimental design. R26-hM4Di/mCitrine mice were crossed with Oxytocin-Ires-Cre mice generating Oxt-hM4Di mice which express inhibitory DREAAD specifically in OXT neurons. (I) Experimental paradigm: Oxt-hM4Di mice were isolated between P28 and P35 and CNO was dissolved in the drinking water. After isolation mice were subjected whole-cell patch-clamp recordings. (J) Left: experimental paradigm, VTA pDA neurons were subjected at multiple depolarizing current steps. Right: example traces from 250 pA depolarizing current injection. (K) Number of APs across increasing depolarizing current steps (Three-way RM ANOVA followed by uncorrected Fisher’s LSD post doc analysis (see Figure 3—source data 1 for details), Current step main effect F_{(1.962, 98.08)}=114.8, p<0.0001, House condition main effect F_{(1, 50)}=3.456, p=0.0689, CNO treatment main effect F_{(1, 50)}=5.826, p=0.0195, Current step × House condition F_{(6, 300)}=1.866, p=0.0865, Current step × CNO treatment F_{(6, 300)}=3.806, p=0.0011, House condition × CNO treatment F_{(1, 50)}=2.293, p=0.1363, Current step × House condition × CNO treatment F_{(6, 300)}=3.459, p=0.0026). (L) Resting membrane potential of recorded cells (Two-way ANOVA, CNO treatment main effect F_{(1, 50)}=0.8745, p=0.3542, House condition main effect F_{(1, 50)}=1.956, p=0.1681, CNO treatment × House condition F_{(1, 50)}=3.531, p=0.0661, Grouped n=12, Grouped +CNO n=12, Isolated n=15, Isolated+CNO n=15 from 3 to 4 mice each group). (M) Experimental design. Oxt-hM4Di mice were isolated or kept grouped from P28 to P35. CNO was dissolved in drinking water and after isolation mice underwent to free direct social interaction task. (N) Social interaction time (Two-way ANOVA followed by Tukey’s multiple comparisons test, CNO treatment main effect F_{(1, 28)}=7.088, p=0.0127, House condition main effect F_{(1, 28)}=9.940, p=0.0038 CNO treatment × House condition F_{(1 ,28)}=4.334, p=0.0466, n=8 each group). Data are represented as mean ± SEM.

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CNO validation.

(A) Experimental design: WT mice were isolated between P28 and P35 or kept in group. After isolation, mice were perfused and brain slices were immunostained with cFOS, OXT, and AVP antibody. (B) PVN cFOS+ cells (absolute value, Unpaired sample t-test, t₍₂₂₎=2.723, p=0.0124). (C) PVN OXT + cells (absolute value, Mann-Whitney sample U-test=35.5, p=0.0344). (D) Representative confocal images of PNV stained with cFOS, OXT, and AVP. (E) PVN AVP+ cells (normalized on DAPI, Mann-Whitney U-test=45, p=0.1277). (F) PVN AVP+ cells (absolute value, Unpaired sample t-test, t₍₂₂₎=1.569, p=0.131). (G) PVN AVP+/cFOS+ cells (Unpaired sample t-test, t₍₂₂₎=1.811, p=0.0838). (H) PVN OXT+/AVP+ cells (Mann-Whitney U-test=61, p=0.7859). (I) Representative confocal images of PVN for OXT-hM4Di expression. (J) Left: experimental paradigm, OXT neurons from Oxt-hM4Di mice were subjected at multiple depolarizing current steps in absence or presence of CNO 10 µM in the recording chamber. Right: example traces from 30 pA depolarizing current injection. (K) Number of action potentials (APs) across increasing depolarizing current steps (Two-way RM ANOVA followed by uncorrected Fisher’s LSD post hoc analysis, CNO main effect F(1, 8)=90.15, p<0.001, Current step main effect F(5, 40)=11.99, p<0.001, CNO × Current step F(5, 40)=7.556, p<0.001). (L) Resting membrane potential of recorded cells (Unpaired samples t-test, t₍₈₎=1.502, p=0.1715, n=5 from one mouse). Data are represented as mean ± SEM.

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Long-lasting effects of adolescence acute social isolation.

