Article | Published:

Inhibitory circuit gating of auditory critical-period plasticity


Cortical sensory maps are remodeled during early life to adapt to the surrounding environment. Both sensory and contextual signals are important for induction of this plasticity, but how these signals converge to sculpt developing thalamocortical circuits remains largely unknown. Here we show that layer 1 (L1) of primary auditory cortex (A1) is a key hub where neuromodulatory and topographically organized thalamic inputs meet to tune the cortical layers below. Inhibitory interneurons in L1 send narrowly descending projections to differentially modulate thalamic drive to pyramidal and parvalbumin-expressing (PV) cells in L4, creating brief windows of intracolumnar activation. Silencing of L1 (but not VIP-expressing) cells abolishes map plasticity during the tonotopic critical period. Developmental transitions in nicotinic acetylcholine receptor (nAChR) sensitivity in these cells caused by Lynx1 protein can be overridden to extend critical-period closure. Notably, thalamocortical maps in L1 are themselves stable, and serve as a scaffold for cortical plasticity throughout life.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

  • 18 June 2018

    In the version of this article initially published online, the wrong version of Fig. 5 was used. There were errors in the statistical comparison brackets in Fig. 5c and the left-hand error bar in Fig. 5f. The errors have been corrected in the print, PDF and HTML versions of this article. In the version of this article initially published online and in print, the wrong version of Fig. 3h was used. There was a slight error in the alignment of the traces in the top right panel. The error has been corrected in the PDF and HTML versions of this article. The original and corrected figures are shown in the accompanying Publisher Correction.


  1. 1.

    Werker, J. F. & Hensch, T. K. Critical periods in speech perception: new directions. Annu. Rev. Psychol. 66, 173–196 (2015).

  2. 2.

    Kuhl, P. K., Tsao, F. M. & Liu, H. M. Foreign-language experience in infancy: effects of short-term exposure and social interaction on phonetic learning. Proc. Natl. Acad. Sci. USA 100, 9096–9101 (2003).

  3. 3.

    de Villers-Sidani, E., Chang, E. F., Bao, S. & Merzenich, M. M. Critical period window for spectral tuning defined in the primary auditory cortex (A1) in the rat. J. Neurosci. 27, 180–189 (2007).

  4. 4.

    Insanally, M. N., Köver, H., Kim, H. & Bao, S. Feature-dependent sensitive periods in the development of complex sound representation. J. Neurosci. 29, 5456–5462 (2009).

  5. 5.

    Barkat, T. R., Polley, D. B. & Hensch, T. K. A critical period for auditory thalamocortical connectivity. Nat. Neurosci. 14, 1189–1194 (2011).

  6. 6.

    Bear, M. F. & Singer, W. Modulation of visual cortical plasticity by acetylcholine and noradrenaline. Nature 320, 172–176 (1986).

  7. 7.

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

  8. 8.

    Cruz-Martín, A. et al. A dedicated circuit links direction-selective retinal ganglion cells to the primary visual cortex. Nature 507, 358–361 (2014).

  9. 9.

    Roth, M. M. et al. Thalamic nuclei convey diverse contextual information to layer 1 of visual cortex. Nat. Neurosci. 19, 299–307 (2016).

  10. 10.

    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).

  11. 11.

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

  12. 12.

    Karnani, M. M. et al. Opening holes in the blanket of inhibition: localized lateral disinhibition by VIP interneurons. J. Neurosci. 36, 3471–3480 (2016).

  13. 13.

    Donato, F., Rompani, S. B. & Caroni, P. Parvalbumin-expressing basket-cell network plasticity induced by experience regulates adult learning. Nature 504, 272–276 (2013).

  14. 14.

    Jiang, X., Wang, G., Lee, A. J., Stornetta, R. L. & Zhu, J. J. The organization of two new cortical interneuronal circuits. Nat. Neurosci. 16, 210–218 (2013).

  15. 15.

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

  16. 16.

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

  17. 17.

    Zhou, M. et al. Scaling down of balanced excitation and inhibition by active behavioral states in auditory cortex. Nat. Neurosci. 17, 841–850 (2014).

  18. 18.

    Winer, J. A., Miller, L. M., Lee, C. C. & Schreiner, C. E. Auditory thalamocortical transformation: structure and function. Trends Neurosci. 28, 255–263 (2005).

  19. 19.

