Sensory inputs control the integration of neurogliaform interneurons into cortical circuits

Abstract

Neuronal microcircuits in the superficial layers of the mammalian cortex provide the substrate for associative cortical computation. Inhibitory interneurons constitute an essential component of the circuitry and are fundamental to the integration of local and long-range information. Here we report that, during early development, superficially positioned Reelin-expressing neurogliaform interneurons in the mouse somatosensory cortex receive afferent innervation from both cortical and thalamic excitatory sources. Attenuation of ascending sensory, but not intracortical, excitation leads to axo-dendritic morphological defects in these interneurons. Moreover, abrogation of the NMDA receptors through which the thalamic inputs signal results in a similar phenotype, as well as in the selective loss of thalamic and a concomitant increase in intracortical connectivity. These results suggest that thalamic inputs are critical in determining the balance between local and long-range connectivity and are fundamental to the proper integration of Reelin-expressing interneurons into nascent cortical circuits.

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Figure 1: Determining the afferent inputs onto developing Re+ interneurons in layers I/III.
Figure 2: Perturbation of sensory inputs during the first postnatal week disrupts the axo-dendritic development of Re+ interneurons.
Figure 3: Severe attenuation of intracortical glutamate release does not interfere with Re+ interneuron morphological development.
Figure 4: Enrichment of NR2B-containing NMDARs activated by thalamic afferents onto Re+ interneurons.
Figure 5: NR2B-containing NMDARs are required for proper Re+ but not VIP+ interneuron development.
Figure 6: Morphological development proceeds normally in NR2A−/− Re+ interneurons.
Figure 7: NMDAR ablation reconfigures afferent connectivity onto Re+ interneurons.

References

  1. 1

    Lien, A.D. & Scanziani, M. Tuned thalamic excitation is amplified by visual cortical circuits. Nat. Neurosci. 16, 1315–1323 (2013).

  2. 2

    Ma, T. et al. Subcortical origins of human and monkey neocortical interneurons. Nat. Neurosci. 16, 1588–1597 (2013).

  3. 3

    Hansen, D.V. et al. Non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences. Nat. Neurosci. 16, 1576–1587 (2013).

  4. 4

    Pfeffer, C.K., Xue, M., He, M., Huang, Z.J. & Scanziani, M. Inhibition of inhibition in visual cortex: the logic of connections between molecularly distinct interneurons. Nat. Neurosci. 16, 1068–1076 (2013).

  5. 5

    Pi, H.J. et al. Cortical interneurons that specialize in disinhibitory control. Nature 503, 521–524 (2013).

  6. 6

    Lee, S., Kruglikov, I., Huang, Z.J., Fishell, G. & Rudy, B. A disinhibitory circuit mediates motor integration in the somatosensory cortex. Nat. Neurosci. 16, 1662–1670 (2013).

  7. 7

    Palmer, L.M. et al. The cellular basis of GABAB-mediated interhemispheric inhibition. Science 335, 989–993 (2012).

  8. 8

    Letzkus, J.J. et al. A disinhibitory microcircuit for associative fear learning in the auditory cortex. Nature 480, 331–335 (2011).

  9. 9

    De Marco García, N.V., Karayannis, T. & Fishell, G. Neuronal activity is required for the development of specific cortical interneuron subtypes. Nature 472, 351–355 (2011).

  10. 10

    Wickersham, I.R., Finke, S., Conzelmann, K.K. & Callaway, E.M. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat. Methods 4, 47–49 (2007).

  11. 11

    Petersen, C.C. The functional organization of the barrel cortex. Neuron 56, 339–355 (2007).

  12. 12

    Xu, X. & Callaway, E.M. Laminar specificity of functional input to distinct types of inhibitory cortical neurons. J. Neurosci. 29, 70–85 (2009).

  13. 13

    Tripodi, M., Stepien, A.E. & Arber, S. Motor antagonism exposed by spatial segregation and timing of neurogenesis. Nature 479, 61–66 (2011).

