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Received 26 Feb 2016 | Accepted 3 Oct 2016 | Published 15 Nov 2016

exhibit increased axonal branching and connectivity, which is accompanied by increased activity in the BLA in response to social stimuli and social behavioural decits. The latter two phenotypes can be suppressed by pharmacological inhibition of S6K1 during early postnatal life or by reducing the activity of mPFCBLA circuitry in adulthood. These ndings 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.

DOI: 10.1038/ncomms13421 OPEN

Hyperconnectivity of prefrontal cortex to amygdala projections in a mouse model of macrocephaly/autism syndrome

Wen-Chin Huang1,2, Youjun Chen1 & Damon T. Page1,2

1 Department of Neuroscience, The Scripps Research Institute, Jupiter, Florida 33458, USA. 2 The Doctoral Program in Chemical and Biological Sciences, The Scripps Research Institute, USA. Correspondence and requests for materials should be addressed to D.T.P. (email: mailto:[email protected]

Web End [email protected] ).

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The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that integrates environmental cues (for example, growth factors and energy status) to

control cell growth and proliferation1. mTOR interacts with several proteins to form at least two distinct multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). When activated, mTORC1 promotes protein synthesis mainly by phosphorylation of ribosomal protein S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein (eIF4E-BP)2. Phosphorylation of eIF4E-BP by mTORC1 releases eIF4E from binding to eIF4E-BP, forming the eIF4F complex to initiate cap-dependent protein translation1. Phosphorylation of ribosomal protein S6 (rpS6) by S6K correlates with the translational efciency of messenger RNAs containing a tract of oligopyrimidine in the 50UTR, named 50TOP messenger

RNA3. Phosphorylated rpS6 (p-S6) is a readout for mTORC1 signalling, and has been shown to regulate cell size4. Moreover, studies in the mammalian nervous system have shown that mTOR-S6K signalling regulates neuronal soma size, dendritic arborization, axonal growth and connectivity57.

Numerous lines of evidence implicate dysregulated mTOR signalling in the pathogenesis of autism spectrum disorder (ASD) and related neurodevelopmental disorders. Genes impinging on the PI3K-Akt-mTOR pathway, for example, phosphatase and tensin homologue (PTEN), TSC1/2, NF1 and FMR1, are highly represented amongst ASD risk genes identied to date8. Elevated mTOR signalling in the cerebral cortex has been reported in postmortem samples from individuals with autism9, and several animal models of risk genes acting in this pathway also show altered mTOR signalling in the brain1014.

PTEN encodes a phosphatase that is a negative regulator of the PI3K-Akt-mTOR pathway15. Germ-line heterozygous mutations in PTEN are found in B717% individuals with autism and macrocephaly16 (head circumference 42 s.d.s above normal), generally reduce PTEN protein levels17,18, and cause macrocephaly/autism syndrome (OMIM #605309) in an autosomal dominant manner. A mouse model that approximates the germ-line heterozygous Pten (Pten / ) mutations found in macrocephaly/autism syndrome displays a dynamic pattern of macroscale brain overgrowth due to altered proliferation and allocation of cell types, as well as selective impairments in ASD-relevant behaviours, particularly social behaviour1922. The widespread macroscale brain overgrowth, together with selective behavioural impairments, suggests that cell types and circuits underlying social behaviour may be more vulnerable or less adaptable to desynchronized patterns of growth caused by Pten mutations20.

This raises a critical problem for understanding the neuro-biology of autism and developing effective treatments: how do mutations in general regulators of growth, such as PTEN and other members of the PI3K-Akt-mTOR pathway, lead to the relatively selective behavioural and cognitive symptoms of ASD? To answer this question, it is necessary to understand the spatiotemporal pattern of activity of the PI3K-Akt-mTOR pathway in the developing brain and how it is impacted by mutations in ASD risk genes at the level of structural and functional connectivity in key circuits underlying ASD-relevant behaviours.

Here we have mapped the activity of mTORC1, using p-S6 as a readout, in the developing brains of normal and germ-line Pten / mice. We report that p-S6 levels are unexpectedly stable in the developing cerebral cortex of germ-line Pten / mice, with the exception of a discreet window during early postnatal life. Among the cell types that display particularly high levels of p-S6 are developing subcortical-projecting layer V neurons. We report that these neurons are hypertrophic in germ-line Pten /

mice and that corticalsubcortical connectivity is altered in these animals. Connectivity between the medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) is a particular focus of this study, as both of these areas are implicated in the processing of social information23 and autism pathophysiology24. Furthermore, structural and functional connectivity between these areas is well conserved between rodents and humans25. Our ndings are consistent with the novel hypothesis that dysregulated mTORC1 signalling during a critical period of development alters neuronal connectivity, including mPFCBLA projections, and social behaviour.

ResultsmTOR activity is dysregulated in Pten mutant mice. To understand whether the laminar distribution of p-S6 in the cerebral cortex is altered in Pten / mice, we performed immunohistochemistry for p-S6 and compared the Pten /

pattern of p-S6 to that of wild type (WT) mice at postnatal day 4 (P4), P8, P14, P28, and adulthood. To our surprise, we found that the spatiotemporal distribution of p-S6 was not grossly altered in the developing cerebral cortex of Pten / mice (Fig. 1a). To compare levels of p-S6 between WT and Pten / mice, we ran western blots on whole cortices from these animals across developmental time points. We found that p-S6 levels were unexpectedly similar between WT and Pten / mice at all stages examined with the exception of P8, when there was more p-S6 detected in the Pten / cortex as compared with WT (Fig. 1b,c).

This indicates that p-S6 levels are transiently dysregulated during early postnatal life (between P4 and P14) in Pten/

mice.

In carrying out immunohistochemistry for p-S6 within the early postnatal cerebral cortex, the non-uniform laminar distribution of p-S6 in WT, as well as Pten / , mice was notable. Plotting the distribution of p-S6 immunoreactivity across layers in the somatosensory cortex at P8 revealed that p-S6 levels are graded, with strongest signal in layer V and moderate signal in layers II/III and VI (Supplementary Fig. 1A). Measuring neuronal soma size and p-S6 immunoreactivity at P8, we found that p-S6 immunoreactivity is correlated with soma size (Supplementary Fig. 1B). Moreover, within cortical layer V, subcortical-projecting neurons have greater soma size and dendritic complexity than callosal-projecting neurons26,27, and we observed that p-S6 immunoreactivity is greater in CTIP2 neurons, which are subcortical-projecting neurons28, than in SATB2 neurons, which are callosal-projecting neurons29 (Supplementary Fig. 1C).

Examining other brain areas, we found that during early postnatal development p-S6 was generally enriched in larger cell types (cell soma Z20 mm in diameter), for example, Purkinje cells in the cerebellum, layer V corticospinal motor neurons (LV neurons), dopaminergic neurons (DA neurons) and noradrenergic neurons (NA neurons; Supplementary Fig. 1D). In contrast, minimal P-S6 was generally detected during development in smaller cell types (cell soma r10 mm in diameter), for example, medium spiny neurons, hippocampal granule cells and cerebellar granule cells (Supplementary Fig. 1D). We interpret this expression pattern as consistent with p-S6 being a positive regulator of cell/neuronal size46,

Given that we detected a change in the amplitude, but not the spatiotemporal distribution, of p-S6 in the developing cortex, we predicted that loss of one copy of Pten (that is, Pten/ ) might particularly impact cell types with high levels of p-S6 in WT animals. As an initial test of this, we examined the size of neurons across layers in the somatosensory cortex. We selected this region because of its relatively well-dened laminar organization within the mouse cerebral cortex. Using uorescent Nissl staining,

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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 mm. 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 mm. (b) Representative images of western blot for p-S6, total S6 and b-actin in WTand Pten / whole cerebral cortex at P0, P4,

P8, P14, P28 and adulthood. (c) Quantication 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. *Po0.05. N 5 animals per genotype for each time point.