    Smith, P. H., Uhlrich, D. J., Manning, K. A. & Banks, M. I. Thalamocortical projections to rat auditory cortex from the ventral and dorsal divisions of the medial geniculate nucleus. J. Comp. Neurol. 520, 34–51 (2012).

  20. 20.

    Ji, X. Y. et al. Thalamocortical innervation pattern in mouse auditory and visual cortex: laminar and cell-type specificity. Cereb. Cortex 26, 2612–2625 (2016).

  21. 21.

    Nahmani, M. & Erisir, A. VGluT2 immunochemistry identifies thalamocortical terminals in layer 4 of adult and developing visual cortex. J. Comp. Neurol. 484, 458–473 (2005).

  22. 22.

    Rose, H. J. & Metherate, R. Auditory thalamocortical transmission is reliable and temporally precise. J. Neurophysiol. 94, 2019–2030 (2005).

  23. 23.

    Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W. & Sanes, J. R. Improved tools for the Brainbow toolbox. Nat. Methods 10, 540–547 (2013).

  24. 24.

    Marlin, J. J. & Carter, A. G. GABA-A receptor inhibition of local calcium signaling in spines and dendrites. J. Neurosci. 34, 15898–15911 (2014).

  25. 25.

    Richardson, R. J., Blundon, J. A., Bayazitov, I. T. & Zakharenko, S. S. Connectivity patterns revealed by mapping of active inputs on dendrites of thalamorecipient neurons in the auditory cortex. J. Neurosci. 29, 6406–6417 (2009).

  26. 26.

    Hackett, T. A., Barkat, T. R., O’Brien, B. M., Hensch, T. K. & Polley, D. B. Linking topography to tonotopy in the mouse auditory thalamocortical circuit. J. Neurosci. 31, 2983–2995 (2011).

  27. 27.

    Kanold, P. O., Nelken, I. & Polley, D. B. Local versus global scales of organization in auditory cortex. Trends Neurosci. 37, 502–510 (2014).

  28. 28.

    Stachniak, T. J., Ghosh, A. & Sternson, S. M. Chemogenetic synaptic silencing of neural circuits localizes a hypothalamus→midbrain pathway for feeding behavior. Neuron 82, 797–808 (2014).

  29. 29.

    Kilgard, M. P. & Merzenich, M. M. Cortical map reorganization enabled by nucleus basalis activity. Science 279, 1714–1718 (1998).

  30. 30.

    Froemke, R. C., Merzenich, M. M. & Schreiner, C. E. A synaptic memory trace for cortical receptive field plasticity. Nature 450, 425–429 (2007).

  31. 31.

    Miwa, J. M. et al. Lynx1, an endogenous toxin-like modulator of nicotinic acetylcholine receptors in the mammalian CNS. Neuron 23, 105–114 (1999).

  32. 32.

    Morishita, H., Miwa, J. M., Heintz, N. & Hensch, T. K. Lynx1, a cholinergic brake, limits plasticity in adult visual cortex. Science 330, 1238–1240 (2010).

  33. 33.

    De Marco García, N. V., Priya, R., Tuncdemir, S. N., Fishell, G. & Karayannis, T. Sensory inputs control the integration of neurogliaform interneurons into cortical circuits. Nat. Neurosci. 18, 393–401 (2015).

  34. 34.

    Mesik, L. et al. Functional response properties of VIP-expressing inhibitory neurons in mouse visual and auditory cortex. Front. Neural Circuits 9, 22 (2015).

  35. 35.

    Muralidhar, S., Wang, Y. & Markram, H. Synaptic and cellular organization of layer 1 of the developing rat somatosensory cortex. Front. Neuroanat. 7, 52 (2014).

  36. 36.

    Knudsen, E. I. Capacity for plasticity in the adult owl auditory system expanded by juvenile experience. Science 279, 1531–1533 (1998).

  37. 37.

    Hangya, B., Ranade, S. P., Lorenc, M. & Kepecs, A. Central cholinergic neurons are rapidly recruited by reinforcement feedback. Cell 162, 1155–1168 (2015).

  38. 38.

    Dahmen, J. C., Hartley, D. E. H. & King, A. J. Stimulus-timing-dependent plasticity of cortical frequency representation. J. Neurosci. 28, 13629–13639 (2008).

  39. 39.

    Tasic, B. et al. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat. Neurosci. 19, 335–346 (2016).

  40. 40.

