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Pyramidal cell regulation of interneuron survival sculpts cortical networks

Naturevolume 557pages668673 (2018) | Download Citation

Abstract

Complex neuronal circuitries such as those found in the mammalian cerebral cortex have evolved as balanced networks of excitatory and inhibitory neurons. Although the establishment of appropriate numbers of these cells is essential for brain function and behaviour, our understanding of this fundamental process is limited. Here we show that the survival of interneurons in mice depends on the activity of pyramidal cells in a critical window of postnatal development, during which excitatory synaptic input to individual interneurons predicts their survival or death. Pyramidal cells regulate interneuron survival through the negative modulation of PTEN signalling, which effectively drives interneuron cell death during this period. Our findings indicate that activity-dependent mechanisms dynamically adjust the number of inhibitory cells in nascent local cortical circuits, ultimately establishing the appropriate proportions of excitatory and inhibitory neurons in the cerebral cortex.

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References

  1. 1.

    Beaulieu, C. Numerical data on neocortical neurons in adult rat, with special reference to the GABA population. Brain Res. 609, 284–292 (1993).

  2. 2.

    Meyer, H. S. et al. Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A. Proc. Natl Acad. Sci. USA 108, 16807–16812 (2011).

  3. 3.

    Gabbott, P. L. & Somogyi, P. Quantitative distribution of GABA-immunoreactive neurons in the visual cortex (area 17) of the cat. Exp. Brain Res. 61, 323–331 (1986).

  4. 4.

    Hendry, S. H., Schwark, H. D., Jones, E. G. & Yan, J. Numbers and proportions of GABA-immunoreactive neurons in different areas of monkey cerebral cortex. J. Neurosci. 7, 1503–1519 (1987).

  5. 5.

    DeFelipe, J., Alonso-Nanclares, L. & Arellano, J. I. Microstructure of the neocortex: comparative aspects. J. Neurocytol. 31, 299–316 (2002).

  6. 6.

    Fishell, G. & Rudy, B. Mechanisms of inhibition within the telencephalon: “where the wild things are”. Annu. Rev. Neurosci. 34, 535–567 (2011).

  7. 7.

    Marín, O. Interneuron dysfunction in psychiatric disorders. Nat. Rev. Neurosci. 13, 107–120 (2012).

  8. 8.

    Nelson, S. B. & Valakh, V. Excitatory/inhibitory balance and circuit homeostasis in autism spectrum disorders. Neuron 87, 684–698 (2015).

  9. 9.

    Yizhar, O. et al. Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477, 171–178 (2011).

  10. 10.

    Hamburger, V. & Levi-Montalcini, R. Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions. J. Exp. Zool. 111, 457–501 (1949).

  11. 11.

    Yuan, J. & Yankner, B. A. Apoptosis in the nervous system. Nature 407, 802–809 (2000).

  12. 12.

    Raff, M. C. et al. Programmed cell death and the control of cell survival: lessons from the nervous system. Science 262, 695–700 (1993).

  13. 13.

    Green, D. R. Apoptotic pathways: the roads to ruin. Cell 94, 695–698 (1998).

  14. 14.

    Southwell, D. G. et al. Intrinsically determined cell death of developing cortical interneurons. Nature 491, 109–113 (2012).

  15. 15.

    Verney, C., Takahashi, T., Bhide, P. G., Nowakowski, R. S. & Caviness, V. S. Jr. Independent controls for neocortical neuron production and histogenetic cell death. Dev. Neurosci. 22, 125–138 (2000).

  16. 16.

    Li, Z. et al. Caspase-3 activation via mitochondria is required for long-term depression and AMPA receptor internalization. Cell 141, 859–871 (2010).

  17. 17.

    Goebbels, S. et al. Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice. Genesis 44, 611–621 (2006).

  18. 18.

    Xu, Q., Tam, M. & Anderson, S. A. Fate mapping Nkx2.1-lineage cells in the mouse telencephalon. J. Comp. Neurol. 506, 16–29 (2008).

  19. 19.

    Price, D. J., Aslam, S., Tasker, L. & Gillies, K. Fates of the earliest generated cells in the developing murine neocortex. J. Comp. Neurol. 377, 414–422 (1997).

  20. 20.