(d) Representative images of uorescent Nissl stain in somatosensory cortical layers IIVI of WT and Pten/ mice at P14. Scale bar, 25 mm.(e) Quantication of cell soma size in somatosensory cortical layers IIVI of WT and Pten / mice at P14, expressed as per cent of WT. Independent-sample t tests were used. *Po0.05. 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 mm. (gk) Quantication 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. *Po0.05, and **Po0.01 and NS indicates no signicant 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, Po0.0001

(interaction); *Po0.05, **Po0.01, and ***Po0.001. N 25 neurons/6 animals each genotype. All mice used in this gure were male.

we detected increased soma size in layer V neurons, but not in layers IIIV or VI neurons, in P14 Pten / mice as compared with WT mice (Fig. 1d,e). This indicates that the growth of cell types enriched for p-S6 (for example, layer V neurons), as reected by cell soma size, is desynchronized relative to neurons

with lower levels of p-S6 (for example, layers IIIV or VI neurons) in Pten / mice.

As we observed enrichment of p-S6 in developing subcortical-projecting layer V neurons, amongst other cell types, and we detected a pronounced change in cell soma size in layer V

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neurons in Pten / mice, we decided to examine these as a representative of cell types whose growth is impacted by Pten haploinsufciency. To understand whether overgrowth of layer V neurons is restricted to the cell soma or also occurs in neurites, we examined dendritic structure. We made use of a genetic reporter for layer V neurons (Etv1-CreERT2; Ai14)30,31 and measured dendritic tree size of these cells in P14 Pten / mice (Etv1-

CreERT2; Ai14; Pten / ). We found that, relative to controls, layer V neurons in Pten / mice exhibited increased cell soma volume and dendritic complexity (Fig. 1fl). Thus, changes in neuronal soma size in layer V neurons caused by Pten haploinsufciency correlate with changes in neurite growth.

To explore whether dysregulated neurodevelopmental mTORC1 signalling might be a point of convergence across multiple mouse models of autism risk genes, we performed these analyses in another mouse model of an autism risk gene,

Fmr1 /y. We found that, similar to Pten / mice, the spatiotemporal distribution of p-S6 in the cerebral cortex is not grossly altered, and p-S6 levels are elevated at P14 as compared with WT, but not other ages (Supplementary Fig. 2AC), indicating that cortical mTORC1 signalling is transiently elevated during early postnatal life. Measuring cell soma size across cortical layers, we found that layer V neurons are enlarged in P14 Fmr1 /y mice as compared with WT mice (Supplementary

Fig. 2D,E). Consistent with overgrowth in layer V neurons, dendritic tree size is also slightly greater in P14 Etv1-CreERT2; Fmr1 /ymice as compared to control littermates (Supplementary

Fig. 2FL). This suggests that multiple autism risk genes may converge on dysregulated neurodevelopmental mTORC1 signalling and desynchronized neuronal growth.

Early inhibition of S6K1 corrects decits in Pten mutants. The observation that p-S6 levels are transiently dysregulated in the cerebral cortex of P8 Pten / mice led us to speculate that this could represent a critical period where normalization of p-S6 levels may suppress the overgrowth of layer V neurons and behaviour decits in these animals. To test this possibility, we treated Pten / mice and WT controls with the S6K1 inhibitor

PF-4708671 (ref. 32), injected daily from P4 to P14 (Fig. 2a). We found that systemic PF-4708671 treatment reduced elevated p-S6 in the cerebral cortex and suppressed the increased soma size of layer V neurons in the somatosensory cortex of P14 Pten/

mice (Fig. 2b,c). The most robust and consistent behavioural phenotype we have observed in Pten/ mice is decits in the three-chamber social approach test in adult females2022. Adult male Pten / mice, in contrast to females, display variable phenotypes in this assay that are highly sensitive to testing conditions20,21 (Supplementary Fig. 3), so three-chamber social approach testing in this study focused on female animals. We found that PF-4708671 treatment in the P4P14 developmental window is sufcient to reverse the three-chamber social approach decit present in vehicle-treated adult female Pten/ mice (Fig. 2d). We also observed that WT mice receiving PF-4708671 treatment displayed social approach decits, consistent with a

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Figure 2 | Inhibition of S6K1 during development corrects cellular and behavioural decits 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 b-actin in the cerebral cortex of P8 WT and Pten/ mice receiving either vehicle or PF-4708671. Quantication 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,

Po0.001 (drug), F(1,16) 25.54, Po0.001 (genotype); ***Po0.001. N 5

animals per group. (c) Representative images of uorescent Nissl staining in the layer V somatosensory cortex and quantication 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 signicant difference between WT and Pten/ mice receiving vehicle, and a signicant 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, Po0.01 (drug), F(1,16) 10.96,

Po0.01 (genotype). *Po0.05. N 5 animals per group. Scale bar, 25 mm.

(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 Sidaks 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, Po0.001 (chamber time).

*Po0.05. N 11 WTreceiving vehicle, 13 Pten / receiving vehicle, 11 WT

receiving PF-4708671, and 10 Pten / receiving PF-4708671. All mice used in this gure were female.

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previous report of reduced social interaction in WT mice receiving rapamycin treatment beginning at 56 weeks of age10. Thus, we speculate that an optimal level of mTORC1 signalling is required during development for normal social behaviour, with too much or too little activity resulting in similar social behavioural decits, in line with a previously proposed model of bidirectional control of protein synthesis and connectivity in ASD33. Taken together, these results point to a developmental window in early postnatal life in which social behaviour is sensitive to modulation of mTOR/S6K1 signalling and normalization of p-S6 levels results in lasting correction of the three-chamber social approach decits in Pten / mice.

In addition to the core symptoms of ASD, some individuals with germ-line heterozygous PTEN mutations also have decits in working memory17. To explore whether germ-line Pten / mice also show working memory decits and whether this phenotype may be responsive to developmental PF-4708671 treatment, we assessed spatial working memory using the non-matching to sample T-maze test (Supplementary Fig. 4A)34. Vehicle and PF-4708671-treated WT and Pten / mice of both sexes showed normal performance in this test (Supplementary

Fig. 4B,C), indicating that the particular aspect of working memory probed by the non-matching to sample T-maze test is not altered in these animals.

Neuronal hyperconnectivity in Pten mutants. Considering that layer V neurons are the primary output neurons of the cerebral cortex, we speculated that corticalsubcortical connectivity between areas important for social behaviour might be altered in these animals. A circuit that is particularly interesting in this context involves projections from the mPFC to the BLA (see Fig. 3a). Both of these areas are implicated in the processing of social information23 and autism pathophysiology24. Thus, we formulated the hypothesis that dysregulated mTORC1 signalling during a critical period of development alters neuronal connectivity, including mPFCBLA projections, and social behaviour.

