Neuronal activity is required for the development of specific cortical interneuron subtypes

Journal name:
Nature
Volume:
472,
Pages:
351–355
Date published:
DOI:
doi:10.1038/nature09865
Received
Accepted
Published online
Corrected online

Electrical activity has been shown to regulate development in a variety of species and in various structures1, including the retina2, 3, 4, spinal cord5, 6 and cortex5. Within the mammalian cortex specifically, the development of dendrites and commissural axons in pyramidal cells is activity-dependent7, 8. However, little is known about the developmental role of activity in the other major cortical population of neurons, the GABA-producing interneurons. These neurons are morphologically and functionally heterogeneous and efforts over the past decade have focused on determining the mechanisms that contribute to this diversity9, 10, 11. It was recently discovered that 30% of all cortical interneurons arise from a relatively novel source within the ventral telencephalon, the caudal ganglionic eminence (CGE)11, 12. Owing to their late birth date, these interneurons populate the cortex only after the majority of other interneurons and pyramidal cells are already in place and have started to functionally integrate. Here we demonstrate in mice that for CGE-derived reelin (Re)-positive and calretinin (Cr)-positive (but not vasoactive intestinal peptide (VIP)-positive) interneurons12, 13, activity is essential before postnatal day 3 for correct migration, and that after postnatal day 3, glutamate-mediated activity controls the development of their axons and dendrites. Furthermore, we show that the engulfment and cell motility 1 gene (Elmo1)14, a target of the transcription factor distal-less homeobox 1 (Dlx1)15, is selectively expressed in Re+ and Cr+ interneurons and is both necessary and sufficient for activity-dependent interneuron migration. Our findings reveal a selective requirement for activity in shaping the cortical integration of specific neuronal subtypes.

At a glance

Figures

  1. Defective morphology of Cr+ and Re+ interneuron subtypes resulting from Kir2.1 expression.
    Figure 1: Defective morphology of Cr+ and Re+ interneuron subtypes resulting from Kir2.1 expression.

    a, Representative examples of P8 VIP+, Cr+ and Re+ interneurons in mice electroporated at E15.5 with Dlx5/6-eGFP (control) or Dlx5/6-eGFP, Dlx5/6-Kir2.1 plasmids at E15.5. Photomicrographs of eGFP expression and corresponding neurolucida reconstructions depicting axons (red), dendrites (blue) and somata (black). Scale bar, 50μm. b, Morphometric analysis of control and Kir2.1-expressing VIP+, Cr+ and Re+ subtypes including the total length of axonal arborizations (top) and number of axonal nodes (bottom). c, Total length of dendritic trees (top) and number of dendritic nodes (bottom) in the same subtypes. Mean values (±s.e.m.) were obtained from >4 reconstructed interneurons each in Dlx5/6-eGFP and Dlx5/6-eGFP, Dlx5/6-Kir2.1 electroporated mice. Paired t-test: *P<0.05, **P<0.01.

  2. Neuronal activity is essential for the proper laminar migration of selective interneuron subtypes.
    Figure 2: Neuronal activity is essential for the proper laminar migration of selective interneuron subtypes.

    a, Laminar positioning of electroporated interneurons in wild-type mice (control) and tetO-Kir2.1.ires.LacZ littermates both co-electroporated with Dlx5/6-Tta and Dlx5/6-eGFP plasmids at E15.5. Tbr1 expression delineates layers II/III and V at P5–P8. Representative examples taken from the analysis of four control and six tetO-Kir2.1.ires.LacZ electroporated mice for each developmental stage. b, Quantification of the distribution of VIP+, Cr+ and Re+ interneuron subtypes across cortical layers at P8. Owing to the lack of selective molecular markers to distinguish between cortical layer II and III at P8–P9, we divided these layers collectively into II/IIIt and II/IIIb (where II/IIIt and II/IIIb refer to II/III top and II/III bottom, respectively). Mean percentage values (±s.e.m.) were obtained from four wild-type and six tetO-Kir2.1.ires.LacZ electroporated mice. Paired t-test: *P<0.05, **P<0.01, ***P<0.001.

  3. Specific interneuron subtypes require activity for migration and morphological maturation at two distinct stages of development.
    Figure 3: Specific interneuron subtypes require activity for migration and morphological maturation at two distinct stages of development.