    Bartolini, G., Ciceri, G. & Marín, O. Integration of GABAergic interneurons into cortical cell assemblies: lessons from embryos and adults. Neuron 79, 849–864 (2013).

  21. 21.

    Ikonomidou, C. et al. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283, 70–74 (1999).

  22. 22.

    Heck, N. et al. Activity-dependent regulation of neuronal apoptosis in neonatal mouse cerebral cortex. Cereb. Cortex 18, 1335–1349 (2008).

  23. 23.

    Léveillé, F. et al. Suppression of the intrinsic apoptosis pathway by synaptic activity. J. Neurosci. 30, 2623–2635 (2010).

  24. 24.

    Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).

  25. 25.

    Bortone, D. & Polleux, F. KCC2 expression promotes the termination of cortical interneuron migration in a voltage-sensitive calcium-dependent manner. Neuron 62, 53–71 (2009).

  26. 26.

    Priya, R. et al. Activity regulates cell death within cortical interneurons through a calcineurin-dependent mechanism. Cell Reports 22, 1695–1709 (2018).

  27. 27.

    Denaxa, M. et al. Modulation of apoptosis controls inhibitory interneuron number in the cortex. Cell Reports 22, 1710–1721 (2018).

  28. 28.

    Anastasiades, P. G. et al. GABAergic interneurons form transient layer-specific circuits in early postnatal neocortex. Nat. Commun. 7, 10584 (2016).

  29. 29.

    Roth, B. L. DREADDs for neuroscientists. Neuron 89, 683–694 (2016).

  30. 30.

    Lindsten, T. et al. The combined functions of proapoptotic Bcl-2 family members Bak and Bax are essential for normal development of multiple tissues. Mol. Cell 6, 1389–1399 (2000).

  31. 31.

    Dudek, H. et al. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275, 661–665 (1997).

  32. 32.

    Datta, S. R. et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231–241 (1997).

  33. 33.

    Stambolic, V. et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29–39 (1998).

  34. 34.

    Backman, S. A. et al. Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte–Duclos disease. Nat. Genet. 29, 396–403 (2001).

  35. 35.

    Fogarty, M. et al. Spatial genetic patterning of the embryonic neuroepithelium generates GABAergic interneuron diversity in the adult cortex. J. Neurosci. 27, 10935–10946 (2007).

  36. 36.

    Lesche, R. et al. Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene. Genesis 32, 148–149 (2002).

  37. 37.

    Grigoriou, M., Tucker, A. S., Sharpe, P. T. & Pachnis, V. Expression and regulation of Lhx6 and Lhx7, a novel subfamily of LIM homeodomain encoding genes, suggests a role in mammalian head development. Development 125, 2063–2074 (1998).

  38. 38.

    Isaacson, J. S. & Scanziani, M. How inhibition shapes cortical activity. Neuron 72, 231–243 (2011).

  39. 39.

    Xue, M., Atallah, B. V. & Scanziani, M. Equalizing excitation–inhibition ratios across visual cortical neurons. Nature 511, 596–600 (2014).

  40. 40.

    Burrone, J., O’Byrne, M. & Murthy, V. N. Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature 420, 414–418 (2002).

  41. 41.

    Maffei, A., Nataraj, K., Nelson, S. B. & Turrigiano, G. G. Potentiation of cortical inhibition by visual deprivation. Nature 443, 81–84 (2006).

  42. 42.

    Butt, S. J. et al. The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes. Neuron 59, 722–732 (2008).

  43. 43.

    Cobos, I. et al. Mice lacking Dlx1 show subtype-specific loss of interneurons, reduced inhibition and epilepsy. Nat. Neurosci. 8, 1059–1068 (2005).

  44. 44.

    Glickstein, S. B. et al. Selective cortical interneuron and GABA deficits in cyclin D2-null mice. Development 134, 4083–4093 (2007).

  45. 45.

    Lui, J. H., Hansen, D. V. & Kriegstein, A. R. Development and evolution of the human neocortex. Cell 146, 18–36 (2011).

  46. 46.

    Florio, M. & Huttner, W. B. Neural progenitors, neurogenesis and the evolution of the neocortex. Development 141, 2182–2194 (2014).

  47. 47.

    Butler, M. G. et al. Subset of individuals with autism spectrum disorders and extreme macrocephaly associated with germline PTEN tumour suppressor gene mutations. J. Med. Genet. 42, 318–321 (2005).