  14. 14

    Miyamichi, K. et al. Cortical representations of olfactory input by trans-synaptic tracing. Nature 472, 191–196 (2011).

  15. 15

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

  16. 16

    Lee, S., Hjerling-Leffler, J., Zagha, E., Fishell, G. & Rudy, B. The largest group of superficial neocortical GABAergic interneurons expresses ionotropic serotonin receptors. J. Neurosci. 30, 16796–16808 (2010).

  17. 17

    Cruikshank, S.J. et al. Thalamic control of layer 1 circuits in prefrontal cortex. J. Neurosci. 32, 17813–17823 (2012).

  18. 18

    Petreanu, L., Mao, T., Sternson, S.M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).

  19. 19

    Mao, T. et al. Long-range neuronal circuits underlying the interaction between sensory and motor cortex. Neuron 72, 111–123 (2011).

  20. 20

    Toda, T. et al. Birth regulates the initiation of sensory map formation through serotonin signaling. Dev. Cell 27, 32–46 (2013).

  21. 21

    Zhang, Y. et al. V3 spinal neurons establish a robust and balanced locomotor rhythm during walking. Neuron 60, 84–96 (2008).

  22. 22

    Xu, W. & Sudhof, T.C. A neural circuit for memory specificity and generalization. Science 339, 1290–1295 (2013).

  23. 23

    Murray, A.J. et al. Parvalbumin-positive CA1 interneurons are required for spatial working but not for reference memory. Nat. Neurosci. 14, 297–299 (2011).

  24. 24

    Dudok, J.J., Groffen, A.J., Toonen, R.F. & Verhage, M. Deletion of Munc18–1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality. PLoS ONE 6, e28137 (2011).

  25. 25

    Arrigoni, E. & Greene, R.W. Schaffer collateral and perforant path inputs activate different subtypes of NMDA receptors on the same CA1 pyramidal cell. Br. J. Pharmacol. 142, 317–322 (2004).

  26. 26

    Wang, C.C. et al. A critical role for GluN2B-containing NMDA receptors in cortical development and function. Neuron 72, 789–805 (2011).

  27. 27

    Kelsch, W., Li, Z., Eliava, M., Goengrich, C. & Monyer, H. GluN2B-containing NMDA receptors promote wiring of adult-born neurons into olfactory bulb circuits. J. Neurosci. 32, 12603–12611 (2012).

  28. 28

    Matta, J.A. et al. Developmental origin dictates interneuron AMPA and NMDA receptor subunit composition and plasticity. Nat. Neurosci. 16, 1032–1041 (2013).

  29. 29

    Sanz-Clemente, A., Nicoll, R.A. & Roche, K.W. Diversity in NMDA receptor composition: many regulators, many consequences. Neuroscientist 19, 62–75 (2013).

  30. 30

    Koch, S.M. et al. Pathway-specific genetic attenuation of glutamate release alters select features of competition-based visual circuit refinement. Neuron 71, 235–242 (2011).

  31. 31

    Xu, H.P. et al. An instructive role for patterned spontaneous retinal activity in mouse visual map development. Neuron 70, 1115–1127 (2011).

  32. 32

    Fu, Y. et al. A cortical circuit for gain control by behavioral state. Cell 156, 1139–1152 (2014).

  33. 33

    Morishita, H. & Hensch, T.K. Critical period revisited: impact on vision. Curr. Opin. Neurobiol. 18, 101–107 (2008).

  34. 34

    Li, H. et al. Laminar and columnar development of barrel cortex relies on thalamocortical neurotransmission. Neuron 79, 970–986 (2013).

  35. 35

    Katz, L.C. & Shatz, C.J. Synaptic activity and the construction of cortical circuits. Science 274, 1133–1138 (1996).

  36. 36

    Li, H. & Crair, M.C. How do barrels form in somatosensory cortex? Ann. NY Acad. Sci. 1225, 119–129 (2011).

  37. 37

    Dunn, F.A., Della Santina, L., Parker, E.D. & Wong, R.O. Sensory experience shapes the development of the visual system's first synapse. Neuron 80, 1159–1166 (2013).