To test this hypothesis, we rst conrmed that BLA-projecting mPFC neurons are hypertrophic in Pten / mice (Supplementary Fig. 5AC), consistent with our observation of layer V neuron hypertrophy in somatosensory cortex (Fig. 1e). Furthermore, we found that protein synthesis and p-S6 levels are dysregulated in the prefrontal cortex of P8 Pten / mice, indicating dysregulated mTORC1 activity (Supplementary

Fig. 5DG). We next injected reporter-expressing (tdTomato) adeno-associated virus (AAV) into the mPFC of adult female mice and visualized axon terminal branching and synaptic boutons within the BLA (Fig. 3a). We found that axons originating from the mPFC in vehicle-treated Pten / mice displayed excessive branching and synaptic boutons within the

BLA, as compared with vehicle-treated WT mice, and that PF-4708671 treatment during development (P4P14) was sufcient to reverse this phenotype in Pten / mice (Fig. 3be). These results indicate that mPFCBLA circuitry is hyperconnected in

Pten / mice, and that this phenotype is sensitive to early postnatal PF-4708671 treatment.

As an initial exploration into the molecular mechanism by which dysregulated mTORC1 signalling might lead to altered axon branching and structural connectivity, we measured levels of CRMP2 (collapsin response mediated protein 2) and Tau (microtubule associated protein), both of which are regulated by mTORC1 signalling and have been shown to inuence axon growth35,36. We observed a signicant increase in CRMP2 levels and a trend towards increased Tau levels in P8 Pten / cerebral cortex (Fig. 3f,g). Actin remodelling is a regulator of axon growth,

and homozygous Pten mutations can alter actin levels37. Thus, we measured b-actin levels and did not observe any differences between WT and Pten / cortex (Fig. 3g), indicating that this is a valid loading control and that heterozygous Pten mutations do not grossly alter b-actin levels, unlike homozygous Pten mutations. Since the excessive axon branching in Pten/

mice was suppressed by pharmacological inhibition of S6K1 during early postnatal life, we suspected that this effect could be due to the inhibition of S6K1-dependent translation of CRMP2 (ref. 36). To test this possibility, we measured CRMP2 levels in the P8 cerebral cortex of Pten / and WT control mice that received PF-4708671 treatment from P4 to P8, by western blot. We found that PF-4708671 treatment suppressed elevated CRMP2 in Pten / mice (Fig. 3h,i). Thus, normalization of excessive axon branching in Pten / mice by PF-4708671 treatment correlates with normalization of CRMP2 levels in these animals.

Hyperactivity in the mPFC and BLA of Pten mutant mice. To test if neuronal activity in response to a social cue is altered in Pten / mice, we measured the number of c-fos neurons in the mPFC and BLA in response to exposure to a novel stimulus mouse (Fig. 4a). Previous work has shown that social exposure induces c-fos activation in the mPFC and BLA, and that the number of c-fos cells in these two regions correlates with the amount of time engaged in social interaction38. Consistent with this, we found that exposure to a novel social stimulus results in a robust induction of c-fos cells in the mPFC and BLA of both

WT and Pten / adult female mice (Fig. 4bd) and that the number of c-fos cells in these two regions correlated with the duration each mouse spent snifng the novel stimulus mouse (Supplementary Fig. 6A). Although the total number of c-fos cells in the mPFC and BLA is not altered in Pten / mice compared with WT mice (Fig. 4c,d), we found that social interaction time, as reected by time spent snifng, varied between and within genotype during the social exposure period (Fig. 4e). Because the induction of c-fos cells in mPFC and BLA is dependent on the amount of social interaction time, we calculated a c-fos activation index (number of c-fos neurons/

time mouse engaged in snifng; Supplementary Fig. 6B). We found that the c-fos activation index is elevated in the mPFC and BLA of vehicle-treated Pten / mice. Furthermore, we found that the elevated c-fos activation index in the mPFC and BLA is normalized in Pten / mice receiving PF-4708671 treatment during early postnatal life (P4P14; Fig. 4f,g). We conrmed this result in a separate cohort of mice in which the animals were allowed to sniff a novel stimulus mouse to a xed criterion of 30 s, thus ensuring equivalent amounts of exposure to the social cue across subjects. In this cohort, we found that c-fos cells in the mPFC and BLA are elevated in Pten / relative to WT mice, and this phenotype is rescued by PF-4708671 treatment during early postnatal life (Supplementary Fig. 7). Thus, with equivalent social exposure, there are more c-fos cells in the mPFC and

BLA of Pten / mice, and this phenotype can be normalized by suppression of S6K1 during development.

Adult S6K1 inhibition does not rescue Pten mutant decits. We next asked whether mPFCBLA connectivity and social phenotypes in Pten / mice might be responsive to PF-4708671 treatment in adulthood, when cortical circuitry has reached a mature state. To address this issue, we treated adult Pten/

mice with the equivalent dosage and duration of PF-4708671 as that of developmental experiments and tested social behaviour 7 days after the nal injection of PF-4708671 (Fig. 5a). We found that the three-chamber social approach decit is not

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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 mm (1), 100 mm (2), 50 mm (3) and 5 mm (right bottom image of (3)). (b,c) Representative images (b) and quantication (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 mm. Two-way ANOVA and Tukey post hoc tests were used, F(1,116) 4.548, Po0.05 (interaction), F(1,116) 6.89, Po0.01

(drug), F(1,116) 16.82, Po0.001 (genotype); **Po0.01, ***Po0.001. N 30 axons from 6 mice in each group. (d,e) Representative images (d) and

quantication (e) of mPFCBLA synaptic boutons in adult WT and Pten/ mice receiving either vehicle or PF-4708671 from P4 to P14. Scale bar, 5 mm. Two-way ANOVA and Tukey post hoc tests were used, F(1,116) 4.094, Po0.05 (interaction), F(1,116) 13.30, Po0.001 (drug), F(1,116) 18.79, Po0.001

(genotype); ***Po0.001. N 30 axon fragments from 6 mice in each group. (f,g) Representative images (f) of western blot for CRMP2, Tau and b-actin in

the cerebral cortex of P8 WTor Pten/ mice. (g) Quantication of CRMP2 and Tau protein levels relative to b-actin, and total b-actin levels, expressed as percentage of WT. Independent-sample t tests were used. *Po0.05. N 5 animals per genotype. (h,i) Representative images (h) of western blot for

CRMP2 and b-actin in the cerebral cortex of P8 WT or Pten / mice injected with either vehicle or PF-4708671 from P4 to P8. (i) Quantication of CRMP2 protein levels relative to b-actin. Planned comparisons revealed a signicant difference between WT and Pten/ mice receiving vehicle, and a signicant 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, Po0.001 (drug), F(1,16) 14.24, Po0.01 (genotype). *Po0.05. N 5 animals in each group.

All mice used in this gure were female.

corrected in Pten/ mice treated with PF-4708671 in adulthood (Fig. 5b). This result prompted us to investigate mPFCBLA structural connectivity and c-fos reactivity in these mice. We found that excessive axon branching and increased density of synaptic boutons in mPFC to BLA projections was not rescued in Pten / mice receiving PF-4708671 treatment in adulthood (Fig. 5cf). The elevated c-fos activation index in the mPFC and

BLA in response to social exposure was also not rescued in Pten / mice treated with PF-4708671 in adulthood (Fig. 5gk).

Taken together, our PF-4708671 treatment results indicate that there is a developmental critical period in which pharmacological suppression of S6K1 can cause a lasting rescue of mPFCBLA hyperconnectivity, excessive c-fos reactivity and three-chamber social approach decits in Pten / mice.

