Hyperconnectivity of prefrontal cortex to amygdala projections in a mouse model of macrocephaly/autism syndrome - PubMed (original) (raw)
Hyperconnectivity of prefrontal cortex to amygdala projections in a mouse model of macrocephaly/autism syndrome
Wen-Chin Huang et al. Nat Commun. 2016.
Abstract
Multiple autism risk genes converge on the regulation of mTOR signalling, which is a key effector of neuronal growth and connectivity. We show that mTOR signalling is dysregulated during early postnatal development in the cerebral cortex of germ-line heterozygous Pten mutant mice (Pten+/-), which model macrocephaly/autism syndrome. The basolateral amygdala (BLA) receives input from subcortical-projecting neurons in the medial prefrontal cortex (mPFC). Analysis of mPFC to BLA axonal projections reveals that Pten+/- mice exhibit increased axonal branching and connectivity, which is accompanied by increased activity in the BLA in response to social stimuli and social behavioural deficits. The latter two phenotypes can be suppressed by pharmacological inhibition of S6K1 during early postnatal life or by reducing the activity of mPFC-BLA circuitry in adulthood. These findings identify a mechanism of altered connectivity that has potential relevance to the pathophysiology of macrocephaly/autism syndrome and autism spectrum disorders featuring dysregulated mTOR signalling.
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Figures
Figure 1. Characterization of mTORC1 activity and neuronal growth in Pten mutant mice.
(a) Images showing phospho-S6 (p-S6), a readout for mTORC1 activity, immunostaining in the cerebral cortex of postnatal day 8 (P8) WT and Pten +/− mice. Phospho-S6 (red), DAPI (blue) and scale bar, 500 μm. Representative somatosensory cortex with p-S6 immunostaining in P4, P8, P14, P28, and adult WT and Pten +/− mice. Phospho-S6 (red), DAPI (blue), and scale bar, 50 μm. (b) Representative images of western blot for p-S6, total S6 and β-actin in WT and Pten +/− whole cerebral cortex at P0, P4, P8, P14, P28 and adulthood. (c) Quantification of p-S6 levels relative to total S6, expressed as fold WT, in the whole cerebral cortex of WT and Pten +/− mice at P0, P4, P8, P14, P28 and adult. Independent-sample t tests were used. *P<0.05. _N_=5 animals per genotype for each time point. (d) Representative images of fluorescent Nissl stain in somatosensory cortical layers II–VI of WT and Pten +/− mice at P14. Scale bar, 25 μm. (e) Quantification of cell soma size in somatosensory cortical layers II–VI of WT and Pten +/− mice at P14, expressed as per cent of WT. Independent-sample t tests were used. *P<0.05. _N_=6 animals in each genotype. (f) Representative images of reconstructed layer V neurons in the somatosensory cortex of P14 Etv1-CreERT2; Ai14 (Control) and Etv1-CreERT2; Ai14; Pten +/− (Pten +/−) mice. Scale bar, 50 μm. (g–k) Quantification of total number of branches (g), cell soma volume (h), and total (i), apical (j) and basal (k) dendrite length of layer V neurons in P14 control and Pten +/− mice. Independent-sample t tests were used. *P<0.05, and **P<0.01 and NS indicates no significant difference. _N_=25 neurons/6 animals per genotype. (l) Sholl analysis of somatosensory cortex layer V neurons in P14 control and Pten +/− mice. Two-way ANOVA with Bonferroni correction, F(50, 2,448)=2.174, P<0.0001 (interaction); *P<0.05, **P<0.01, and ***P<0.001. _N_=25 neurons/6 animals each genotype. All mice used in this figure were male.
Figure 2. Inhibition of S6K1 during development corrects cellular and behavioural deficits in Pten mutant mice.