  38. 38

    Espinosa, J.S., Wheeler, D.G., Tsien, R.W. & Luo, L. Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B. Neuron 62, 205–217 (2009).

  39. 39

    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).

  40. 40

    Vue, T.Y. et al. Thalamic control of neocortical area formation in mice. J. Neurosci. 33, 8442–8453 (2013).

  41. 41

    Joshi, P.S. et al. Bhlhb5 regulates the postmitotic acquisition of area identities in layers II–V of the developing neocortex. Neuron 60, 258–272 (2008).

  42. 42

    Karayannis, T., De Marco Garcia, N.V. & Fishell, G.J. Functional adaptation of cortical interneurons to attenuated activity is subtype-specific. Front. Neural Circuits 6, 66 (2012).

  43. 43

    De Marco Garcia, N.V. & Fishell, G. Subtype-selective electroporation of cortical interneurons. J. Vis. Exp. e51518 (2014).

  44. 44

    Miyamichi, K. & Luo, L. Neuroscience. Brain wiring by presorting axons. Science 325, 544–545 (2009).

  45. 45

    Wickersham, I.R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).

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Acknowledgements

We are grateful to S. Arber, B. Benedetti, J. Burrone, X. Jaglin, J. Kaltschmidt, S. Lee, D. Pisapia and S. Shi for comments on the manuscript. We thank E. Callaway (Salk Institute for Biological Sciences) for providing the recombinant rabies virus; M. Tripodi, A. Ponti and S. Arber for guidance with the rabies method analysis; and L. Yin, J. Deng and J. Dai for technical assistance. N.V.D.M.G. is a recipient of a NARSAD Young Investigator Award and is also supported by grants from the US National Institutes of Health (5 K99 MH095825-02; 3 K99 MH095825-02S1). T.K. has been supported by the Patterson Trust postdoctoral fellowship in brain circuitry and a Roche postdoctoral fellowship. Research in the Fishell laboratory is supported by the US National Institutes of Health, National Institute of Mental Health, National Institute of Neurological Disorders and Stroke, New York State Stem Cell Science and the Simons Foundation.

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N.V.D.M.G., T.K. and G.F. conceived the project. N.V.D.M.G., T.K. and R.P. performed the experiments. S.N.T. prepared the rabies virus. N.V.D.M.G. wrote the manuscript with the help of all authors.

Corresponding author

Correspondence to Gord Fishell.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Model.

Superficial Re+ interneurons receive abundant thalamic innervation during development. These afferents preferentially activate NR2B-containing NMDARs. Different manipulations interfering with the signaling through these receptors cause a loss of thalamic innervation. Despite preserving their net number of excitatory afferents, the normal balance is skewed such that there is a net gain of intracortical innervation onto Re+ interneurons at the expense of thalamic innervation.

Supplementary Figure 2 Analysis of the identity of presynaptic targets of of Re+ cells revealed by rabies tracing.

a. Representative examples of single Re+ starter cells in 3 independent experiments. Brains 1 and 2 were obtained from Cre-, NR1fl/fl mice whereas Brain 3 was obtained from a Cre+, NR1fl/fl mouse (Dlx5/6.Cre; NR1fl/fl). Note that the starter cells in these brains reside in layer II of the SSBF1. b. Representative examples of the distribution of presynaptic targets at different antero-posterio levels. c and d. Representative examples of immunolabeled excitatory neurons (Ecx)(c) and interneurons (Icx) (d) (see Methods for details). e-g. Viral mCherry expression allows for the morphological identification of pyramidal cells (e-f) and local Re+ interneurons (g). Scale bar, 50 μm.

Supplementary Figure 3 Tracing of cholinergic projections onto non-Re+ superficial interneuron subtypes.

a. Schematic representation of the genetic strategy to target superficial interneurons. The 5HT3ACre driver line restricts the expression of Cre to superficial layer interneurons. b. i. A low-power image showing rabies-traced neurons in the nucleus basalis (NB). ii. A high-power image of the same section shown in panel i. iii. An additional example of rabies-traced neurons in the nucleus basalis. NB, nucleus basalis; Rt, reticular nucleus; GP, globus pallidus; ic, internal capsule.