    a, Laminar positioning of P8 electroporated interneurons in wild-type mice (control) and tetO-Kir2.1.ires.LacZ mice both co-electroporated with Dlx5/6-Tta and Dlx5/6-eGFP plasmids at E15.5. Mice received either no treatment (Kir2.1(on)); or were treated with doxycycline at E16.5 (Kir2.1(off) at P0 onwards); or with doxycycline at P0 (Kir2.1(off) at P3 onwards). b, β-Galactosidase (β-Gal) activity in P8 tetO-Kir2.1.ires.LacZ mice co-electroporated with Dlx5/6-Tta and Dlx5/6-eGFP plasmids either untreated or treated with doxycycline at E16.5 (Kir2.1(off) at P0 onwards). c, Neurolucida reconstructions of Cr+ and Re+ interneurons in wild-type (control) and tetO-Kir2.1.ires.LacZ mice both co-electroporated with Dlx5/6-Tta and Dlx5/6-eGFP plasmids. Mice received either no doxycycline treatment (Kir2.1(on)) or doxycycline at P0 (Kir2.1(off) at P3 onwards). Axons are shown in red, dendrites in blue and somata in black. Scale bar, 50μm. d, Quantification of dendritic and axonal morphology in control and experimental Cr+ and Re+ interneurons in tetO-Kir2.1.ires.LacZ mice after doxycycline administration at P0. Mean percentage values (± s.e.m.) were obtained from >3 reconstructed interneurons each in doxycycline-treated wild-type and tetO-Kir2.1.ires.LacZ mice for each subtype analysed at P8.

  4. Ionotropic glutamate receptor blockade mimics the effects of Kir2.1 expression on Cr+ and Re+ interneuron morphology.
    Figure 4: Ionotropic glutamate receptor blockade mimics the effects of Kir2.1 expression on Cr+ and Re+ interneuron morphology.

    a, Representative examples of P8 VIP+, Cr+ and Re+ interneurons in Dlx5/6-eGFP electroporated mice at E15.5 injected with PBS (control) or kynurenic acid (Kyn) at P3 and corresponding neurolucida reconstructions depicting axons (red), dendrites (blue) and somata (black). Scale bar, 50μm. b, c, Morphometric analysis of control and kynurenic-acid-treated neurons including the total length of axonal arborizations (b, top) and the number of axonal nodes (b, bottom), and the total length of dendritic trees (c, top) and the number of dendritic nodes (c, bottom) in VIP+, Cr+ and Re+ subtypes. Mean percentage values (± s.e.m.) were obtained from three electroporated interneurons each in control and kynurenic-acid-treated mice for each subtype. Paired t-test: *P<0.05, **P = 0.05, ***P<0.01.

  5. Activity-dependent expression of ELMO1 regulates CGE-derived interneuron migration.
    Figure 5: Activity-dependent expression of ELMO1 regulates CGE-derived interneuron migration.

    a, Expression of Dlx genes and ELMO1 at P5 in Dlx5/6-eGFP and Dlx5/6-Kir2.1 electroporated interneurons at E15.5. b, Expression of ELMO1 in GAD67-GFP transgenic mice at P2 and P5. Selective expression of ELMO1 in CGE-derived interneuron subtypes at P9. Quantification of ELMO1 expression in Re+, Cr+ and VIP+ interneurons (IN) at P9 (right). Mean percentage values (± s.e.m.) were obtained from >70 interneurons for each subtype. c, Quantification of DLXHi (where Hi refers to high level of DLX protein expression) and ELMO1 expression in Dlx5/6-eGFP (control) and Dlx5/6-Kir2.1 Re+ E15.5 electroporated interneurons at P5. Mean percentage values (± s.e.m.) were obtained from >20 interneurons each in control and Kir2.1 electroporated mice for each quantification. d, Electroporation of Dlx5/6-Elmo1_TN558.Flag plasmid at E15.5. Flag immunoreactivity is detected in electroporated interneurons at P9 (inset). Neuronal morphology of a Re+ interneuron and laminar distribution of electroporated interneurons at P9. Representative examples from four electroporated mice. e, Co-electroporation of Dlx5/6-Elmo1 and Dlx5/6-Kir2.1 plasmids at E15.5. ELMO1 expression in electroporated interneurons at P9. Morphological defects of an electroporated Re+ interneuron and laminar distribution of electroporated interneurons. Representative examples from six electroporated mice. Scale bars for d and e, 50μm. f, Quantification of the distribution of Re+ interneurons across cortical layers at P9 on expression of different plasmids. Mean percentage values (± s.e.m.) were obtained from >80 interneurons for each group. Values for control and Dlx5/6-Kir2.1 alone groups are repeated from Fig. 2 to facilitate comparison between groups. The large bracket indicates comparison between the control and Dlx5/6-Elmo1_TN558.Flag electroporated internerneurons. Paired t-test: *P<0.05, **P<0.01, ***P<0.0001.

Change history

Corrected online 21 April 2011
Reference 9 was substituted.

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Author information

  1. These authors contributed equally to this work.

    • Natalia V. De Marco García &
    • Theofanis Karayannis

Affiliations

  1. Smilow Neuroscience Program, Departments of Cell Biology and Neural Science, New York University Langone Medical Center, New York, New York 10016, USA

    • Natalia V. De Marco García,
    • Theofanis Karayannis &
    • Gord Fishell

Contributions

N.V.D.G. and G.F. conceived the project. N.V.D.G. and T.K. designed and carried out the experiments. N.V.D.G. wrote the manuscript with the help of all authors.

Competing financial interests

The authors declare no competing financial interests.

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