(A) Experimental design: WT mice were isolated between P28 and P35 and regrouped until P60 or always kept in group. Mice (different cohorts) were subjected to different behavioral tasks. (B) Free-interaction task paradigm. (C) Time exploring social stimulus (Unpaired-samples t-test, t₍₁₈₎=3.004, p=0.0076, n=10 mice each group). (D) Time exploring object (Unpaired-samples t-test, t₍₁₈₎=3.3717, p=0.7144, n=10 mice each group). (E) Habituation task paradigm. (F) Interaction time on Day 1 (Unpaired-samples t-test, t₍₁₄₎=3.553, p=0.0032, n=8 mice each group). (G) Interaction time across 4 days (Two-way RM ANOVA, Day × House condition F_{(3, 42)}=2.607, p=0.064, Day main effect F_{(2.840, 39.76)}=32.66, p<0.001, House condition main effect F_{(1, 14)}=8.240, p=0.0123, n=8 mice each group). (H) Interaction time during Day 1 and Day 4 for Grouped mice (Paired samples t-test, t₍₇₎=3.923, p=0.0057). (I) Interaction time during Day 1 and Day 4 for Regrouped mice (Paired samples t-test, t₍₇₎=7.621, p<0.001). (J) Experimental design. Oxt-hM4Di mice were isolated from P28 to P35 and regrouped until P60 or kept always grouped. CNO was dissolved in drinking water and administered during social isolation. At P60 mice underwent to free direct social interaction task. (K) Social interaction time (One-way ANOVA followed by Tukey’s multiple comparisons test, F_{(2, 19)}=9.430, p=0.0014, Grouped (CNO) n=7, Regrouped n=7, Regrouped (CNO) n=8). Data are represented as mean ± SEM.

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Long-lasting effects of acute social isolation in adolescence.

(A) Experimental design: WT mice were isolated between P28 and P35 and regrouped until P60 or always kept in group. (B) Three-chamber task experimental paradigm. (C) Time in chamber during social preference phase (One-way RM-ANOVA followed by Tukey’s multiple comparisons test. Grouped: chamber main effect F_{(1.684, 18.52)}=111.4, p<0.001. Regrouped: chamber main effect F_{(1.390, 5.30)}=79.34, p<0.001, n=12 mice each group). (D) Interaction time with object or social stimulus 1 (### Grouped: Paired sample t-test t₍₉₎=4.941, p<0.001; ## Regrouped: Paired sample t-test t₍₉₎=4.082, p=0.0027. Two-way RM-ANOVA, Target main effect F_{(1, 22)}=0.3602, p=0.5545; House condition main effect F_{(1, 22)}=0.8850, p=0.3571; Target × House condition F_{(1, 22)}=0.3602, p=0.5545, n=12 mice each group). (E) Preference index calculated as object interaction time/(object+stimulus 1) or stimulus 1 interaction time/(object+stimulus 1) (One-sample t-test against chance level 0.5; ### Grouped t₍₁₁₎=6.611, p<0.001; ### Regrouped t₍₁₁₎=5.698, p<0.001). (F) Time in chamber during social novelty phase (One-way RM-ANOVA followed by Tukey’s multiple comparisons test. Grouped: chamber main effect F_{(1.416, 15,57)}=16.18, p<0.001. Regrouped: chamber main effect F_{(1.768, 19.450)}=15.61, p<0.001, n=12 mice each group). (G) Interaction time with stimulus 1 (familiar) and stimulus 2 (unfamiliar) (### Grouped: Paired sample t-test t₍₉₎=4.736, p<0.001; ### Regrouped: Paired sample t-test t₍₉₎=4.939, p<0.001. Two-way RM-ANOVA, Target main effect F_{(1, 22)}=25.71, p<0.001; House condition main effect F_{(1, 22)}=2.949, p=0.1; Target × House condition F_{(1, 22)}=1.927, p=0.1790, n=12 mice each group). (H) Preference index calculated as stimulus 1 interaction time/(stimulus 1+stimulus 2) or stimulus 2 interaction time/(stimulus 1+stimulus 2) (One-sample t-test against chance level 0.5; ### Grouped t₍₁₁₎=4.694, p<0.001; ## Regrouped t₍₁₁₎=3.480, p=0.0051). (I) Distance moved during the three-chamber (Social preference: Mann-Withney U-test=30, p=0.0145. Social novelty: Unpaired samples t-test t₍₁₈₎=2.260, p=0.0364, n=12 mice each group). Data are represented as mean ± SEM.