Supplementary Figure 4 Genetic strategies for activation of cortical and thalamic afferents.

a-b. Representative sections depicting cortical EYFP expression at P8. c. Absence of eGFP labeled somata in the thalamus of Emx1Cre/+, RCEfl/+ mice. d. Representative sections depicting thalamic EYFP expression in Olig3Cre/+, Rosa26LSL-ChR2-EYFP/+ mice. e. The presence of eGFP labeled neuronal somata in the VPM (e1) but not in SSBF1 (e2) of Olig3Cre/+,, RCEfl/+ mice. f. EYFP expression in VGlut2Cre/+, Rosa26LSL-ChR2-EYFP/+ miceg. The presence of GFP labeled neuronal somata in the VPM (g1, note this image is also shown in Figure 4a) and in some layer IV neurons (g2) of VGlut2Cre/+, RCEfl/+ mice. Scale bar, 50 μm.

Supplementary Figure 5 Developing Re+ interneurons receive functional monosynaptic inputs from thalamic afferents.

a.The latency between light presentation (1) and onset of AMPAR-mediated EPSCs (2) is 7 ms with very little variability from trial to trial (black traces) in Olig3Cre/+; Rosa26LSL-ChR2-EYFP/+ mice. The red trace is the average of the 10 sweeps shown. b. Average traces of light-evoked monosynaptic NMDAR mediated EPSCs in the presence of just CNQX and bicuculine (black) and after adding 4-AP (red). c. Synaptic responses recorded in a P10 layer I interneuron at – 70m V, evoked with a 470nm LED light stimulation of ChR2 terminals in the absence (control) or presence of TTX/4–AP. The traces show that drug application leads to a significant reduction in the peak amplitude and a large prolongation of the latency of the EPSCs, as well as the kinetics. d. Representative example of an evoked trace of an EPSC recorded at – 70 mV using a different 470nm LED light source to “a-c” for a layer IV stellate cell and a layer I interneuron located in the same vertical axis. It demonstrates that the EPSC onset latency is comparable between the two cell types. Blue lines indicate the duration of light stimulation.

Supplementary Figure 6 The time course of thalamic innervation of the SSBF1 cortex.

Thalamic terminals delineated by VGlut2 expression invade the superficial layers of the cortex around P3. By P8, these afferents coalesce into defined barrels in layer IV. Cortical layers are delineated by Tbr1 expression. Scale bar, 50 μm.

Supplementary Figure 7 Cortical layering after early sensory deprivation.

The number and distribution of pyramidal cells (Satb2+) in layers I-III and V is comparable in control (a) and sensory-deprived (b) mice at P8. Thalamic terminals are shown in red (VGlut2 staining). c and d. Quantification of complexity of dendritic arbors. Mean values (± SEM) were obtained from reconstructed Re+ interneurons each in Dlx5/6-eGFP electroporated control (ctrl, n = 7), sensory-deprived (depr n = 5; P = 0.0017) and TelC (n=6; P = 0.0007) mice (c) and VIP+ interneurons each in Dlx5/6-eGFP electroporated control (ctrl = 12) and sensory-deprived (depr n = 4; P = 0.262) (d). **, P < 0.01; ***, P < 0.001. Scale bar, 50 μm.

Supplementary Figure 8 Reduction of VAMP2 staining upon thalamic expression of tetanus toxin.

a and b. VAMP2 levels are significantly reduced in Olig3Cre/+; Rosa26LSL.TeLC (B) compared to control Rosa26LSL.TeLC (a) mice at P8.

Supplementary Figure 9 In Re+ cells, NMDAR-mediated responses in VGlut2.ChR2 activated terminals possess a prominent NR2B-component vs intracortical inputs.

