SHANK3 Downregulation in the Ventral Tegmental Area Accelerates the Extinction of Contextual Associations Induced by Juvenile Non-familiar Conspecific Interaction

Abstract

Haploinsufficiency of the SHANK3 gene, encoding for a scaffolding protein located in the postsynaptic density of glutamatergic synapse, has been linked to forms of autism spectrum disorders (ASDs). It has been shown that SHANK3 controls the maturation of social reward circuits in the ventral tegmental area (VTA). Whether the impairments in associative learning observed in ASD relate to SHANK3 insufficiency restricted to the reward system is still an open question. Here, we first characterize a social-conditioned place preference (CPP) paradigm based on the direct and free interaction with a juvenile and non-familiar conspecific. In both group- and single-housed C57Bl6/j late adolescence male mice, this CPP protocol promotes the formation of social-induced contextual associations that undergo extinction. Interestingly, the downregulation of Shank3 expression in the VTA altered the habituation to a non-familiar conspecific during conditioning and accelerated the extinction of social-induced conditioned responses. Thus, inspired by the literature on drugs of abuse-induced contextual learning, we propose that acquisition and extinction of CPP might be used as behavioral assays to assess social-induced contextual association and "social-seeking" dysfunctions in animal models of psychiatric disorders.

Keywords: Shank3; VTA; conditioned place preference; isolation; social novelty.

Figure 1

Non-familiar conspecific-induced conditioned place preference (CPP): a variation of social-induced CPP to assess the reinforcing properties of social interaction. Schematic 3D representation of the CPP procedure and apparatus. The protocol consisted of: (A) 15 min pre-TEST, (B) 15 min post-TEST and (C) conditioning phase (30 min per day for four consecutive days). (D) Schematic 3D representation of conditioning phase for non-familiar vs. familiar social stimuli protocol, during which the experimental mouse interacted with a non-familiar social stimulus in one chamber and with a familiar (cage-mate) stimulus in the other one.

Figure 2

Acquisition of non-familiar conspecific-induced CPP does not require single-housing. (A) Left: schematic representation of CPP time-course. Right: schematic representation of a single CPP conditioning alternation. (B) Representative apparatus occupancy heat-maps for single-housed mice during pre-TEST and (C) post-TEST. (D) Preference index calculated at pre- and post-TEST for single-housed mice (N = 12; paired t-test: t₍₁₁₎ = 2.934; P = 0.0136). (E) Time spent in non-social and non-familiar conspecific-paired chambers for single-housed mice during pre- and post-TEST (N = 12; two-way RM ANOVA: time × chamber interaction: F_(1,11) = 4.021, P = 0.0702; main effect chamber: F_(1,11) = 9.85, P = 0.0094; main effect time: F_(1,11) = 6.363, P = 0.0284; followed by Bonferroni’s multiple comparisons test). (F) Representative apparatus occupancy heat-maps for group-housed animals at pre-TEST and (G) post-TEST. (H) Preference index calculated at pre- and post-TEST for group-housed mice (N = 11; paired t-test: t₍₁₀₎ = 2.366; P = 0.0396). (I) Time spent in the non-social and non-familiar conspecific-associated chambers for group-housed mice during pre- and post-TEST (N = 11; two-way RM ANOVA: time × chamber interaction: F_(1,10) = 5.115, P = 0.0472; main effect chamber: F_(1,10) = 3.019, P = 0.1129; main effect time: F_(1,10) = 0.2936, P = 0.5998; followed by Bonferroni’s multiple comparisons test). (J) Representative apparatus occupancy heat-maps of group-housed mice subject to CPP contingency break at pre-TEST and (K) post-TEST. (L) Preference index of group-housed mice subjected to CPP contingency break calculated at pre- and post-TEST (N = 9; paired t-test: t₍₈₎ = 0.6537; P = 0.8080). (M) Time spent in the two chambers of the apparatus for group-housed mice subjected to CPP contingency break during pre- and post-TEST (the chambers were assigned a non-social and non-familiar property for analyses purposes; N = 9; two-way RM ANOVA: time × chamber interaction: F_(1,8) = 0.1864, P = 0.6773; main effect chamber: F_(1,8) = 0.1024, P = 0.7572; main effect time: F_(1,8) = 2.401, P = 0.1599; followed by Bonferroni’s multiple comparisons test). (N) Learning index calculated for group-housed mice subjected to either a contingency break (N = 9) or a paired protocol CPP (N = 11; unpaired t-test with Welch’s correction: t_(14.4) = 2.175; P = 0.0467). N indicates number of mice. Abbreviations: US, unconditioned stimulus.