    Batista-Brito, R. et al. Developmental dysfunction of VIP interneurons impairs cortical circuits. Neuron 95, 884–895 (2017).

  41. 41.

    Blundon, J. A. et al. Restoring auditory cortex plasticity in adult mice by restricting thalamic adenosine signaling. Science 356, 1352–1356 (2017).

  42. 42.

    Bennett, C., Arroyo, S., Berns, D. & Hestrin, S. Mechanisms generating dual-component nicotinic EPSCs in cortical interneurons. J. Neurosci. 32, 17287–17296 (2012).

  43. 43.

    Brown, C. E., Sweetnam, D., Beange, M., Nahirney, P. C. & Nashmi, R. α4* nicotinic acetylcholine receptors modulate experience-based cortical depression in the adult mouse somatosensory cortex. J. Neurosci. 32, 1207–1219 (2012).

  44. 44.

    Yazaki-Sugiyama, Y., Kang, S., Câteau, H., Fukai, T. & Hensch, T. K. Bidirectional plasticity in fast-spiking GABA circuits by visual experience. Nature 462, 218–221 (2009).

  45. 45.

    Beurdeley, M. et al. Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex. J. Neurosci. 32, 9429–9437 (2012).

  46. 46.

    Engineer, N. D. et al. Reversing pathological neural activity using targeted plasticity. Nature 470, 101–104 (2011).

  47. 47.

    Polley, D. B., Steinberg, E. E. & Merzenich, M. M. Perceptual learning directs auditory cortical map reorganization through top-down influences. J. Neurosci. 26, 4970–4982 (2006).

  48. 48.

    Fu, Y., Kaneko, M., Tang, Y., Alvarez-Buylla, A. & Stryker, M. P. A cortical disinhibitory circuit for enhancing adult plasticity. eLife 4, e05558 (2015).

  49. 49.

    Derégnaucourt, S., Poirier, C., Kant, A. V., Linden, A. V. & Gahr, M. Comparisons of different methods to train a young zebra finch (Taeniopygia guttata) to learn a song. J. Physiol. Paris 107, 210–218 (2013).

  50. 50.

    Mowery, T. M., Kotak, V. C. & Sanes, D. H. The onset of visual experience gates auditory cortex critical periods. Nat. Commun. 7, 10416 (2016).

  51. 51.

    Hippenmeyer, S. et al. A developmental switch in the response of DRG neurons to ETS transcription factor signaling. PLoS. Biol. 3, e159 (2005).

  52. 52.

    Taniguchi, H. et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71, 995–1013 (2011).

  53. 53.

    Madisen, L. et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat. Neurosci. 15, 793–802 (2012).

  54. 54.

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

  55. 55.

    Kobayashi, A. et al. Lynx1 supports neuronal health in the mouse dorsal striatum during aging: an ultrastructural investigation. J. Mol. Neurosci. 53, 525–536 (2014).

  56. 56.

    Meyer, A. H., Katona, I., Blatow, M., Rozov, A. & Monyer, H. In vivo labeling of parvalbumin-positive interneurons and analysis of electrical coupling in identified neurons. J. Neurosci. 22, 7055–7064 (2002).

  57. 57.

    Ichinohe, N., Hyde, J., Matsushita, A., Ohta, K. & Rockland, K. S. Confocal mapping of cortical inputs onto identified pyramidal neurons. Front. Biosci. 13, 6354–6373 (2008).

  58. 58.

    Chattopadhyaya, B. et al. Experience and activity-dependent maturation of perisomatic GABAergic innervation in primary visual cortex during a postnatal critical period. J. Neurosci. 24, 9598–9611 (2004).

  59. 59.

    Takesian, A. E., Kotak, V. C. & Sanes, D. H. Presynaptic GABA(B) receptors regulate experience-dependent development of inhibitory short-term plasticity. J. Neurosci. 30, 2716–2727 (2010).

  60. 60.

    Alkondon, M. & Albuquerque, E. X. Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes. J. Pharmacol. Exp. Ther. 265, 1455–1473 (1993).

  61. 61.

    Porter, J. T. et al. Selective excitation of subtypes of neocortical interneurons by nicotinic receptors. J. Neurosci. 19, 5228–5235 (1999).

  62. 62.

    Lucas-Meunier, E. et al. Involvement of nicotinic and muscarinic receptors in the endogenous cholinergic modulation of the balance between excitation and inhibition in the young rat visual cortex. Cereb. Cortex 19, 2411–2427 (2009).