  48. 48.

    Buxbaum, J. D. et al. Mutation screening of the PTEN gene in patients with autism spectrum disorders and macrocephaly. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 144B, 484–491 (2007).

  49. 49.

    Blanquie, O. et al. Electrical activity controls area-specific expression of neuronal apoptosis in the mouse developing cerebral cortex. eLife 6, e27696 (2017).

  50. 50.

    DeFelipe, J. Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex. J. Chem. Neuroanat. 14, 1–19 (1997).

  51. 51.

    Mort, R. L. et al. Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice. Cell Cycle 13, 2681–2696 (2014).

  52. 52.

    Fazzari, P. et al. Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling. Nature 464, 1376–1380 (2010).

  53. 53.

    West, M. J. & Gundersen, H. J. Unbiased stereological estimation of the number of neurons in the human hippocampus. J. Comp. Neurol. 296, 1–22 (1990).

  54. 54.

    Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

  55. 55.

    Green, D. M. & Swets, J. A. Signal Detection Theory and Psychophysics. (Wiley, Oxford, 1966).

  56. 56.

    Krashes, M. J. et al. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. J. Clin. Invest. 121, 1424–1428 (2011).

  57. 57.

    Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

  58. 58.

    Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

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Acknowledgements

We thank S. Bae for laboratory support, I. Andrew for management of mouse colonies, V. van den Berghe for help with breeding strategies, S. A. Anderson, N. Kessaris, R. L. Mort and K. A. Nave for mouse lines, N. Flames, C. Houart and M. Maravall for critical reading of the manuscript, and members of the Marín and Rico laboratories for stimulating discussions and ideas. This work was supported by a grant from the Wellcome Trust (103714MA) to O.M. F.K.W. was supported by an EMBO postdoctoral fellowship and is currently a Marie Skłodowska-Curie Fellow from the European Commission under the H2020 Programme. K.B. is a Henry Wellcome Postdoctoral Fellow and O.M. is a Wellcome Trust Investigator.

Author information

Author notes

  1. These authors contributed equally: Fong Kuan Wong, Kinga Bercsenyi.

Affiliations

  1. Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK

    • Fong Kuan Wong
    • , Kinga Bercsenyi
    • , Varun Sreenivasan
    • , Adrián Portalés
    • , Marian Fernández-Otero
    •  & Oscar Marín
  2. Medical Research Council Centre for Neurodevelopmental Disorders, King’s College London, London, UK

    • Fong Kuan Wong
    • , Kinga Bercsenyi
    • , Varun Sreenivasan
    • , Adrián Portalés
    • , Marian Fernández-Otero
    •  & Oscar Marín

Authors

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Contributions

F.K.W., K.B., V.S., and O.M. designed experiments. F.K.W., K.B., A.P. and M.F.-O. carried out stereology quantifications. V.S. performed and analysed in vivo imaging experiments. F.K.W. performed and analysed DREADDs experiments, except for the analysis of PTEN levels, which was carried out by K.B. F.K.W. analysed Bax/Bak mutant mice. K.B. performed western blots, examined interneuron PTEN levels and analysed Pten mutant mice. F.K.W. performed in vivo pharmacological PTEN inhibition experiments. F.K.W., K.B., V.S., and O.M. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Oscar Marín.

Extended data figures and tables

  1. Extended Data Fig. 1 Extensive cell death in layer 2–6 pyramidal cells.

    a, Coronal sections through the S1 cortex of P4 NexCre/+;Fucci2 (left) and P7 Nkx2-1-Cre;RCLtdTomato (right) mice immunostained for cleaved caspase-3 (yellow) and mCherry (green, left) or tdTomato (magenta, right). b, Quantification of density of cleaved caspase-3 cells in pyramidal neurons (left, green) and MGE interneurons (right, magenta) during postnatal development (for pyramidal neurons, ANOVA, F = 73.6, ***P = 0.003 (P2 versus P4), ***P = 0.00006 (P4 versus P7), n = 3 mice for all ages; for MGE interneurons, ANOVA, F = 16.91, *P = 0.027 (P5 versus P7), **P = 0.0029 (P7 versus P10), n = 3 animals for all ages). c, Coronal sections through the barrel cortex of NexCre/+;Fucci2 mice during postnatal development immunostained for mCherry (green) and CTGF (yellow). d, Total number of pyramidal cells excluding subplate cells in the neocortex of NexCre/+;Fucci2 mice (ANOVA, F = 4.83 and *P = 0.03; n = 3 mice for P2 and P5, and 4 mice for P3, P4 and P21). e, Temporal variation in the percentage of pyramidal cells excluding the subplate contribution during postnatal development. Data are shown as mean ± s.e.m. Scale bars, 100 µm.