Figure 3

The interaction with a juvenile non-familiar conspecific is reinforcing. (A) Left: schematic representation of CPP time-course. Right: schematic representation of a single CPP conditioning alternation between a familiar session and a non-familiar conspecific session. (B) Representative apparatus occupancy heat-maps during pre-TEST and (C) post-TEST for group-housed mice subjected to this protocol. (D) Preference index calculated at pre- and post-TEST for group-housed mice subjected to this protocol (N = 15; paired t-test: t₍₁₄₎ = 2.389; P = 0.0315). (E) Time spent in familiar and non-familiar conspecific-paired chambers for group-housed mice subjected to this protocol during pre- and post-TEST (N = 15; two-way RM ANOVA: time × chamber interaction: F_(1,14) = 4.525, P = 0.0517; main effect chamber: F_(1,14) = 3.416, P = 0.0858; main effect time: F_(1,14) = 1.657, P = 0.2189). (F) Learning index calculated for group-housed mice subjected to either the protocol reported in Figure 2A (N = 11) or the protocol reported here in (A) (N = 15; Mann-Whitney test; P = 0.5830). (G) Left: schematic representation of CPP time-course. Right: schematic representation of a single CPP conditioning alternation between a familiar session and a non-familiar conspecific session. (H) Representative apparatus occupancy heat-maps during pre-TEST and (I) post-TEST for group-housed mice subjected to this protocol. (J) Preference index calculated at pre- and post-TEST for group-housed mice subjected to this protocol (N = 12; paired t-test: t₍₁₁₎ = 0.0358; P = 0.9721). (K) Time spent in familiar and non-familiar conspecific-paired chambers for group-housed mice subjected to this protocol during pre- and post-TEST (N = 12; two-way RM ANOVA: time × chamber interaction: F_(1,11) = 0.0157, P = 0.9026; main effect chamber: F_(1,11) = 0.0927, P = 0.7665; main effect time: F_(1,11) = 11.1, P = 0.0067). N indicates number of mice. Abbreviations: US, unconditioned stimulus.