  63. 63.

    Krause, B. M., Raz, A., Uhlrich, D. J., Smith, P. H. & Banks, M. I. Spiking in auditory cortex following thalamic stimulation is dominated by cortical network activity. Front. Syst. Neurosci. 8, 170 (2014).

  64. 64.

    Saxena, A. et al. Trehalose-enhanced isolation of neuronal sub-types from adult mouse brain. Biotechniques 52, 381–385 (2012).

  65. 65.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods 25, 402–408 (2001).

  66. 66.

    Cruikshank, S. J., Rose, H. J. & Metherate, R. Auditory thalamocortical synaptic transmission in vitro. J. Neurophysiol. 87, 361–384 (2002).

  67. 67.

    Zhou, X., Nagarajan, N., Mossop, B. J. & Merzenich, M. M. Influences of un-modulated acoustic inputs on functional maturation and critical-period plasticity of the primary auditory cortex. Neuroscience 154, 390–396 (2008).

  68. 68.

    Kim, H. & Bao, S. Selective increase in representations of sounds repeated at an ethological rate. J. Neurosci. 29, 5163–5169 (2009).

  69. 69.

    Geissler, D. B. & Ehret, G. Auditory perception vs. recognition: representation of complex communication sounds in the mouse auditory cortical fields. Eur. J. Neurosci. 19, 1027–1040 (2004).

  70. 70.

    Guo, Z. V. et al. Flow of cortical activity underlying a tactile decision in mice. Neuron 81, 179–194 (2014).

  71. 71.

    Tsuriel, S., Gudes, S., Draft, R. W., Binshtok, A. M. & Lichtman, J. W. Multispectral labeling technique to map many neighboring axonal projections in the same tissue. Nat. Methods 12, 547–552 (2015).

Download references


We thank J. Miwa (Lehigh University, Bethlehem, PA, USA) for providing the original Lynx1-knockout breeder mice and Lynx1-knockout × α7-nAChR-knockout mice (care of A. Beaudet, Baylor University, Waco, TX, USA); H. Monyer (Heidelberg University, Heidelberg, Germany) for the original PV–GFP mice; M. Fagiolini for comments and discussions; A. Covello and M. Snyder for assistance with Brainbow tracing; A. Castros and E. He for assistance with immunocytochemistry quantification and in situ hybridization; A. Galvin for ABR measurements to test DHβE effects; T. Barkat, E. Diel, H. Lee, R. Reh, S. Mierau, A. Patrizi, D. Cai, G. Corfas, J. Holt, G. Géléoc, A. Thompson, and C. Chen for technical assistance and discussions; and N. De Souza, M. Nakamura, H. Bond, E. Centofante, and M. Marcotrigiano for animal care. This work was supported by the Nancy Lurie Marks Family Foundation and the Canadian Institute for Advanced Research (CIFAR; to A.E.T.), the Ellison Medical Foundation, and the NIMH Silvio O Conte Center (P50MH094271 to T.K.H.).

Author information

A.E.T. and T.K.H. designed the study and wrote the manuscript. A.E.T. conducted the experiments and analyzed the data. J.W.L. and L.J.B. assisted with the Brainbow experiments, imaging and analysis.

Competing interests

The authors declare no competing financial interests.

Correspondence to Takao K. Hensch.

Integrated supplementary information

Supplementary Figure 1 MGB projects to cortical layer 1.

Projections from the medial geniculate body (MGB; red) onto 5-HT3AR interneurons within layer 1 of primary auditory cortex (A1) colocalize with vesicular glutamate transporter 2 (vGluT2; green). Representative images from one of 3 mice. Scale bar = 5 µm.

Supplementary Figure 2 5-HT3AR+ interneurons receive direct MGB inputs.