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Regrouping after adolescence social isolation restore PVN and VTA excitability.

(A) Experimental design: WT mice were isolated between P28 and P35 and regrouped until P60 or always kept in group. CTB-488 was injected at p45-50. (B) Left: experimental paradigm, PVN neurons were subjected at multiple depolarizing current steps. Right: example traces from 250 pA depolarizing current injection. (C) Number of action potentials (APs) across increasing depolarizing current steps (Two-way RM ANOVA, Current step × House condition F_{(6, 132)}=0.2047, p=0.9748, current step main effect F_{(2.764, 60.82)}=39.55, p<0.001, House condition main effect F_{(1, 22)}=0.006080, p=0.9386). (D) Resting membrane potential of recorded cells (Unpaired samples t-test, t₍₂₂₎=0.4549, p=0.6536. Grouped n=11, Isolated n=13 from three mice each group). (E) Left: experimental paradigm, VTA pDA neurons were subjected at multiple depolarizing current steps. Right: example traces from 250 pA depolarizing current injection. (F) Number of APs across increasing depolarizing current steps (Two-way RM ANOVA, Current step × House condition F_{(6, 126)}=0.1048, p=0.9957, Current step main effect F_{(1.983, 41.64)}=50.14, p<0.001, House condition main effect F_{(1, 21)}=0.05862, p=0.8110). (G) Resting membrane potential of recorded cells (Unpaired samples t-test, t₍₂₁₎=3.538, p=0.0019. Grouped n=11, Isolated n=12 from three mice each group). Data are represented as mean ± SEM.

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Adolescence acute social isolation induces synaptic scaling in adulthood mice.

(**A, B, G**) Experimental paradigm. WT mice were isolated between P28 and P35. Then mice were regrouped, injected with 488-CTB in the mPFC (B) or NAc (G) between P45–P50 and at P60 were subjected at whole-cell patch-clamp recording. (C) Example traces of isolated AMPA and NMDA currents recorded at +40 mV. (D) AMPA-NMDA ratio of VTA-DA:mPFC projecting neurons (Unpaired samples t-test, t₍₈₎=0.07544, p=0.9417, Grouped n=5, Isolated n=5 from two mice each group). (E) Example traces of Isolated AMPA current recorded at +40, 0, and –60 mV. (F) Rectification index (RI) of VTA-pDA:mPFC projecting neurons (Unpaired samples t-test, t₍₁₂₎=3.545, p=0.004, Grouped n=8, Isolated n=8 from two mice each group). (H) Example traces of isolated AMPA and NMDA currents recorded at +40 mV. (I) AMPA-NMDA ratio of VTA-pDA:NAc projecting neurons (Mann-Whitney U-test=7, p=0.1602, Grouped n=5, Isolated n=6 from two mice each group). (J) Example traces of Isolated AMPA current recorded at +40, 0, and –60 mV. (K) RI of VTA-pDA:NAc projecting neurons (Unpaired samples t-test, t₍₁₀₎=0.3720, p=0.7176, Grouped n=5, Isolated n=7 from two mice each group). (L). Experimental paradigm: Oxt-hM4Di mice were isolated between P28 and P35 or kept always grouped and CNO was dissolved in drinking water. Then mice were regrouped, injected with CTB at P45–P50, and kept group-housed until P60 when were subjected at whole-cell patch-clamp recording. (M) Example traces of isolated AMPA current recorded at +40, 0, and –60 mV. (N) RI of VTA-pDA:mPFC projecting neurons (Two-way ANOVA followed by Tukey’s multiple comparisons test, CNO treatment main effect F_{(1, 23)}=11.19, p=0.0028, House condition main effect F_{(1, 23)}=5.459, p=0.0285, CNO treatment × Hous condition F_{(1, 23)}=11.22, p=0.028, Grouped n=8, Grouped CNO n=7, Regrouped n=7, Regrouped CNO n=5 from two mice each group). Data are represented as mean ± SEM.

Figure 5—source data 1 Figure 5 - raw data and statistical output.: https://cdn.elifesciences.org/articles/73421/elife-73421-fig5-data1-v2.xlsx
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Figure 5—figure supplement 1

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Adolescence acute social isolation induces synaptic scaling in VTA pDA-mPFC projecting neruons.