Hyperactivity of mPFC-BLA circuitry in Pten mutants. To test whether Pten / mice exhibit hyperactive mPFCBLA circuitry in response to social cues, we used c-fos expression as a measure of neuronal activity in BLA-projecting mPFC neurons in WT and Pten / mice. To label BLA-projecting mPFC neurons, we injected an AAV vector carrying the reporter mCherry, expressed in a Cre-dependent manner (double-oxed inverted open reading frame) into the mPFC, and a retrograde-transporting canine adenovirus carrying Cre recombinase (CAV2-Cre) into the BLA of adult female WT and Pten / mice (Fig. 6a). Each subject mouse was allowed to sniff a novel stimulus mouse to a xed criterion of 30 s so that each subject mouse engaged in an approximately equal amount of social interaction. We found that a greater percentage of BLA-projecting mPFC neurons (mCherry

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WT + vehicle Pten+/ + vehicleWT + PF-4708671 Pten+/ + PF-4708671

100

mPFC

0 Control Social exposure

BLA

Number of c-fos+

neurons

Control

Social exposure

e f

Social exposure

mPFC

BLA

** *

Time spent sniffing (s)

c-fos activation index (%)

200

400

Pten+/

150

100

c-fos activation index (%)

150

Pten+/

300

100

200

100

0 Vehicle PF-4708671

0 Vehicle PF-4708671 Vehicle PF-4708671

Figure 4 | mPFC and BLA hyperactivity in Pten mutant mice is corrected by inhibition of S6K1 during development. (a) Diagram showing the workow 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. (bd) Representative images (b) and quantication of c-fos staining in the mPFC (c) and the basal lateral amygdala (BLA) (d) of control or social exposed WTand Pten/ mice receiving either vehicle or PF-4708671 from postnatal day 4 (P4) to P14. Scale bar, 100 mm. N 4 animals in each group. (e) Graph showing social interaction time, reected as time spent snifng, in WTand Pten/ mice receiving either

vehicle or PF-4708671 from P4 to P14. N 4 animals in each group. (f,g) Quantication of c-fos activation index (number of c-fos neurons/time spend

snifng) 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 signicant difference between WT and Pten / mice receiving vehicle, and a signicant 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,

Po0.05 (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, Po0.01

(interaction), F(1,12) 0.571, P 0.464 (drug), F(1,12) 4.901, Po0.05 (genotype). *Po0.05 and **Po0.01. N 4 animals in each group. All mice used

in this gure were female.

neurons) were c-fos in Pten/ mice as compared with WT mice (Fig. 6b,c), suggesting that mPFCBLA circuitry is hyper-active in response to social exposure in Pten/ mice.

To test whether decreasing activity in mPFC to BLA projections may normalize three-chamber social approach decits in Pten / mice, we reduced excitability in mPFC neurons projecting to the BLA using retro-DREADD (Designer Receptor

Exclusively Activated by Designer Drugs) technology39. An AAV vector carrying hM4Di and mCherry expressed in a Cre-dependent manner was bilaterally injected into the mPFC, and a CAV2-Cre vector was bilaterally injected into the BLA of adult female Pten / mice (Fig. 6d,e). Clozapine n-oxide (CNO) or vehicle was injected into these mice, and we then tested social

behaviour using the three-chamber social approach assay. We found that the three-chamber social approach decit present in vehicle injected hM4Di expressing Pten / mice or vehicle and CNO injected mCherry expressing Pten/

mice was corrected in CNO injected hM4Di expressing Pten / mice (Fig. 6f).

We next measured c-fos cells in the mPFC and BLA of these mice in response to snifng a novel stimulus mouse to a xed criterion of 30 s. We found that the number of c-fos cells in the mPFC and BLA in response to social exposure was reduced in

CNO injected compared with vehicle injected hM4Di expressing Pten / mice, and we found no change in the number of c-fos cells in either vehicle or CNO injected mCherry expressing

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% chamber time in

female

Vehicle

PF-4708671

Social behaviour

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Mouse Center Object

Pten+/

PF-4708671

Adult

Day 1 Day 11

WT + vehiclePten+/ + vehicle

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Number of axon branches

2.5

1.0

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10 m

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2.0

Vehicle

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Pten+/

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0.6

1.0

0.4

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PF-4708671

0.2

Pten+/

0.0 Vehicle PF-4708671

g h j

mPFC

* NS

Vehicle PF-4708671

Vehicle

PF-4708671

Social exposure

Number of c-fos+ neurons

c-fos activation index (%)

Pten+/

250

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WT + vehicle Pten+/ + vehicleWT + PF-4708671 Pten+/ + PF-4708671

200

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mPFC

Pten+/

mPFC BLA

Vehicle PF-4708671

i k

200

Time spent sniffing (s)

BLA

* NS

200

150

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c-fos activation index (%)

100

BLA

Pten+/

Social exposure

Vehicle PF-4708671

Figure 5 | Inhibition of S6K1 in adulthood does not rescue cellular and behavioural decits 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 Sidaks 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, Po0.001 (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 quantication (d) of reconstructed mPFC axon terminals in the BLA. Scale bar, 50 mm. Planned comparisons revealed a signicant 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, Po0.001 (genotype). N 30 axons from 6 mice in each group. (e,f) Representative images (e) and

quantication (f) of mPFCBLA synaptic boutons. Scale bar, 5 mm. Planned comparisons revealed a signicant 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, Po0.001 (genotype). N 30 axon fragments from 6 mice in each group. (g,h) Representative images (g) and quantication (h) of c-fos

staining in the mPFC and BLA of social exposed mice. Scale bar, 100 mm. N 4 animals in each group. (i) Graph showing time mice spent snifng, reecting

social interaction time. N 4 animals in each group. (j,k) Quantication of c-fos activation index (number of c-fos neurons/time spend snifng) in the

mPFC (j) and BLA (k). Planned comparisons revealed a signicant 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, Po0.05 (interaction), F(1,12) 1.008, P 0.335 (drug),

F(1,12) 44.45, Po0.001 (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, Po0.001 (genotype). For all panels, *Po0.05, **Po0.01, ***Po0.001 and NS indicates not signicantly

different. N 4 animals in each group. All mice used in this gure were female adult mice.

Pten / mice (Fig. 6g,h). We interpret these results as consistent with a causal relationship between hyperactivity in mPFCBLA circuitry and three-chamber social approach decits in Pten / mice.

Reducing mTOR activity corrects decits in Pten mutant mice. To determine whether isolating Pten / mutations to the cerebral cortex will lead to three-chamber social approach decits, we generated conditional Pten/ mutant mice using Emx1-Cre,

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CAV2-Cre

AAV-DIO-mCherry c-fos

mPFC

30 s sniffing time

2 h

WT or Pten+/

c-fos mCherry Merge c-fos mCherry Merge

c-fos+ /mCherry+ (%)

Pten+/

AAV-DIO-mCherry

0 Vehicle CNO

Bregma 1.98

AAV-DIO-hM4Di-mCherry

CAV2-Cre

Pten+/

f g h

mCherry

Vehicle CNO

hM4Di

Pten+/ (mCherry)

Pten+/ (hM4Di)

mPFC (mCherry) BLA (mCherry)

NS NS

% chamber time in

females

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Number of c-fos

neurons

Mouse Centre Object

Number of c-fos

neurons

NS NS

100

mPFC

Vehicle CNO

0 Vehicle CNO

0 Vehicle CNO Vehicle CNO

20 Vehicle CNO

mPFC (hM4Di)

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150

Number of c-fos

neurons

***

% chamber time

in females

Number of c-fos

neurons

100

BLA

Figure 6 | Reducing activity in the mPFC to BLA projections corrects social behavioural decits 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 snifng time. (b) Representative images of mCherry (red) and c-fos (green) double labelling in the mPFC of WTand Pten / mice. Scale bar, 100 mm. (c) Quantication of the percentage of c-fos neurons within the mCherry neuronal population. Independent-sample t tests were used. *Po0.05. 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 magnied images in (e0). Scale bar, 500 mm (left and middle) and 100 mm (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 Sidaks 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, Po0.001 (interaction), F(1,28) 0.00068, P 0.9794 (treatment) and F(1,28) 63.12,

Po0.001 (chamber time). ***Po0.001 and NS indicates not signicant difference. (g,h) Images (g) and quantication (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 mm. 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, *Po0.05. All mice used in this gure were female.