(a) Layer (L) 1 interneurons and L4 pyramidal cells in primary auditory cortex (A1) show similar responses to medial geniculate body (MGB) stimulation. Left; Schematic illustrating recording configuration. L1 interneurons and L4 pyramidal cells were recorded during electrical stimulation of the MGB fibers. Right; Individual (gray) and mean (bolded) MGB-evoked EPSPs recorded in representative L1 late-spiking (LS) or non-LS cell subtypes and a L4 pyramidal cell. (b) Left; Mean (± SEM) EPSP amplitudes evoked by maximal MGB stimulation (EPSP maximum amplitude (mV): L1 LS = 7.34 ± 1.41, n = 7 cells/4 mice; L1 non-LS = 6.16 ± 2.14, n = 4 cells/4 mice; L4 = 7.33 ± 1.96, n = 11 cells/4 mice; Kruskal-Wallis test, χ2(2) = 0.47, P = 0.79). Right; Mean (± SEM) EPSP onset time, EPSP 10–90% rise time, and width at half maximum EPSP amplitude recorded in L1 interneurons and L4 pyramidal cells (onset time (ms): L1 = 11.36 ± 0.80, n = 11 cells/4 mice; L4 = 8.11 ± 0.84, n = 11 cells/4 mice; unpaired t test, two-tailed, t(20) = −2.80, P = 0.011, CL = −5.66, −0.83; rise time (ms): L1 = 8.18 ± 1.39, n = 11 cells/4 mice; L4 = 7.45 ± 1.35, n = 11 cells/4 mice; Mann-Whitney U test, two-tailed, z = −0.53, P = 0.599, CL = −3.13, 1.97; half-width (ms): L1 = 50.95 ± 4.96, n = 11 cells/4 mice; L4 = 57.64 ± 8.09, n = 11 cells/4 mice; Mann-Whitney U test, two-tailed, z = 0.33, P = 0.743, CL = −15.55, 32.66). (c) L1 interneurons and L4 pyramidal cells exhibit similar short-term depression of MGB-evoked EPSPs. Left; Schematic illustrating recording configuration. Trains of electrical stimuli were delivered to the MBG fibers during recordings in L1 interneurons or L4 pyramidal cells. Right; Mean (± SEM, shaded region) EPSPs recorded in all L1 interneurons (green, n = 8 cells/3 mice) and all L4 pyramidal cells (gray, n = 10 cells/3 mice) during repetitive electrical stimulation of the MGB fibers (10Hz). (d) Left; Mean (± SEM) EPSP amplitudes normalized to the first EPSP (EPSPN/EPSP1) evoked by each of 10 stimuli applied to the MGB fibers at 10 or 20Hz (L1, n = 8 cells/3 mice; L4, n = 10 cells/3 mice). Box indicates EPSP10/EPSP1. Right; Mean (± SEM) EPSP10/EPSP1 shows no significant differences between L1 interneurons and L4 pyramidal cells (10 Hz, L1 = 0.36 ± 0.06, L4 = 0.66 ± 0.18, z = −1.64, P = 0.10, CL = −0.06, 0.43; 20 Hz, L1 = 0.20 ± 0.04, L4 = 0.23 ± 0.04, z = −0.49, P = 0.63, CL = −0.11, 0.15, Mann-Whitney U tests, two-tailed, L1, n = 8 cells/3 mice; L4, n = 10 cells/3 mice). n.s. (not significant), P > 0.05; *P < 0.05.

Supplementary Figure 3 5-HT3AR+ interneurons differentially affect MGB-evoked EPSPs in PV and pyramidal cells.

(a) Schematic of recording configuration. (b) Example of ventral medial geniculate body (MGBv)-evoked subthreshold EPSPs recorded in L4 PV cells alone (red) or during optogenetic activation of 5HT3AR interneurons (blue). (c) Mean (± SEM) effects of 5-HT3AR interneuron activation on MGBv-evoked EPSPs in PV interneurons (red) and pyramidal cells (black) within L4 at varying intervals between 5-HT3AR interneuron activation and MGB stimulation (PV cell, n = 4 cells/4 mice; PYR cell, n = 12 cells/9 mice, Mann-Whitney U tests, two-tailed, *P < 0.05).

Supplementary Figure 4 5-HT3AR+ interneurons provide spatially and temporally precise inhibition.

(a) Averaged responses (normalized ΔF/F) in primary auditory cortex (A1) to stimulation of ventral medial geniculate body (MGBv) using voltage-sensitive dye imaging (VSDI) within layers (L) 2/3, 4 and 5 of P12–15 mice (n = 10 mice) at baseline (black) or during 5-HT3AR cell optogenetic silencing (blue). (b) 5-HT3AR cell silencing decreased the early (0–40ms) peak response within L2/3/4 of activated columns (normalized ΔF/F: baseline = 0.68 ± 0.11; 5-HT3AR cell silencing = 0.49 ± 0.06, n = 10 mice; paired t test, two tailed, t(9) = −2.40, P = 0.0396, CL = −0.37, −0.01), but increased the late (200–450ms) response in neighboring columns (normalized ΔF/F: baseline = 0.51 ± 0.11; 5-HT3AR cell silencing = 0.68 ± 0.09, n = 10 mice; paired t test, two tailed, t(9) = 4.00, P = 0.0031, CL = 0.07, 0.27). Mean ± SEM, *P < 0.05, **P < 0.01.