(a) Diagram showing time line of PF-4708671 treatment and analysis of layer V cell soma size and social behaviour. (b) Representative images of western blot for p-S6, total S6 and β-actin in the cerebral cortex of P8 WT and Pten +/− mice receiving either vehicle or PF-4708671. Quantification of p-S6 levels relative to total S6, expressed as per cent of WT. Two-way ANOVA and Tukey post hoc tests were used. F(1,16)=4.488, _P_=0.05 (interaction), F(1,16)=102.2, P<0.001 (drug), F(1,16)=25.54, P<0.001 (genotype); ***P<0.001. _N_=5 animals per group. (c) Representative images of fluorescent Nissl staining in the layer V somatosensory cortex and quantification of layer V cell soma size in WT and Pten +/− mice receiving either vehicle or PF-4708671 from P4 to P14. Planned comparisons revealed a significant difference between WT and Pten +/− mice receiving vehicle, and a significant difference between vehicle and PF-4708671-treated Pten +/− mice, with two-way ANOVA showing main effects of drug and genotype. F(1,16)=2.541, _P_=0.131 (interaction), F(1,16)=10.96, P<0.01 (drug), F(1,16)=10.96, P<0.01 (genotype). *P<0.05. _N_=5 animals per group. Scale bar, 25 μm. (d) Per cent time WT and Pten +/− mice receiving either vehicle or PF-4708671 from P4 to P14 spent in each chamber during the three-chamber social approach test. Two-way ANOVA and Sidak's post hoc tests were used. F(3,82)=3.334, _P_=0.0234 (interaction), F(3,82)=0.2417, _P_=0.867 (genotype) and F(1,82)=28.87, P<0.001 (chamber time). *P<0.05. _N_=11 WT receiving vehicle, 13 Pten +/− receiving vehicle, 11 WT receiving PF-4708671, and 10 Pten +/− receiving PF-4708671. All mice used in this figure were female.
Figure 3. Hyperconnectivity of mPFC to BLA circuit in Pten mutant mice is corrected by inhibition of S6K1 during development.
(a) Images showing tdTomato expression in coronal sections of mPFC and basal lateral amygdala (BLA), and sagittal section showing mPFC-originating axon terminals and synaptic boutons in the BLA. Scale bar, 500 μm (1), 100 μm (2), 50 μm (3) and 5 μm (right bottom image of (3)). (b,c) Representative images (b) and quantification (c) of reconstructed mPFC axon terminals in the BLA of adult WT and Pten +/− mice receiving either vehicle or PF-4708671 from postnatal day 4 (P4) to P14. Scale bar, 50 μm. Two-way ANOVA and Tukey post hoc tests were used, F(1,116)=4.548, P<0.05 (interaction), F(1,116)=6.89, P<0.01 (drug), F(1,116)=16.82, P<0.001 (genotype); **P<0.01, ***P<0.001. _N_=30 axons from 6 mice in each group. (d,e) Representative images (d) and quantification (e) of mPFC–BLA synaptic boutons in adult WT and Pten +/− mice receiving either vehicle or PF-4708671 from P4 to P14. Scale bar, 5 μm. Two-way ANOVA and Tukey post hoc tests were used, F(1,116)=4.094, P<0.05 (interaction), F(1,116)=13.30, P<0.001 (drug), F(1,116)=18.79, P<0.001 (genotype); ***P<0.001. _N_=30 axon fragments from 6 mice in each group. (f,g) Representative images (f) of western blot for CRMP2, Tau and β-actin in the cerebral cortex of P8 WT or Pten +/− mice. (g) Quantification of CRMP2 and Tau protein levels relative to β-actin, and total β-actin levels, expressed as percentage of WT. Independent-sample t tests were used. *P<0.05. _N_=5 animals per genotype. (h,i) Representative images (h) of western blot for CRMP2 and β-actin in the cerebral cortex of P8 WT or Pten +/− mice injected with either vehicle or PF-4708671 from P4 to P8. (i) Quantification of CRMP2 protein levels relative to β-actin. Planned comparisons revealed a significant difference between WT and Pten +/− mice receiving vehicle, and a significant difference between vehicle and PF-4708671-treated Pten +/− mice, with two-way ANOVA showing main effects on drug and genotype. F(1,16)=0.089, _P_=0.77 (interaction), F(1,16)=16.82, P<0.001 (drug), F(1,16)=14.24, P<0.01 (genotype). *P<0.05. _N_=5 animals in each group. All mice used in this figure were female.
Figure 4. mPFC and BLA hyperactivity in Pten mutant mice is corrected by inhibition of S6K1 during development.