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200

* *

150

p-S6/S6 (%)

100

Emx1-Cre+ ;Rptorloxp/+ ;Pten+/

Mouse

Object

Emx1-Cre ;

Rptor

Emx1-Cre ; Rptor ;

Pten

Rptorloxp/+

Rptorloxp/+ ;Pten+/

Emx1-Cre+ ;Rptorloxp/+

% chamber time in

females

Centre

p-S6

Control Pten

-actin

Control

Emx1-Cre+ ; Ptenloxp/+

f g

Layer V cell soma size

(m2 )

Layer V cells

350

Mouse

Object

% chamber time in

females

** **

60 ** ** *

Centre

250

Control

Pten+/

Emx1-Cre+; Rptor loxp/+

Emx1-Cre+; Rptor loxp/+;

Pten+/

150

Rptorloxp/+

Rptorloxp/+ ; Pten+/

Emx1-Cre+ ; Rptorloxp/+

Emx1-Cre+ ;Rptorloxp/+ ; Pten+/

Rptorloxp/+

Rptorloxp/+ ; Pten+/

Emx1-Cre+ ; Rptorloxp/+

Emx1-Cre+ ;Rptorloxp/+ ; Pten+/

Figure 7 | Suppression of mTOR activity corrects cellular and behavioural decits 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; Ptenloxp/ 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 ; Ptenloxp/, mice exhibited a signicant preference for the social chamber. **Po0.01. 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 b-actin in the cerebral cortex of control (Rptorloxp/), Pten/ (Rptorloxp/; Pten / ), Emx1-Cre; Rptorloxp/ , and Emx1-Cre; Rptorloxp/ ; Pten/ mice. (e) Quantication 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, Po0.05 (genotype), *Po0.05.

N 8 control, 8 Pten / , 6 Emx1-Cre ; Rptorloxp/ , and 7 Emx1-Cre; Rptorloxp/; Pten/ mice. (f) Representative images of uorescent Nissl staining

in somatosensory cortical layer V, and quantication of layer V cell soma size in control (Rptorloxp/ ), Pten / (Rptorloxp/ ; Pten/ ), Emx1-Cre ; Rptorloxp/ and Emx1-Cre; Rptorloxp/ ; Pten / mice. One-way ANOVA and Tukey post hoc tests were used. F(3,16) 9.183, Po0.001 (genotype),

**Po0.01. N 5 animals per genotype. Scale bar, 25mm. (g) Per cent time mice spent in each chamber. Two-way ANOVA and Sidaks 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, Po0.001 (chamber time). *Po0.05, and

**Po0.01. N 8 control (Rptorloxp/ ), 8 Pten/ (Rptorloxp/ ; Pten / ), 10 Emx1-Cre ; Rptorloxp/ , and 8 Emx1-Cre ; Rptorloxp/ ; Pten / mice. All

mice used in this gure were female.

which is expressed primarily in cortical pyramidal cells throughout development40 (Fig. 7a). We used a Cre driver that is expressed broadly in the cortex, rather than a Cre driver that is restricted to subcortical-projecting pyramidal cells, because we do not know at present if the effects of Pten /

mutations on the development of this neuronal population are cell autonomous or non-cell autonomous. We have previously shown that Emx1-Cre ; Ptenloxp/ mice recapitulate the cortical overgrowth phenotype seen in germ-line Pten /

mice19. Testing social behaviour using three-chamber social approach test, we found that although adult female Emx1-Cre ;

Ptenloxp/ mice did not display a signicant difference

in allocation of chamber time as compared with controls, a within-group analysis revealed a lack of signicant difference in time spent in the stimulus mouse versus object chambers (Fig. 7b).

To test whether reducing mTORC1 signalling in the cerebral cortex of germ-line Pten / mice is sufcient to correct three-chamber social approach decits, we generated Emx1-Cre ;

Rptorloxp/ ; Pten / mice (Fig. 7c). We found that dysregulated p-S6 in P8 Pten / cerebral cortex was normalized in Emx1-

Cre ; Rptorloxp/ ; Pten / mice (Fig. 7d,e). We measured cell soma size as a marker for layer V neuron structural changes in

Pten / mice and found that layer V neuronal hypertrophy was

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suppressed in Emx1-Cre ; Rptorloxp/ ; Pten / mice in adulthood (Fig. 7f). Moreover, the three-chamber social approach decit present in adult female Pten / mice was also corrected in Emx1-Cre ; Rptorloxp/ ; Pten/ mice (Fig. 7g). Taken together, our results indicate that dysregulated mTOR signalling in the developing cerebral cortex contributes to social behavioural decits in Pten / mice. Future experiments will examine whether conditional Pten / mutation or modulation of mTORC1 signalling specically in subcortical-projecting pyramidal cells is sufcient to alter three-chamber social approach behaviour.

DiscussionThe genetic causes of ASD are heterogeneous. Studying a signalling pathway regulated by multiple autism risk genes at the level of circuit function will provide insights into common biological mechanisms underling ASD pathogenesis and pathophysiology. Here we have presented evidence that dysregulated mTOR signalling impacts the assembly of circuitry underlying social behaviour in germ-line Pten /

mice, a mouse model for macrocephaly/autism syndrome. We have mapped the spatiotemporal activity of mTORC1 signalling in developing mouse brain, and found that a subset of cells, including subcortical-projecting layer V neurons in the cerebral cortex, exhibit enrichment for p-S6, a readout for mTORC1 signalling. We further investigated corticalsubcortical connectivity, which is largely mediated by subcortical-projecting layer V neurons, between two brain areas that are implicated in the processing of social information and ASD pathophysiology, the mPFC and BLA. We show that Pten / mice exhibit hyperconnectivity and hyperactivity of mPFCBLA circuitry, excessive c-fos reactivity in response to social cues, and three-chamber social approach decits. Correcting structural connectivity by treatment with a pharmacological inhibitor of S6K1 during early postnatal life or modulating neuronal activity by DREADDs in adulthood can suppress these phenotypes.

We nd that Pten / mice display non-uniform overgrowth across neuronal cell types in the developing cerebral cortex, indicating that the growth of some neuronal populations (for example, subcortical-projecting layer V neurons) is desynchronized relative to others. This nding has implications for information processing within cortical microcircuitry. Fmr1 /y mice, which model fragile X syndrome, display a similar phenotype. One possible explanation for this nding is that mutations in genes that encode negative regulators of mTORC1 signalling, such as Pten and Fmr1, have the greatest effect on growth in cell types with high baseline levels of mTORC1 activity (for example, layer V neurons) because these cells are more dependent on negative regulation by repressive factors. It is worth contrasting the effects of heterozygous and homozygous Pten mutations in light of this explanation. Studies from Pten conditional knockout mice have shown that even relatively small neural cell types, such as hippocampal granule cells, exhibit overgrowth on conditional homozygous mutation of Pten (ref. 11). Indeed, we have found that conditional homozygous mutation of Pten leads to massively increased p-S6 levels and decreased cell density throughout the newborn cerebral cortex, consistent with widespread hypertrophy, whereas we did not observe this phenotype upon conditional heterozygous mutation of Pten (ref. 19). We attribute this to an effect of Pten dosage: homozygous mutation of Pten shifts a wide variety of neural cell types towards a pathological state, with strong upregulation of mTORC1 activity and hypertrophy. In contrast, most neural cell types appear to maintain more or less normal

physiological levels of mTORC1 signalling upon heterozygous mutation of Pten.