Supplementary Figure 5 Silencing of 5-HT3AR+ cells abolishes a critical period for auditory thalamocortical plasticity.

Normalized (norm.) ∆F/F (mean ± SEM) across primary auditory cortex (A1) locations in response to ventral medial geniculate body (MGBv) stimuli imaged in slices from naïve C57 mice (P20–25) raised in a normal acoustic environment (a, n = 15 mice), 7kHz-exposed controls (b, n = 14 mice), and 7kHz-exposed mice with silenced 5-HT3AR cells using hM4D (c, n = 28 mice). Colors indicate MGBv positions. Insets show the MGBv positions that elicited the maximal average responses across A1.

Supplementary Figure 6 Developmental changes in genes encoding 5-HT3AR and nAChRs within 5-HT3AR+ interneurons.

Normalized expression (mean ± SEM) of Htr3a, Chrna4, Chrna7, and Chrnb2 genes encoding 5-HT3AR and nAChR subunits (α4, α7, and β2) within 5-HT3AR-expressing interneurons from primary auditory cortex (A1) in mice aged postnatal day (P) 11 and P20. (Normalized quantity Htr3a: P11 = 0.74 ± 0.12 (95% CI, 0.37, 1.11), P20 = 1.87 ± 0.58 (95% CI, 0.01, 3.73), z = 1.01, P = 0.31; Normalized quantity Chrna4: P11 = 1.52 ± 0.03 (95% CI, 1.43, 1.62), P20 = 1.27 ± 0.07 (95% CI, 1.05, 1.48), z = −2.17, P = 0.03; Normalized quantity Chrna7: P11 = 1.08 ± 0.07 (95% CI, 0.85, 1.32), P20 = 1.22 ± 0.05 (95% CI, 1.06, 1.37), z = 1.30, P = 0.19; Normalized quantity Chrnb2: P11 = 1.02 ± 0.09 (95% CI, 0.73, 1.31), P20 = 1.00 ± 0.05 (95% CI, 0.83, 1.18), z = 0, P = 1.00; Mann-Whitney U tests, two-tailed, n = 4 mice each). *P < 0.05.

Supplementary Figure 7 Lynx1 expression in cortical interneurons.

Expression of Lynx1 (mean ± SEM) measured within inhibitory interneuron subtypes from primary auditory cortex (A1) in adult mice. (Normalized quantity Lynx1: 5-HT3AR = 0.90 ± 0.13 (95% CI, 0.55, 1.26), n = 5 mice; non-5-HT3AR = 1.38 ± 0.13 (95% CI, 1.01, 1.74), n = 5 mice; PV = 2.10 ± 0.15 (95% CI, 1.68, 2.52), n = 5 mice; SOM = 1.22 ± 0.02 (95% CI, 1.16, 1.29), n = 4 mice; Kruskal-Wallis, χ2(3) = 14.25, P = 0.003, compared to 5-HT3AR using Steel test, non-5-HT3AR, P = 0.15; PV, P = 0.03; SOM, P = 0.10). n.s. (not significant), P > 0.05; *P < 0.05.

Supplementary Figure 8 Nicotine induces a barrage of IPSCs in PV and pyramidal cells from Lynx1-knockout mice.