(**A, B, G**) Experimental paradigm. WT mice were injected with 488-CTB in the mPFC (B) or NAc (G) at P21 and isolated between P28 and P35. Then mice were subjected at whole-cell patch-clamp recording. (C) Example traces of isolated AMPA and NMDA currents recorded at +40 mV. (D) AMPA-NMDA ratio of VTA-pDA:mPFC projecting neurons (Mann-Whitney U-test=56, p=0.0945, Grouped n=13, Isolated n=14 from three mice each group). (E) Example traces of Isolated AMPA current recorded at +40, 0, and –60 mV. (F) Rectification index (RI) of VTA-pDA:mPFC projecting neurons (Unpaired samples t-test, t(₂₀)=3.964, p<0.001, Grouped n=8, Isolated n=14 from three mice each group). (H) Example traces of isolated AMPA and NMDA currents recorded at +40 mV. (I) AMPA-NMDA ratio of VTA-pDA:NAc projecting neurons (Mann-Whitney U-test=29, p=0.4252, Grouped n=7, Isolated n=11 from three mice each group). (J) Example traces of Isolated AMPA current recorded at +40, 0, and –60 mV. (K) RI of VTA-pDA:NAc projecting neurons (Unpaired samples t-test, t(₁₂)=0.1811, p=0.0953, Grouped n=6, Isolated n=8 from three mice each group). (L) Example traces of sIPSCs recorded from VTA-pDA neurons after adolescence social isolation. (M) sIPSCs frequency (Mann-Whitney U-test=29, p=0.5414, Grouped n=9, Isolated n=8 from two mice each group). (M) sIPSCs amplitude (Mann-Whitney U-test=33, p=0.8148, Grouped n=9, Isolated n=8 from two mice each group). (M) sIPSCs rise (Mann-Whitney U-test=34, p=0.8884, Grouped n=9, Isolated n=8 from two mice each group). (M) sIPSCs decay (Unpaired samples t-test, t₍₁₅₎=1.994, p=0.0647, Grouped n=9, Isolated n=8 from two mice each group). Data are represented as mean ± SEM. sIPSC, spontaneous inhibitory postsynaptic current.

Figure 5—figure supplement 1—source data 1 Figure 5—figure supplement 1 - raw data and statistical output.: https://cdn.elifesciences.org/articles/73421/elife-73421-fig5-figsupp1-data1-v2.xlsx
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Figure 5—figure supplement 2

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Immunohistochemical validation of VTA-DA neurons.

(A) Example of VTA horizontal slice. (B) Confocal images of post hoc immunohistochemical validation of VTA neurons filled with GFP (Alexa Fluor 488) and stained with TH-antibody. The white arrow indicates a neuron with capacitance of 32pF which is positive for TH staining (DA neuron). The pink arrow indicates a neuron with capacitance of 22pF which is negative for TH staining (non-DA neuron).

Figure 6

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CP-AMPARs are responsible of increased social interaction during adulthood.

(A) Experimental paradigm: WT mice were isolated between P28 and P35. Then mice were regrouped until P53 and canulated over the VTA. (B) Mice underwent to direct free interaction task after infusion of CP-AMPARs antagonist NASPM. (C) Social interaction time (Two-way ANOVA followed by Tukey’s multiple comparisons test, NASPM treatment main effect F _{(1, 24)}=13.78, p=0.0011, House condition main effect F_{(1, 24)}=2.626, p=0.1182, NASPM × House condition F_{(1, 24)}=6.099, p=0.021, Grouped Veh n=6, Grouped NASPM n=7, Regrouped Veh n=8, Regropued NASPM n=7). Data are represented as mean ± SEM.

Figure 6—source data 1 Figure 6 - raw data and statistical output.: https://cdn.elifesciences.org/articles/73421/elife-73421-fig6-data1-v2.xlsx
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Figure 7

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Graphical summary.

One week of social isolation during adolescence in mice increased social interaction at the expense of social habituation and social novelty preference. Behavioral changes were accompanied by the acute hyperexcitability of PVN-VTA projecting neurons, VTA putative dopamine (pDA) neuron, and long-lasting expression of GluA2-lacking AMPARs at excitatory inputs onto pDA neurons that project to the prefrontal cortex (PFC).