The normal developmental trajectory of mTORC1 activity in the brain may help to explain our nding of transient changes in cortical p-S6 levels in early postnatal life in germ-line Pten /

and Fmr1 /y mice. One study that examined mTORC1 signalling in the cerebral cortex of WT mice in both early postnatal life and adulthood found that mTORC1 signalling is most active during early postnatal life, especially the rst two postnatal weeks41. Just as cell types with high baseline levels of mTORC1 signalling may be particularly vulnerable to the effects of dominant-acting mutations in genes that encode negative regulators of mTORC1 signalling, neurodevelopmental time windows of peak mTORC1 activity (for example, early postnatal life) might represent critical periods of vulnerability to such mutations, as well as targets for therapeutic intervention.

We found that in the mature brain, axonal projections from the mPFC to the BLA have excessive branches and synaptic boutons. Given that elevated cortical p-S6 and CRMP2, and rescue by S6K1 inhibitor treatment, all occur during the early postnatal life, we predict that the initial growth of subcortical-projecting neurons is over-exuberant in Pten/ mice. This would be consistent with reports of Pten and mTOR-S6K signalling acting as regulators of axonal growth7,11. Another factor that could be at play is reduced axonal pruning of subcortical-projecting neurons in Pten / mice. This is an interesting possibility given that a recent study has reported reduced dendritic spine pruning in cortical layer V cells of individuals with ASD and in Tsc2 mutant mice, with this phenotype resulting from a disruption of mTOR-dependent macroautophagy9. The contribution of mTORC1-mediated axonal branching to the white matter abnormalities reported in individuals with ASD17,42,43 will be an interesting area of future research.

This study has focused on connectivity between the mPFC and BLA, as this circuit is implicated in ASD pathophysiology and has relevance for the three-chamber social approach decit observed in germ-line Pten/ mice. However, we anticipate that altered connectivity is unlikely to be restricted to this circuit, given that we have observed p-S6 enrichment and neuronal overgrowth outside of the prefrontal cortex (for example, in layer V neurons in the somatosensory cortex). Given the relatively selective behavioural decits in germ-line Pten / mice20, we predict that the behavioural prole of these animals is a product of altered hard-wired connectivity across multiple circuits, together with the capacity of specic circuits to adapt to these changes during development via synaptic plasticity mechanisms. Indeed, we have found that dopaminergic neurons are enriched for p-S6 during development (Supplementary Fig. 1D). Considering that dopaminergic neurons in the ventral tegmental area encode and modulate features of social interaction44, dopamine signalling plays a key neuromodulatory role in the PFC and limbic system45,46, and conditional deletion of Pten in dopaminergic neurons results in three-chamber social approach decits20, we speculate that mesocorticolimbic circuitry may be especially vulnerable to the effects of abnormal structural and functional connectivity resulting from Pten haploinsufciency.

We note that both increased and decreased brain network connectivity has been reported in individuals with ASD47. While we have found evidence for hyperconnectivity in our model of macrocephaly/autism syndrome caused by germ-line heterozygous PTEN mutations, it is likely that distinct subgroups of individuals with ASD may display different connectivity endophenotypes that correlate with underlying risk factors. It is possible that either increased or decreased connectivity may cause similar behavioural and cognitive decits, potentially

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resulting from imbalanced excitation and inhibition48 across brain circuits and areas critical for processing of social information.

An important problem for developing therapeutic approaches to psychiatric disorders is dening critical periods of phenotypic reversibility. Previous studies have shown that the mTORC1 inhibitor rapamycin, when given to Pten conditional knockout animals at different time points, is able to prevent or arrest, but not reverse, altered neurite projection patterns10,49. Our results show that suppression of S6K1 by PF-4708671 during early postnatal life, but not in adulthood, results in a lasting correction of mPFCBLA hyperconnectivity, excessive c-fos reactivity to social cues, and three-chamber social approach decits in Pten / mice. This indicates that suppression of mTOR-S6K signalling in adulthood is not sufcient to reverse structural connectivity changes (i.e. hyperconnectivity of mPFCBLA circuitry) in Pten / mice. Our experimental design included a washout period for PF-4708671 because we wanted to test whether treatment with this drug during development or in adulthood results in lasting changes in structural connectivity. We cannot exclude the possibility that overlapping PF-4708671 treatment with behavioural testing may result in modication of behavioural decits through a mechanism independent of changes in structural connectivity.

While our data indicate that abnormal mPFCBLA structural connectivity is hard-wired in adulthood, we nd that reducing activity in mPFCBLA projections via DREADD technology is capable of normalizing both c-fos reactivity and the three-chamber social approach decits. Thus, we propose that there are at least two distinct windows and strategies whereby social behavioural decits in Pten / mice can be modulated.

The rst window is in early postnatal life, when suppression of mTORC1-S6K1 can prevent or arrest the development of altered mPFCBLA structural connectivity. The second window is in adulthood, where modulation of activity in mPFCBLA circuitry can offset the consequences of pathological structural and functional connectivity. Future work will explore the specic nature of altered structural and functional connectivity between the mPFC and BLA at the level of cell types within the BLA in Pten / mice and whether modulating activity of this circuit is capable of inuencing social behaviour in

WT animals. It will also be interesting to investigate whether the modulation of amygdala reactivity, habituation and connectivity with the prefrontal cortex that has been associated with genetic variants inuencing serotonin signalling5052 might be a substrate for modication of social behavioural endophenotypes in Pten / mice by second-site mutations in serotonin transporter21 and pharmacological targeting of serotonin receptor signalling22.

An emerging theme in the eld of psychiatric disorders is a shift towards stratifying populations based on common underlying risk factors and pathophysiology, and aligning preclinical research to investigate circuit and neurophysiological correlates of these risk factors53. We have shown that haploinsufciency for Pten leads to dysregulation of a disease-implicated signalling pathway (the mTOR signalling pathway) that regulates neuronal connectivity, and altered connectivity in an autism-relevant neural circuit (mPFC-amygdala) affects activity in a key brain region (amygdala) in response to social cue, as well as social behaviour. Given that both mPFCBLA connectivity and mTOR-S6K signalling are highly evolutionarily conserved between rodents and humans25,54, we anticipate that the mechanism of pathogenesis and corresponding therapeutic strategy presented here may be relevant for individuals with ASD who have been exposed to risk factors associated with dysregulated mTOR signalling.

Methods

Mice. B6.129-Ptentm1Rps (Pten / )55 were obtained from the repository at the National Cancer Institute at Frederick, where they were already backcrossed onto a congenic C57BL/6J background by the Donating Investigator. The line has been maintained by backcrossing to C57BL/6J mice for more than 10 generations.

All other mouse lines were obtained from the Jackson Laboratory: Etv1tm1.1 (cre/ERT2)Zjh(Etv1-CreERT2, stock number 013048), Gt (ROSA)26Sortm14

(CAG tdTomato)Hze (Ai14, stock number 007914), Rptortm1.1Dmsa(Rptorloxp/loxp, stock number 013188), Emx1tm1 (cre)Krj (Emx1-Cre , stock number 005628),

Ptentm1Hwu(PtenloxP/loxP, stock number 006440), and Fmr1tm1Cgr (Fmr1 /y, stock number 003025). To obtain Emx1-Cre ; Rptorloxp/ ; Pten / mice, Rptorloxp/loxp;

Pten / male mice were mated with Emx1-Cre female mice. Rptorloxp/ mice were used as controls. All animal experiments were conducted in accordance with

NIH and AAALAC guidelines and were approved by The Scripps Research Institutes Institutional Animal Care and Use Committee.