(a) Diagram showing the workflow of social exposure and c-fos staining. Adult mice were exposed to a novel mouse for 20 min, perfused 2 h later, and then underwent c-fos staining to measure neuronal activity in the mPFC and BLA. (b–d) Representative images (b) and quantification of c-fos staining in the mPFC (c) and the basal lateral amygdala (BLA) (d) of control or social exposed WT and Pten +/− mice receiving either vehicle or PF-4708671 from postnatal day 4 (P4) to P14. Scale bar, 100 μm. _N_=4 animals in each group. (e) Graph showing social interaction time, reflected as time spent sniffing, in WT and Pten +/− mice receiving either vehicle or PF-4708671 from P4 to P14. _N_=4 animals in each group. (f,g) Quantification of c-fos activation index (number of c-fos+ neurons/time spend sniffing) in the mPFC (f) and BLA (g) of social exposed WT and Pten +/− mice receiving either vehicle or PF-4708671 from P4 to P14. Planned comparisons revealed a significant difference between WT and Pten +/− mice receiving vehicle, and a significant difference between vehicle and PF-4708671-treated Pten +/− mice. For (f) two-way ANOVA revealed a main effect on drug. F(1,12)=3.194, _P_=0.099 (interaction), F(1,12)=7.069, P<0.05 (drug), F(1,12)=3.947, _P_=0.07 (genotype). For (g) two-way ANOVA revealed main effects on genotype and interaction. F(1,12)=16.8, P<0.01 (interaction), F(1,12)=0.571, _P_=0.464 (drug), F(1,12)=4.901, P<0.05 (genotype). *P<0.05 and **P<0.01. _N_=4 animals in each group. All mice used in this figure were female.
Figure 5. Inhibition of S6K1 in adulthood does not rescue cellular and behavioural deficits in Pten mutant mice.
(a) Diagram showing time line of PF-4708671 treatment and testing of social behaviour. (b) Graph showing per cent time mice spent in each chamber. Two-way ANOVA and Sidak's post hoc tests were used. F(3,88)=3.59, _P_=0.0168 (interaction), F(3,88)=0.0573, _P_=0.9819 (genotype), and F(1,88)=33.20, P<0.001 (chamber time). _N_=12 WT receiving vehicle, 12 Pten +/− receiving vehicle, 13 WT receiving PF-4708671 and 11 Pten +/− receiving PF-4708671. (c,d) Representative images (c) and quantification (d) of reconstructed mPFC axon terminals in the BLA. Scale bar, 50 μm. Planned comparisons revealed a significant difference between WT and Pten +/− mice receiving vehicle, with two-way ANOVA showing a main effect on genotype. F(1,116)=0.006, _P_=0.94 (interaction), F(1,116)=0.006, _P_=0.94 (drug), F(1,116)=28.54, P<0.001 (genotype). _N_=30 axons from 6 mice in each group. (e,f) Representative images (e) and quantification (f) of mPFC–BLA synaptic boutons. Scale bar, 5 μm. Planned comparisons revealed a significant difference between WT and Pten +/− mice receiving vehicle, with two-way ANOVA showing a main effect on genotype. F(1,116)=0.066, _P_=0.798 (interaction), F(1,116)=1.301, _P_=0.256 (drug), F(1,116)=40.33, P<0.001 (genotype). _N_=30 axon fragments from 6 mice in each group. (g,h) Representative images (g) and quantification (h) of c-fos staining in the mPFC and BLA of social exposed mice. Scale bar, 100 μm. _N_=4 animals in each group. (i) Graph showing time mice spent sniffing, reflecting social interaction time. _N_=4 animals in each group. (j,k) Quantification of c-fos activation index (number of c-fos+ neurons/time spend sniffing) in the mPFC (j) and BLA (k). Planned comparisons revealed a significant difference between WT and Pten +/− mice receiving vehicle. For (j) two-way ANOVA revealed main effects on genotype and interaction. F(1,12)=5.964, P<0.05 (interaction), F(1,12)=1.008, _P_=0.335 (drug), F(1,12)=44.45, P<0.001 (genotype). For (k) two-way ANOVA revealed a main effect on genotype. F(1,12)=0.002, _P_=0.967 (interaction), F(1,12)=0.287, _P_=0.602 (drug), F(1,12)=24.87, P<0.001 (genotype). For all panels, *P<0.05, **P<0.01, ***P<0.001 and NS indicates not significantly different. _N_=4 animals in each group. All mice used in this figure were female adult mice.
Figure 6. Reducing activity in the mPFC to BLA projections corrects social behavioural deficits in Pten mutant mice.