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Chemical compound, drug	Choline chloride	Sigma-Aldrich	C7527
Chemical compound, drug	D-(+)-Glucose	Sigma-Aldrich	G8270
Chemical compound, drug	D(+)-Saccharose	Roth	4621.1
Chemical compound, drug	NaHCO₃	Sigma-Aldrich	S5761
Chemical compound, drug	MgCl₂	Sigma-Aldrich	63068
Chemical compound, drug	Ascorbic acid	Roth	6288.1
Chemical compound, drug	Sodium pyruvate	Roth	8793.1
Chemical compound, drug	KCl	Sigma-Aldrich	60130
Chemical compound, drug	NaH₂PO₄	Sigma-Aldrich	S0751
Chemical compound, drug	CaCl2	Sigma-Aldrich	21097
Chemical compound, drug	NaCl	Sigma-Aldrich	31434
Chemical compound, drug	K-Gluconate	Sigma-Aldrich	G4500
Chemical compound, drug	EGTA	Sigma-Aldrich	E4378
Chemical compound, drug	HEPES	Sigma-Aldrich	H3375
Chemical compound, drug	Na2ATP	Sigma-Aldrich	A2383
Chemical compound, drug	Na3GTP	Sigma-Aldrich	G8877
Chemical compound, drug	Creatine-phosphate	Sigma-Aldrich	P7936
Chemical compound, drug	CsCl	Sigma-Aldrich	20966
Chemical compound, drug	Sodium creatine phosphate	Sigma-Aldrich	27920
Chemical compound, drug	Spermine	Sigma-Aldrich	S4264
Chemical compound, drug	Lidocaine N-ethyl bromide (QX-314)	Sigma-Aldrich	L5783
Chemical compound, drug	NaOH	Sigma-Aldrich	S5881
Chemical compound, drug	Clozapine N-oxide	Enzo Life	BML-NS-105-0025	5 mg/200 ml
Chemical compound, drug	Saccharin	Sigma-Aldrich	240931
Chemical compound, drug	NASPM	Tocris	2766
Chemical compound, drug	NGS	Sigma-Aldrich	AB7481
Chemical compound, drug	Triton X-100	Sigma-Aldrich	X100
Chemical compound, drug	Kynurenic acid	Sigma-Aldrich	K3375
Chemical compound, drug	Picrotoxin	Tocris	1128
Chemical compound, drug	D-APV	Tocris	106
Antibody	Oxytocin antibody (guinea pig polyclonal)	Synaptic Systems	#408004	(1:500)
Antibody	cFOS antibody (rabbit monoclonal)	Synaptic Systems	#226008	(1:5000)
Antibody	Neurophysin 2/NP-AVP antibody (mouse monoclonal)	Merck Millipore	#MABN856	(1:250)
Antibody	HA-tag (6E2) antibody (mouse monoclonal)	Cell Signaling Technology	#2367	(1:200)
Antibody	Oxytocin antibody (rabbit polyclonal)	Merck Millipore	#AB911	(1:500)
Antibody	Tyrosine Hydroxylase antibody (mouse monoclonal)	Merck Millipore	#MAB318	(1:500)
Antibody	Goat anti-mouse Alexa Fluor 555 (polyclonal)	Thermo Fisher Scientific	A21422	(1:500)
Antibody	Goat anti-rabbit Alexa Fluor 488 (polyclonal)	Thermo Fisher Scientific	A11008	(1:500)
Antibody	Goat anti-mouse Alexa Fluor 594 (polyclonal)	Thermo Fisher Scientific	A11005	(1:500)
Antibody	Goat anti-mouse Alexa Fluor 647 (polyclonal)	Thermo Fisher Scientific	A21235	(1:500)
Antibody	Goat anti-guinea pig Alexa Fluor 555 (polyclonal)	Thermo Fisher Scientific	A21435	(1:500)
Other	Mounting medium with DAPI	Abcam	ab104139	Mounting media used for immunofluorescence
Other	Alexa Fluor-488 hydrazydes	Thermo Fisher Scientific	#A10436	10 mM—GFP tracer used for DA neurons validation (Figure 5—figure supplement 2)
Other	Cholera Toxin Subunit B, Alexa Fluor 488 Conjugate	Thermo Fisher Scientific	#C34775	Retrograde tracer used in Figures 3 and 5 and Figure 5—figure supplement 1