Immunohistochemistry. Mouse brains were xed in 4% PFA, incubated in a 20% sucrose/PBS solution at 4 C for 2 days, and embedded in Tissue-Tek OCT compound (Sakura). Coronal or sagittal sections were collected on Superfrost/Plus slides and immunostained with the following antibodies. Primary antibodies used in this study include phospho-S6 (Ser235/236; 1:2,000, Cell Signaling, #4858), CTIP2 (1:2,000, Abcam, ab18465), SATB2 (1:500, Abcam, ab51502) and c-fos (1:1,000, Millipore, ABE457). Alexa Fluor 488, 594 and 647 conjugated secondary antibodies (Life Technologies) were used in this study. Immunouorescent brain sections were mounted and counterstained with VectaShield with DAPI (Vector Laboratories, H-1500). For c-fos staining, ABC (avidin-biotin complex) kit (Vector Laboratories, PK-6101) and DAB (3,30 Diaminobenzidine; Life Technologies, #34001) were used. Images were obtained with a Olympus VS120 microscope and processed using the VS-DESKTOP software (Olympus).c-fos immunoreactive neurons were quantied manually using the VS-DESKTOP software (Olympus) in the mPFC, including the prelimbic and infralimbic cortices (PL and IL; bregma 1.98, 1.94 and 1.78 mm), and in the BLA (bregma 1.46, 1.58 and 1.70 mm) of coronal sections. For each region, the

number of c-fos neurons was quantied in three coronal brain sections from both left and right hemispheres, and averaged within each animal. The c-fos activation index was calculated as the average number of c-fos neurons in each region divided by the time that the mouse spent snifng the novel mouse during the social exposure period.

Cell size analysis. Brain sections were stained with NeuroTrace green uorescent Nissl stain (1:100 dilution). Regions of interest (ROIs) were cropped from primary somatosensory cortex barrel eld (coronal sections) using Photoshop (Adobe). Each layer (layers IIVI) was further cropped according the anatomical boundaries as judged by uorescent Nissl stain. Cell soma size of all neurons in each layer was measured56 using ImageJ (NIH). For BLA-projecting mPFC neurons, mCherry cells were measured. The freehand tool in ImageJ was used to contour the boundary of neuronal soma. Three ROIs were measured in each animal.

Western blot analysis. Whole cerebral corticies or prefrontal cortices were extracted from mice, snap frozen in liquid nitrogen and stored at 80 C until

used. Tissue was homogenized in radioimmunoprecipitation buffer containing the Protease Inhibitor Cocktail (Roche) and phosphatase inhibitors (Sigma-Aldrich). Total protein concentrations were measured using the Pierce BCA Protein Assay Kit (ThermoScientic), and samples were diluted for equal amounts of protein in each lane. Proteins were electrophoresed onto NuPAGE 412% Bis-Tris Gel (Novex, Life Technologies) and were subsequently transferred to polyvinylidene diuoride membranes (Immobilon, Millipore). Primary antibodies used in this study were phospho-S6 (S235/236; 1:2,000, Cell Signaling, #4858), S6 (1:2,000, cell signaling, #2217), CRMP2 (1:1,000, Cell Signaling, #9393), Tau (1:1,000, Cell Signaling, #4019) and b-actin (1:2,000, Cell Signaling, #4970). Peroxydase-conjugated anti-rabbit IgG secondary antibody (1:5,000, Jackson ImmunoResearch Laboratories, Inc.) was used. Proteins were visualized with enhanced chemiluminescence using the WesternBright Quantum kit (Advansta) and quantied using ImageJ software. Phospho-S6 level was normalized to total S6 level. Graphs represent an average of at least 5 animals. Images have been cropped for presentation. Full size images are presented in Supplementary Figs 8 and 9.

Pharmacological S6K1 inhibitor treatment. The S6K1 inhibitor PF-4708671 was dissolved in DMSO. Both vehicle and PF-4708671 were formulated in 10% DMSO, 10% tween 80 and 80% water. PF-4708671 (75 mg kg 1) or vehicle were injected subcutaneously into pups from P4 to P14. For adult female mice (24 month of age), intraperitoneal injection of PF-4708671 (75 mg kg 1) was performed for 11 consecutive days. 75 mg kg 1 was selected based on prior studies reporting that doses of

PF-4708671 in this range can suppress p-S6 levels in the adult mouse brain57.

3D neuronal reconstruction. Etv1-CreERT2 ; Ai14 / ; Pten / and Etv1-CreERT2 ; Ai14 / ; Fmr1 /y mice, as well as controls, received subcutaneous tamoxifen injections (3 mg kg 1) at P7, and were perfused at P14.

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Brains were xed in 4% PFA, incubated in a 20% sucrose/PBS solution at 4 C for 3 days, and embedded in Tissue-Tek OCT compound. Coronal sections (500 mm thickness) in the primary somatosensory cortex were collected and incubated in Scale A2 solution58 for at least 14 days, making the tissue transparent. Layer V neurons were imaged using a Confocal Laser Scanning Microscope Leica TCS SP8, and reconstructed and analysed using Neuromantic reconstruction software59, with Sholl analysis performed using Neurolucida.

Axon tracing of mPFC to BLA projections. Axon tracing in this study followed a previously published method60. AAV-tdTomato was obtained from Penn Vector Core. For injections, mice were anaesthetized under isourane and placed onto a stereotaxic frame. Bregma and lambda were used to nd the coordinates of the mPFC (1.9 mm anterior to bregma, 0.4 mm lateral to midline and 2.3 mm ventral to bregma). The virus, diluted to 10% with water, was injected by a syringe pump at a rate of 100 nl min 1, with 200 nl volume injected per site in each mouse. Mice were perfused 2 weeks later, allowing for the expression of tdTomato. 100 mm-thick sagittal brain sections were collected to image mPFC axon terminals in the BLA (based on mouse brain atlas sagittal sections from the Allen Brain Atlas), with setting as z-series (stacks at 1 mm depth interval) using a 25 Leica objective with

constant parameter for laser power and gain. tdTomato expressing axons (300350 mm length including the axon terminal) in the BLA were traced and analysed using Neuromantic reconstruction software59. Protrusions more than6 mm long were characterized as axon branches. Boutons were dened as at least two times wider than the width of axons, with uorescence intensity at least two times that in axon backbones.

Mouse behaviour tests. For three-chamber social approach test, mice were tested as previously described20,21. Briey, mice were housed under a reversed 12:12 h light cycle for at least 2 weeks before testing. Mice were put into the apparatus for 5 min to habituate on each of the 2 days preceding the test, and then returned to the housing room. During the test, each mouse was put into the three-chamber apparatus for 5 min for habituation, followed by 10 min of testing with a novel stimulus mouse inside a tube of one chamber and an empty tube in the chamber of the other side (the centre chamber was left empty). The per cent time spent in each of the three chambers, containing either a stimulus mouse in a tube, an empty tube or nothing (in the centre) was recorded during the 10-min social approach trial.