(a) Diagrams showing experimental design. To label BLA-projecting mPFC neurons, CAV2-Cre was injected into the BLA, and AAV-DIO-mCherry was injected into the mPFC. Social exposure was controlled for 30 s sniffing time. (b) Representative images of mCherry (red) and c-fos (green) double labelling in the mPFC of WT and Pten +/− mice. Scale bar, 100 μm. (c) Quantification of the percentage of c-fos+ neurons within the mCherry+ neuronal population. Independent-sample t tests were used. *P<0.05. _N_=6 animals per genotype. (d) Diagrams showing the injection of CAV2-Cre into the BLA and AAV-DIO-mCherry or AAV-DIO-hM4Di-mCherry into the mPFC of Pten +/− mice. (e) Images showing reporter (mCherry) expression in mPFC. PrL: prelimbic cortex. IL: infralimbic cortex, with magnified images in (e′). Scale bar, 500 μm (left and middle) and 100 μm (right). (f) Per cent time spent in each chamber during the three-chamber social approach test. _N_=8 mice in Pten +/− (hM4Di) group and 9 mice in Pten +/− (mCherry) group. Two-way ANOVA and Sidak's post hoc tests were used. For mCherry group, F(1,32)=0.0569, _P_=0.813 (interaction), F(1,32)=0.02535, _P_=0.8745 (treatment) and F(1,32)=0.1798, _P_=0.6744 (chamber time). For hM4Di group, F(1,28)=32.40, P<0.001 (interaction), F(1,28)=0.00068, _P_=0.9794 (treatment) and F(1,28)=63.12, P<0.001 (chamber time). ***P<0.001 and NS indicates not significant difference. (g,h) Images (g) and quantification (h) of c-fos staining in the mPFC and BLA of social exposed Pten +/− mice that expressed either hM4Di plus mCherry or mCherry alone in BLA-projecting mPFC neurons and received either vehicle or CNO injections. Scale bar, 100 μm. _N_=4 mice in hM4Di expressing Pten +/− mice receiving either vehicle or CNO, _N_=5 mice in vehicle injected, and 4 mice in CNO injected, mCherry expressing Pten +/− mice. Independent-sample t tests were used, *P<0.05. All mice used in this figure were female.
Figure 7. Suppression of mTOR activity corrects cellular and behavioural deficits in Pten mutant mice.
(a) Schema showing the brain regions where one copy of Pten is deleted (green colour, primarily in the cerebral cortex). (b) Per cent time control and Emx1-Cre + ; Pten loxp/+ mice spent in each chamber. Two-way ANOVA was used. F(1,56)=0.8877, _P_=0.3502 (interaction), F(1,56)=0.3242, _P_=0.5714 (genotype) and F(1,56)=11.16, _P_=0.0015 (chamber time). Paired t tests showed that control, but not Emx1-Cre + ; Pten loxp/+, mice exhibited a significant preference for the social chamber. **P<0.01. _N_=15 animals in each genotype. (c) Schema showing the brain regions where one copy of Rptor is deleted in Pten +/− mice (red colour, primarily in the cerebral cortex). (d,e) Representative images (d) of western blot for p-S6, total S6 and β-actin in the cerebral cortex of control (Rptor loxp/+), Pten +/− (Rptor loxp/+ ; Pten +/−), Emx1-Cre + ; Rptor loxp/+, and Emx1-Cre + ; Rptor loxp/+ ; Pten +/− mice. (e) Quantification of p-S6 levels relative to total S6, expressed as per cent of control. One-way ANOVA and Tukey post hoc tests were used. F(3,25)=4.153, P<0.05 (genotype), *P<0.05. _N_=8 control, 8 Pten +/−, 6 Emx1-Cre + ; Rptor loxp/+, and 7 Emx1-Cre + ; Rptor loxp/+ ; Pten +/− mice. (f) Representative images of fluorescent Nissl staining in somatosensory cortical layer V, and quantification of layer V cell soma size in control (Rptor loxp/+), Pten +/− (Rptor loxp/+ ; Pten +/−), Emx1-Cre + ; Rptor loxp/+ and Emx1-Cre + ; Rptor loxp/+ ; Pten +/− mice. One-way ANOVA and Tukey post hoc tests were used. F(3,16)=9.183, P<0.001 (genotype), **P<0.01. _N_=5 animals per genotype. Scale bar, 25μm. (g) Per cent time mice spent in each chamber. Two-way ANOVA and Sidak's post hoc tests were used. F(3,60)=3.902, _P_=0.013 (interaction), F(3,60)=0.0842, _P_=0.9684 (genotype) and F(1,60)=60.65, P<0.001 (chamber time). *P<0.05, and **P<0.01. _N_=8 control (Rptor loxp/+), 8 Pten +/− (Rptor loxp/+ ; Pten +/−), 10 Emx1-Cre + ; Rptor loxp/+, and 8 Emx1-Cre + ; Rptor loxp/+ ; Pten +/− mice. All mice used in this figure were female.
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