The percentage reduction of NMDAR-dependent current amplitude in Re+ interneurons after ifenprodil (3 μM) application and intracortical electrode stimulation (Ecx; n = 4 interneurons) or light stimulation of terminals in VGlut2.ChR2 mice (n = 12 interneurons; P = 0.0011, Mann-Whitney test). **, P < 0.01

Supplementary Figure 10 Unaffected intrinsic electrophysiological properties in Re+ interneurons and normal development of VIP+ interneurons in the absence of NMDARs.

a. Example a. Example traces of passive and active electrophysiological properties.Top panel shows the characteristic sub- (red) and supra-threshold (black) membrane step depolarization with the late spiking pattern of a Re+ interneuron. Bottom trace shows the resulting membrane deflection to a series of sub-threshold current steps and a suprathreshold one that leads to non-accommodating sustained spiking activity. b. An array of measures of passive and active electrophysiological properties show the absence of major differences between NMDAR-lacking and control Re+ interneurons. Spike width 1.30±1.46 ms (ctrl) and 1.30±1.46 ms (Dlx5/6.Cre, NR1fl/fl). Mean values (±SEM) were obtained from 6 ctrl and 5 Dlx5/6.Cre, NR1fl/fl interneurons (Mann-Whitney test, P = 0.1399 Vrm, P = 0.2468 Rin, P = 0.9004 Cm, P = 0.5281 tau, P = 0.2468 spike threshold, P = 0.1775 spike amplitude, P = 0.0866 spike width, P = 0.0762 max firing) c and d. Quantification of complexity of axonal (c) and dendritic (d) nodes. P > 0.05, ns (control n = 9 interneurons; Dlx5/6.Cre, NR1fl/fl n = 6 interneurons; axonal nodes: P = 0.543; dendritic nodes: P = 0.277). P > 0.05, ns, Scale bar, 50 μm. Axons are shown in red; dendrites are shown in blue.

Supplementary Figure 11 In vivo pharmacological blockade of NR2B-containing NMDARs disrupts the morphological development of Re+ cells

a. Experimental strategy (see also). b. Neurolucida reconstructions of Re+ interneurons in control and ifenprodil injected brains. c. Quantification of length and complexity of dendritic arbors and axonal trees. (control n = 8 interneurons; Ifen n = 7 interneurons; axonal length: P = 0.0467, axonal nodes: P = 0.0523; dendritic length: P = 0.0074). *, P < 0.05; **, P<0.01. Scale bar, 50 μm. Axons are shown in red; dendrites are shown in blue.

Supplementary Figure 12 In vivo pharmacological blockage of AMPA signaling (by subdural administration of DNQX at P3) does not perturb the morphological development of Re+ interneurons.

a. Experimental strategy (see also ). b-c. Neurolucida reconstructions of Re+ interneurons in mice electroporated with a Dlx5/6-eGFP plasmid and treated with vehicle (control) or DNQX (2-4 nM). d. Quantification of length and complexity of dendritic arbors and axonal trees (ctrl n=4 interneurons; DNQX n=4 interneurons; axonal length: P = 0.885; axonal nodes: P = 0.319; dendritic length: P = 0.613; dendritic nodes: P = 0.263). P > 0.05, ns. Scale bar, 50 μm. Axons are shown in red; dendrites are shown in blue.

Supplementary Figure 13 The critical period for NMDAR-dependent morphological development of Re+ cells occurs around P3 and is complete by P6

a-d. Representative reconstructions of Re+ interneurons electroporated with a Dlx5-CreER plasmid in control (a and c; no tamoxifen, n = 7 interneurons) and tamoxifen-treated NR1fl/fl mice at P3 (b, n = 7 interneurons) and P6 (d, n = 5 interneurons). Note that the loss of NMDA-signaling perturbs Re+ development if done at P3 but not at P6.

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De Marco García, N., Priya, R., Tuncdemir, S. et al. Sensory inputs control the integration of neurogliaform interneurons into cortical circuits. Nat Neurosci 18, 393–401 (2015). https://doi.org/10.1038/nn.3946

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