For non-matching to sample T-maze task, mice were food restricted to 8590% of ad libitum body weight, to increase motivation for this test. The behaviour protocol consisted of 3 days of habituation, followed by alternation training and then delay testing. During habituation, mice were allowed to explore the maze for 4 min per trial, with a total of 4 trials per day. All mice learned to obtain food reward from the trough after 3 days of habituation. During the alternation training, each trial consisted of one forced and one free-choice run. In the forced run, the mouse ran down the centre arm of the maze and was directed into one of the arms. The mouse was then returned to the start box. In the free-choice run, the mouse was required to select the arm opposite to that visited in the forced run to receive a food reward. Mice were given daily training of four trials until they reached criterion, dened as performance of at least 75% correct per day for 2 consecutive days. Both WT and Pten / mice reached criterion after 7 days of training.

During delay testing, each day consisted of two non-delay trials and two delay trials. During delay trials, the mouse was kept in the start box for 60 s between the forced and free-choice runs. Mice received 5 days of delay testing, for a total of 10 trials each of delay and non-delay testing. The non-delay tests were used to determine if the mice maintained criterion performance.

For social exposure without xed interaction time, female mice were housed individually in a home cage containing a perforated acrylic tube for 7 days before the test. On the test day, a novel conspecic female stimulus mouse was placed in the tube within the home cage of subject female mice. The subject mouse was allowed to explore the stimulus mouse for 20 min (social exposed group) and then the stimulus mouse was removed. The behaviour was video-recorded and manually scored to determine the amount of time that the subject mouse spent snifng the stimulus mouse. After the stimulus mouse was removed, the subject mouse remained in the home cage for an additional 2 h before being perfused. For the control group, mice were individually housed in a home cage containing a perforated acrylic tube for 7 days and then perfused.

For social exposure with xed interaction time, female mice were housed individually in a home cage containing a perforated acrylic tube for 7 days before the test. On the test day, a novel conspecic female stimulus mouse was placed in the tube within the home cage of subject female mice. The subject mouse was allowed to explore the stimulus mouse until it reached a xed criterion of 30 s of snifng time (manually scored during the social exposure period). In this way, each subject mouse received roughly equivalent social cue exposure.

DREADD experiments. Mice were anaesthetized with isourane in the stereotaxic frame for the entire surgery and their body temperature was maintained with a heating pad. To reduce activity in mPFC neurons projecting to the BLA, 350 nl of canine adenovirus carrying Cre recombinase (CAV2-Cre)61,62 was bilaterally injected into the BLA (1.45 mm posterior to bregma, 3.3 mm lateral to midline and

5.2 mm ventral to bregma), and 400 nl of AAV carrying Cre-dependent hM4Di and mCherry (AAV-DIO-hM4Di-mCherry) or mCherry alone (AAV-DIO-mCherry) were bilaterally injected into mPFC (1.9 mm anterior to bregma, 0.4 mm lateral to midline, and 2.3 mm ventral to bregma). Injections were performed at a rate of 50 nl per minute. The needle was allowed to sit in the target location for 3 min before the start of viral injection and for 7 min after injection was complete. Social behaviour was tested 4 and 6 weeks after the surgery, with each mouse being tested following vehicle and CNO injections in a counterbalanced design. Mice were giveni.p. injections of either vehicle or CNO (3 mg kg 1 body weight) 30 min before social approach test. CNO was dissolved in saline to a working solution of0.5 mg ml 1.

Laminar plot analysis. After image acquisition, ROIs were cropped from the primary somatosensory cortex across the radial thickness using Photoshop. Cerebral cortex thickness was normalized, and images were then auto-adjusted for each channel and saved. Plot proles for each channel of each image were obtained using ImageJ. Three brains of WT mice were analysed, and 3 ROIs per brain were used for the laminar plot analysis.

SUnSET method for measurement of protein synthesis. We obtained 400 mm coronal prefrontal cortical slices from postnatal day 8 (P8) WT and Pten / mice.

Slices were allowed to recover in articial cerebrospinal uid (in mM: 124 NaCl, 2.8 KCl, 2 CaCl2, 1.25 NaH2PO4, 1.25 MgSO4, 26 NaHCO3, 10 Dextrose) for 2 h at 32 C. Puromycin (5 mg ml 1) was applied to slices for 40 min at 32 C to label newly synthesized proteins. Slices were then snap frozen on dry ice and stored at

80 C until used. Protein (30 mg) was loaded on 412% gradient gels, transferred onto polyvinylidene diuoride membranes, and probed with anti-puromycin antibody (1:5,000, Millipore, MABE343). Anti-mouse HRP-tagged secondary antibody was used and chemiluminiscence was developed using standard ECL reagents. Slices that were not exposed to puromycin were used to control for nonspecic labelling. Quantication was done by measuring intensity of each lane from 14 to 191 kDa, subtracting it from the intensity of the unlabelled lane, normalized to loading control and expressed relative to WT.

Statistical analysis. Prior studies using similar experiments or power analysis were used to determine sample size. Planned comparisons between WT and mutant mice were performed for all assays using independent-sample t tests. One-way analyses of variance (ANOVAs) were used to assess genotype effect in experiments containing control, Pten / , Emx1-Cre ; Rptorloxp/ , and

Emx1-Cre ; Rptorloxp/ ; Pten / mice. Tukeys post hoc tests were used where appropriate. Two-way ANOVAs were used to assess drug and genotype effects and their interactions in experiments containing WT and Pten / mice with vehicle or PF-4708671 treatment; Tukeys post hoc tests were used where appropriate. Planned comparison independent-sample t tests compared WT and Pten / with vehicle treatment, and compared vehicle and PF-4708671-treated

Pten / mice. For the three-chamber social approach test, two-wayANOVAs were used to assess the interaction between chamber time and genotype; Sidaks post hoc tests were used where appropriate. Paired t tests were also used within genotype to determine if mice showed a preference for the social chamber. All statistics were performed using Graphpad, with signicance set at Po0.05. Throughout the manuscript, values represent means; error bars indicate s.e.m.s; N values refer to biological replicates. All measurements and testing were performed blind to the genotype or experimental manipulations.

Data availability. All relevant data are available upon request.

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Acknowledgements

We thank members of the Page laboratory (Amy Clipperton-Allen, Julien Sejourne and Ori Cohen) for technical assistance and comments on this manuscript. We alsothank Massimiliano Aceti, Chien-Cheng, Juanji An, Clint Kenney and Thomas Vaissiere for technical assistance. This research was supported by gift funds from Ms. Nancy Lurie Marks, The American Honda and Childrens Healthcare Charity Inc., the Fraternal Order of Eagles, and an anonymous donor, as well as National Instituteof Health (NIH) grants R01MH105610 and R01MH108519, and Simons Foundation Grant Award 360712.

Author contributions

W.-C.H. and D.T.P. conceived and designed the experiments. W.-C.H. executed experiments and data analysis, and performed and analysed all the experiments in this study except the three-chamber social approach test on Emx1-Cre ; Ptenloxp/ mice, which was performed and analysed by Y.C. Data interpretation by W.C.H.,

Y.C. and D.T.P.; W.-C.H. and D.T.P. wrote the paper. W.-C.H., Y.C. and D.T.P. edited the paper.

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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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Title

Hyperconnectivity of prefrontal cortex to amygdala projections in a mouse model of macrocephaly/autism syndrome

Author

Huang, Wen-chin; Chen, Youjun; Page, Damon T

Pages

13421

Publication year

2016

Publication date

Nov 2016

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Nature Publishing Group

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20411723

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Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms13421

ProQuest document ID

1839102310

Hyperconnectivity of prefrontal cortex to amygdala projections in a mouse model of macrocephaly/autism syndrome

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