Figure 4

Extinction of non-familiar conspecific-induced CPP is affected by housing condition. (A) Schematic representation of CPP acquisition and extinction. (B) Time spent in non-social and non-familiar conspecific-paired chambers across extinction sessions for group-housed mice subjected to CPP (N = 10; two-way RM ANOVA: time × chamber interaction: F_(5,45) = 0.4486, P = 0.8120; main effect chamber: F_(1,9) = 8.484, P = 0.0172; main effect time: F_(5,45) = 1.25, P = 0.3021; followed by Bonferroni’s multiple comparisons test). (C) Mean visit time spent in non-social and non-familiar conspecific-paired chambers across extinction sessions for group-housed mice subjected to CPP (N = 10; two-way RM ANOVA: time × chamber interaction: F_(5,45) = 2.642, P = 0.0354; main effect chamber: F_(1,9) = 9.002, P = 0.0149; main effect time: F_(5,45) = 1.589, P = 0.1827; followed by Bonferroni’s multiple comparisons test). (D) Frequency to enter in non-social and non-familiar conspecific-paired chambers across extinction sessions for group-housed mice subjected to CPP (N = 10; two-way RM ANOVA: time × chamber interaction: F_(5,45) = 1.215, P = 0.3175; main effect chamber: F_(1,9) = 2.086, P = 0.1826; main effect time: F_(5,45) = 1.361, P = 0.2568). (E) Time spent in non-social and non-familiar conspecific-paired chambers across extinction sessions for single-housed mice (N = 8) subjected to a paired protocol (two-way RM ANOVA: time × chamber interaction: F_(5,35) = 1.037, P = 0.4113; main effect chamber: F_(1,7) = 78.38, P < 0.0001; main effect time: F_(5,35) = 0.997, P = 0.4339; followed by Bonferroni’s multiple comparisons test). (F) Mean visit time spent in non-social and non-familiar conspecific-paired chambers across extinction sessions for single-housed mice (N = 8) subjected to a paired protocol (two-way RM ANOVA: time × chamber interaction: F_(5,35) = 0.6993, P = 0.6268; main effect chamber: F_(1,7) = 11.16, P = 0.0086; main effect time: F_(5,35) = 9.897, P < 0.0001; followed by Bonferroni’s multiple comparisons test). (G) Frequency to enter in non-social and non-familiar conspecific-paired chambers across extinction sessions for single-housed mice (N = 8) subjected to a paired protocol (two-way RM ANOVA: time × chamber interaction: F_(5,35) = 0.6539, P = 0.6600; main effect chamber: F_(1,7) = 5.746, P = 0.0401; main effect time: F_(5,35) = 18.08, P < 0.0001; followed by Bonferroni’s multiple comparisons test). (H) Time spent in non-social or non-familiar conspecific-paired chamber at extinction session 6 for single-housed (N = 8) and group-housed (N = 10) mice (two-way ANOVA: time × group: F_{(1, 32)} = 3.316, P = 0.0780; main effect group: F_{(1, 32)} = 1.149, P = 0.2917; main effect time: F_{(1, 32)} = 23.09, P < 0.0001; followed by Bonferroni’s multiple comparisons test). N indicates number of mice. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Ventral tegmental area (VTA) SHANK3 downregulation alters the exploration of non-familiar conspecific without affecting CPP. (A) Experimental time course of AAV-scrShank3/AAV-shShank3 VTA infection, CPP acquisition, extinction and post hoc histological verification of viral infection and effect. (B) Histological validation of VTA infection (scale bar: left panel: 150 μm, right panel: 50 μm; white arrows indicate tyrosine hydroxylase (TH)-shShank3 colocalization). (C) Relative expression of Shank3a after infection of AAV-scrShank3/AAV-shShank3 in the VTA (unpaired t-test: t₍₁₀₎ = 2.972; P = 0.0140). (D) Ratio between Shank3a and TH expression after AAV-scrShank3/AAV-shShank3 infection in the VTA (Mann-Whitney test: P = 0.0152). (E) Body and nose-to-nose contact time during conditioning days for group-housed VTA-scrShank3 mice (N = 10) and VTA-shShank3 (N = 9) mice (two-way ANOVA: time × group interaction: F_(3,51) = 0.4552, P = 0.7148; group main effect: F_(1,17) = 4.894, P = 0.0409; main effect time: F_(3,51) = 4.363, P = 0.0082; followed by Bonferroni’s multiple comparisons test). (F) Distance moved in the arena during conditioning days for group-housed VTA-scrShank3 mice (N = 10) and VTA-shShank3 (N = 9) mice (two-way ANOVA: time × group interaction: F_(3,51) = 0.2209, P = 0.8814; group main effect: F_(1,17) = 3.388, P = 0.0832; main effect time: F_(3,51) = 3.707, P = 0.0173). (G) Representative occupancy heat-maps for group-housed VTA-scrShank3 mice at pre-TEST and (H) post-TEST. (I) Preference index calculated at pre- and post-TEST for group-housed VTA-scrShank3 mice (N = 10; paired t-test: t₍₉₎ = 4.827; P = 0.0009). (J) Time spent in non-social and non-familiar conspecific-paired chamber for group-housed VTA-scrShank3 mice during pre- and post-TEST (N = 10; two-way RM ANOVA: time × chamber interaction: F_(1,9) = 25.35, P = 0.0007; main effect chamber: F_(1,9) = 1.918, P = 0.1994; main effect time: F_(1,9) = 0.4628, P = 0.5134; followed by Bonferroni’s multiple comparisons test). (K) Representative occupancy heat-maps for group-housed VTA-shShank3 mice at pre-TEST and (L) post-TEST. (M) Preference index calculated at pre- and post-TEST for group-housed VTA-shShank3 mice (N = 9; paired t-test: t₍₈₎ = 2.103; P = 0.0687). (N) Time spent in non-social and non-familiar conspecific-paired chamber for group-housed VTA-shShank3 mice during pre- and post-TEST (N = 9; two-way RM ANOVA: time × chamber interaction: F_(1,8) = 4.943, P = 0.0329; main effect chamber: F_(1,8) = 2.651, P = 0.1027; main effect time: F_(1,8) = 0.119, P = 0.7322; followed by Bonferroni’s multiple comparisons test). (O) Learning index calculated for VTA-scrShank3 (N = 10) and VTA-shShank3 (N = 9; unpaired t-test: t₍₁₇₎ = 0.3468; P = 0.7330). N indicates number of mice. Abbreviations: US, unconditioned stimulus; fr, fasciculus retroflexus; SNc, substantia nigra pars compacta; RN, red nucleus. *p < 0.05, ***p < 0.001.

