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Pyramidal cell regulation of interneuron survival sculpts cortical networks

Nature volume 557, pages 668–673 (2018)Cite this article

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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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Fig. 1: Consecutive waves of programmed cell death of pyramidal cells and interneurons in the early postnatal cortex.
Fig. 2: Interneuron activity levels predict cell death.
Fig. 3: Bidirectional modulation of pyramidal cell activity regulates the extent of interneuron cell death.
Fig. 4: Survival of pyramidal cells rescues interneuron cell death.
Fig. 5: Pyramidal cell activity controls interneuron cell survival through PTEN inhibition.

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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.

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Author notes

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

Authors and 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

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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.

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Correspondence to Oscar Marín.

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Extended data figures and tables

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.

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.

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.

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.

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.

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.

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.

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.

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.

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Supplementary Table 1

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

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Wong, F.K., Bercsenyi, K., Sreenivasan, V. et al. Pyramidal cell regulation of interneuron survival sculpts cortical networks. Nature 557, 668–673 (2018). https://doi.org/10.1038/s41586-018-0139-6

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