  2. Extended Data Fig. 2 Interneuron cell loss in the barrel field during postnatal development.

    a, Coronal sections through S1BF of Nkx2-1-Cre;RCLtdTomato mice (magenta, MGE interneurons) during postnatal development counterstained with DAPI (grey). b, Total number of MGE and POA interneurons in S1BF of Nkx2-1-Cre;RCLtdtomato mice during postnatal development (ANOVA, F = 6.40 and *P = 0.03; n = 4 animals for each age). Data are shown as mean ± s.e.m. Scale bar, 100 µm.

  3. Extended Data Fig. 3 Alteration of pyramidal cell activity affects interneuron density but not distribution.

    a, Coronal sections through S1BF cortex immunostained for GABA (magenta) and NeuN (green) and counterstained with DAPI (grey) from P21 NexCre/+ mice injected with hM3Dq-mCherry virus followed by vehicle or CNO treatment. b, Quantification of the density of GABA (left) and NeuN+ but GABA (right) cells in P21 mice injected with hM3Dq-mCherry followed by vehicle (grey) or CNO (magenta) treatment (two-tailed Student’s unpaired t-test, **P = 0.005 (GABA), P = 0.68 (NeuN+ GABA), n = 4 animals for vehicle, n = 3 animals for CNO conditions). c, d, Quantification of the distribution of PV+ (left) and SST+ neurons (right) in P21 NexCre/+ mice injected at P0 with hM3Dq-mCherry (c) or hM4Di-mCherry (d) and treated with vehicle (grey) or CNO (magenta) during P5–P8 (two-way ANOVA, Ftreatment = 0.48, P = 0.50 (hM3Dq PV), Ftreatment = −0.04, P = 0.99 (hM3Dq SST), Ftreatment = 0.88, P = 0.37 (hM4DI PV), Ftreatment = 0.79, P = 0.39 (hM4DI SST); for PV, n = 7 animals for hM3Dq and hM4DI −CNO, 6 animals for hM3Dq +CNO, and 5 animals for hM4DI +CNO; for SST, n = 9 animals for hM3Dq −CNO, 7 animals for hM3Dq +CNO and hM4Di −CNO, and 5 animals for hM4DI +CNO). e, Coronal sections through auditory cortex immunostained for PV (magenta) or SST (magenta) and counterstained with DAPI (grey) from P21 NexCre/+ mice injected with hM3Dq-mCherry virus followed by vehicle or CNO treatment. f, Quantification of the density of PV+ (right) and SST+ neurons (left) in auditory cortex in P21 mice injected with hM3Dq-mCherry followed by vehicle (grey) or CNO (magenta) treatment (two-tailed Student’s unpaired t-test, P = 0.574 (PV), P = 0.419 (SST), n = 4 animals for both). Data are shown as mean ± s.e.m. Scale bars, 100 µm.

  4. Extended Data Fig. 4 CNO control experiments.

    a, Schematic of experimental design. b, Coronal sections through S1 of P8 NexCre mice injected with AAV8-dio-hM4Di-mCherry at P0 and treated with (+) or without (−) CNO between P5 and P8, immunostained for cleaved caspase-3 (magenta) and counterstained with DAPI (grey). c, Quantification of the density of cleaved caspase-3 cells in P8 mice injected with AAV8-dio-hM4Di-mCherry and treated (magenta) or not treated (grey) with CNO between P5 and P8 (two-tailed Student’s unpaired t-test, ***P = 0.009, n = 8 animals for −CNO, and n = 7 animals for +CNO). d, Schematic of experimental design for CNO control experiments. e, Quantification of the density of PV+ (left) and SST+ (right) cells in P21 mice injected with hM3Dq-mCherry or hM4Di-mCherry and not treated with CNO (grey), or not injected with viruses and treated with CNO (magenta) between P5 and P8 (ANOVA, P = 0.24 (PV+) and P = 0.65 (SST+); for PV, n = 7 animals for hM3Dq and hM4DI −CNO, 4 animals for non-injected +CNO; for SST, n = 9 animals for hM3Dq −CNO, 7 animals for hM4Di −CNO, and 4 animals for non-injected +CNO). Data are shown as mean ± s.e.m. Scale bar, 100 µm.