Figure 6

VTA SHANK3 downregulation alters the extinction of non-familiar conspecific-induced CPP. (A) Experimental time course of AAV-scrShank3/AAV-shShank3 VTA infection, CPP acquisition and extinction. (B) Time spent in non-social and non-familiar conspecific-paired chamber for group-housed VTA-scrShank3 mice across extinction sessions (N = 10; two-way RM ANOVA: time × chamber interaction: F_(4,36) = 4.624, P = 0.0041; main effect chamber: F_(1,9) = 18.32, P = 0.0020; main effect time: F_(4,36) = 1.126, P = 0.3595; followed by Bonferroni’s multiple comparisons test). (C) Mean visit time spent in non-social and non-familiar conspecific-paired chambers across extinction sessions for group-housed VTA-scrShank3 mice subjected to CPP (N = 10; two-way RM ANOVA: time × chamber interaction: F_(4,36) = 2.097, P = 0.1043; main effect chamber: F_(1,9) = 23.93, P = 0.0012; main effect time: F_(4,36) = 0.889, P = 0.4817; followed by Bonferroni’s multiple comparisons test). (D) Frequency to enter in non-social and non-familiar conspecific-paired chambers across extinction sessions for group-housed VTA-scrShank3 mice subjected to CPP (N = 10; two-way RM ANOVA: time × chamber interaction: F_(4,36) = 1.443, P = 0.2399; main effect chamber: F_(1,9) = 0.3101, P = 0.5912; main effect time: F_(4,36) = 0.5925, P = 0.6703). (E) Time spent in non-social and non-familiar conspecific-paired chambers for group-housed VTA-shShank3 mice during extinction (N = 9; two-way ANOVA: time × chamber interaction: F_(4,32) = 1.453, P = 0.2394; main effect chamber: F_(1,8) = 6.002, P = 0.0399; main effect time: F_(4,32) = 4.226, P = 0.0074; followed by Bonferroni’s multiple comparisons test). (F) Mean visit time spent in non-social and non-familiar conspecific-paired chambers across extinction sessions for group-housed VTA-shShank3 mice subjected to CPP (N = 9; two-way RM ANOVA: time × chamber interaction: F_(4,32) = 1.168, P = 0.3433; main effect chamber: F_(1,8) = 4.496, P = 0.0671; main effect time: F_(4,32) = 8.131, P = 0.0001; followed by Bonferroni’s multiple comparisons test). (G) Frequency to enter in non-social and non-familiar conspecific-paired chambers across extinction sessions for group-housed VTA-shShank3 mice subjected to CPP (N = 9; two-way RM ANOVA: time × chamber interaction: F_(4,32) = 1.708, P = 0.1725; main effect chamber: F_(1,8) = 0.6025, P = 0.4600; main effect time: F_(4,32) = 9.554, P < 0.0001). (H) Preference index calculated during pre-TEST, post-TEST and extinction sessions for group-housed VTA-scrShank3 mice (N = 10) and VTA-shShank3 (N = 9) mice (two-way ANOVA: time × group interaction: F_(6,102) = 2.118, P = 0.0575; group main effect: F_(1,17) = 5.436, P = 0.0323; main effect time: F_(6,102) = 4.953, P = 0.0002; followed by Bonferroni’s multiple comparisons test). (I) Distance moved in the arena during pre-TEST, post-TEST and extinction sessions for group-housed VTA-scrShank3 mice (N = 10) and VTA-shShank3 (N = 9) mice (two-way ANOVA: time × group interaction: F_(6,102) = 3.056, P = 0.0086; group main effect: F_(1,17) = 4.211, P = 0.0550; main effect time: F_(6,102) = 7.497, P < 0.0001; followed by Bonferroni’s multiple comparisons test). N indicates number of mice. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.