  5. Extended Data Fig. 5 Alteration of pyramidal cell activity beyond the normal period of interneuron cell death does not affect interneuron survival or distribution.

    a, Schematic of experimental design. b, c, Coronal sections through S1BF immunostained for PV (b) or SST (c) and counterstained with DAPI (grey) from P21 NexCre/+ mice injected with hM3Dq-mCherry (left) or hM4Di-mCherry (right) viruses followed by vehicle or CNO treatment. d, g, Quantification of the density of PV+ (d) and SST+ (g) cells in P21 hM3Dq-mCherry injected mice (left bars) and hM4Di-mCherry injected mice (right bars) followed by vehicle (grey bars) and CNO (magenta bars) treatment at P10–P13 (for PV, two-tailed unpaired Student’s t-test, P = 0.99 and P = 0.087, respectively; for SST, two-tailed unpaired Student’s t-test, P = 0.56 and P = 0.37, respectively; n = 4 animals for hM3Dq –CNO and 3 animals for all other groups). e, f, h, i, Quantification of the distribution of PV+ (e, f) and SST+ cells (h, i) in mice injected with hM3Dq-mCherry (e, h) or hM4Di-mCherry (f, i) followed by vehicle (grey bars) or CNO (magenta bars) treatment at P10–P13 (two-way ANOVA, Ftreatment = 0.15, P = 0.71 (hM3Dq PV), Ftreatment = 0.60, P = 0.48 (hM3Dq SST), Ftreatment = 1.00, P = 0.37 (hM4DI PV), Ftreatment = 1.78, P = 0.25 (hM4DI SST); n = 4 animals for hM3Dq –CNO and 3 animals for all other groups). Data are shown as mean ± s.e.m. Scale bar, 100 µm.

  6. Extended Data Fig. 6 Loss of BAK and BAX prevents programmed cell death in pyramidal cells.

    a, Coronal sections through S1BF from P2 and P21 NexCre/+;Bak−/−;Baxfl/fl;Fucci2 mice immunostained for mCherry (green) and CTGF (yellow). b, Total number of pyramidal cells (excluding subplate cells) in the neocortex of P2 and P21 NexCre/+;Bak−/−;Baxfl/fl;Fucci2 mice (two-tailed Student’s unpaired t-test, P = 0.30; n = 3 animals for both ages). Data are shown as mean ± s.e.m. Scale bar, 100 µm.

  7. Extended Data Fig. 7 Loss of BAK and BAX in pyramidal cells or MGE and POA interneurons affects densities but not lamination of MGE and POA interneurons.

    a, Quantification of the distribution of PV+ (left) and SST+ (right) interneurons in P30 control (grey), NexCre/+;Bak−/−;Baxfl/fl (dark magenta) and Nkx2-1-Cre;Bak−/−;Baxfl/fl (light magenta) mice (two-way ANOVA, Ftreatment = 3.56, P = 0.10 (NexCre/+ PV), Ftreatment = 0.44, P = 0.53 (Nkx2-1-Cre PV), Ftreatment = 0, P = 0.99 (NexCre/+ SST), Ftreatment = 0.44, P = 0.54 (Nkx2-1-Cre SST), n = 4 animals for NexCre/+;Bak−/−;Baxfl/fl (PV) and 5 animals for all other groups). b, Quantification of the fold change in the density of PV+ (top) and SST+ (bottom) interneurons in NexCre/+;Bak−/−;Baxfl/fl (dark magenta) and Nkx2-1-Cre;Bak−/−;Baxfl/fl (light magenta) mice compared to their respective controls (two-tailed Student’s unpaired t-test, P = 0.90 (PV), P = 0.67 (SST); for PV, n = 4 animals for NexCre/+;Bak−/−;Baxfl/fl, 6 animals for Nkx2-1-Cre;Bak−/−;Baxfl/fl; for SST, n = 5 animals for both NexCre/+;Bak−/−;Baxfl/fl and Nkx2-1-Cre;Bak−/−;Baxfl/fl). c, Coronal sections through the motor cortex of P30 Bak+/+;Baxfl/fl and NexCre/+;Bak−/−;Baxfl/fl mice immunostained for parvalbumin (PV, left) and somatostatin (SST, right) and counterstained with DAPI (grey). d, Quantification of the density of PV+ (left) and SST+ (right) cells in the motor cortex of control and pyramidal cell-specific Bax/Bak double mutant mice at P30 (two-tailed Student’s unpaired t-test, *P = 0.02 (PV), *P = 0.01 (SST); for PV, n = 4 animals for both; for SST, n = 3 animals for both). Data are shown as mean ± s.e.m. Scale bar, 100 µm.

  8. Extended Data Fig. 8 PTEN expression in deep layer cortical interneurons and effects of loss of PTEN function on neurons and blood vessels.

    a, Coronal sections through layer 5 of S1BF from Nkx2-1-Cre;RCLtdTomato mice at P5, P7, P8 and P10, immunostained for PTEN and counterstained with DAPI (grey). PTEN expression is shown as a custom LUT in tdTomato-masked cells. b, Cumulative distribution of mean PTEN intensity in layer 5 and 6 MGE and POA interneurons (Kruskal–Wallis test, ***P = 0; n = 7,270 cells (P5), 4,544 cells (P7), 6,780 cells (P8) and 5,043 cells (P10) from 3 mice at each age). c, Coronal sections through S1BF from Ptenfl/fl and Lhx6-Cre;Ptenfl/fl mice at P16 immunostained for GABA (red, left), NeuN (green, middle) and isolectin B4 (IB4, cyan, right) and counterstained with DAPI (grey). d, Quantification of the density of GABA+ (far left) and NeuN+ GABA (left) cells and vessel area (right) and diameter (far right) in P16 Ptenfl/fl (grey) and Lhx6-Cre;Ptenfl/fl (magenta) mice (two-tailed unpaired Student’s t-test, **P = 0.0035 (GABA), *P = 0.0326 (vessel area), P = 0.0810 (vessel diameter); Kolmogorov–Smirnov test, P = 0.1000 (NeuN+ GABA cells), n = 3 mice for both genotypes). e, Quantification of the distribution of PV+ (left) and SST+ (right) cells in P16 Ptenfl/fl (grey) and Lhx6-Cre;Ptenfl/fl (magenta) mice (two-way ANOVA, Fgenotype = 0.29, P = 0.61 (PV); Fgenotype = 0.0004, P = 0.98 (SST); n = 4 Ptenfl/fl mice and 3 Lhx6-Cre;Ptenfl/fl mice). Data are shown as mean ± s.e.m. Scale bars, 100 µm.

  9. Extended Data Fig. 9 Pharmacological inhibition of PTEN during the interneuron cell death period increases interneuron survival.

    a, f, Schematics of experimental design. b, Coronal sections through S1BF from P10 mice injected at P7–P8 with vehicle (left) or BpV(pic) (right) stained for isolectin B4 (IB4, cyan) and DAPI (grey). c, Quantification of blood vessel area (left) and diameter (right) in P10 mice treated with vehicle (grey) or BpV(pic) (magenta) (Kolmogorov–Smirnov test (vessel area), P = 0.60; two-tailed unpaired Student’s t-test (vessel diameter), P = 0.58, n = 3 animals for each group). d, g, Coronal sections through S1BF from P21 mice injected at P7–P8 (d) or P12–P13 (g) with vehicle (left) or BpV(pic) (right) and immunostained for PV and SST and counterstained with DAPI. e, h, Quantification of the density of PV+ (left) and SST+ (right) cells in S1BF from P21 mice injected at P7–P8 (e) or P12–P13 (h) with vehicle (grey) or BpV(pic) (magenta) (P7–P8 groups: two-tailed unpaired Student’s t-test, *P = 0.04 (PV), *P = 0.03 (SST); n = 7 mice for each group; P12–P13 groups: two-tailed unpaired Student’s t-test, P = 0.84 (PV), P = 0.82 (SST), n = 5 animals for each group). Data are shown as mean ± s.e.m. Scale bars, 100 µm.

Supplementary information

  1. Supplementary Table 1

    Summary of data and statistical analyses reported in Figures 1-5 and Extended Data Figures 1–9.

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