(a) Schematic of recordings from PV and pyramidal (PYR) cells in primary auditory cortex (A1) from P25–45 wild-type (WT) and Lynx1 KO mice. These cell types are targeted by 5HT3AR inhibitory interneurons that express α4β2- and α7-containing nAChRs. (b, c) Examples of spontaneous inhibitory postsynaptic currents (sIPSCs) before (b) and after (c) bath application of nicotine (10 µM). Insets show expanded sIPSCs from gray region. (d) Mean (± SEM, shaded region) sIPSC frequency following nicotine application in pyramidal and PV cells from Lynx1 KO mice. Nicotine-induced increase in sIPSC frequency persisted in α7-nAChR/Lynx1 KO mice or during application of the α7-nAChR selective antagonist methyllycaconitine (MLA; bath-applied, 10 nM), but was abolished by pharmacological blockade of α4-containing nAChRs with DHβE (bath-applied, 500 nM). (PV cells: WT, n = 11 cells/10 mice; Lynx1 KO, n = 9 cells/6 mice; PYR cells: WT, n = 10 cells/7 mice; Lynx1 KO, n = 9 cells/9 mice; α7-nAChR/Lynx1 KO, n = 6 cells/6 mice; MLA, n = 10 cells/7 mice; DHβE, n = 8 cells/7 mice). (e) Mean (± SEM) nicotine effect (peak sIPSC frequency during nicotine application divided by baseline) in pyramidal and PV cells from WT and Lynx1 KO mice (nicotine effect on PV cells: WT = 1.55 ± 0.29, n = 11 cells/10 mice; Lynx1 KO = 4.98 ± 1.33, n = 9 cells/6 mice, Mann-Whitney U test, two-tailed, z = 2.74, P = 0.006, CL = 0.61, 6.74; nicotine effect on PYR cells: WT = 1.50 ± 0.18, n = 10 cells/7 mice; Lynx1 KO = 3.29 ± 0.47, n = 9 cells/9 mice; α7-nAChR/Lynx1 KO = 6.15 ± 2.80, n = 6 cells/6 mice; MLA = 4.82 ± 1.17, n = 10 cells/7 mice; DHβE = 1.35 ± 0.36, n = 8 cells/7 mice; Kruskal-Wallis, χ2(4) = 12.31, P = 0.015, compared to Lynx1 KO using Steel test, WT, P = 0.028, CL = 0.67, 3.50; α7-nAChR/Lynx1 KO, P = 1.0, CL = −3.66, 12.39; MLA, P = 0.989, CL = −2.83, 5.94; DHβE, P = 0.049, CL = −3.84, 0.09). n.s. (not significant), P > 0.05; *P < 0.05; **P < 0.01.

Supplementary Figure 9 Lynx1-knockout mice exhibit normal peripheral hearing thresholds.

(a, b) Representative auditory brainstem responses (ABR) recorded from C57 and Lynx1 KO adult mice during 11.3 kHz tone pips of increasing sound pressure level (decibels; dB). Circles represent location of the five positive (P) wave peaks (1–5). (c) C57 and Lynx1 KO mice showed similar ABR thresholds (mean ± SEM) across tone frequencies (MANOVA, ABR threshold changes by genotype, P = 0.51, frequency, P = 0.0018, genotype x frequency, P = 0.77). (d) C57 and Lynx1 KO mice showed similar Distortion Product Otoacoustic Emissions (DPOAE) thresholds (mean ± SEM) across tone frequencies (MANOVA, threshold changes by genotype, P = 0.10, frequency, P < 0.0001, genotype x frequency, P = 0.50, C57, n = 4 mice; Lynx1 KO, n = 6 mice). (e, f) Representative ABR waves recorded from Lynx1 KO adult mice before and 30 minutes after administration of DHβE (i.p. 1mg/kg) during 11.3 kHz tone pips of increasing dB. (g, h) DHβE treatment did not impact ABR or DPOAE thresholds (mean ± SEM) across tone frequencies (MANOVA, ABR threshold by treatment condition, P = 0.54, frequency, P = 0.0116, treatment x frequency, P = 0.98; DPOAE threshold by treatment condition, P = 0.26, frequency, P = 0.13, treatment × frequency, P = 0.70, n = 4 mice).

Supplementary Figure 10 A topographic organization of 5-HT3AR+ interneurons.

Imaging of 5-HT3AR interneurons expressing the genetically-encoded calcium indicator (GCaMP6f) using 5-HT3AR-Cre x Ai95 mice. (a) Schematic of an auditory thalamocortical slice illustrates the lateral (1) and medial (6) positions stimulated within the ventral medial geniculate body (MGBv). (b) Average normalized traces (ΔF/F) across all mice (n = 3 mice) within layer (L) 1 locations of primary auditory cortex (A1) to stimulation of MGBv positions 1 and 6. (c) Mean peak responses (norm. ∆F/F) within L1 of A1 to stimulation of MGBv positions 1 and 6 across all mice (n = 3 mice). Black arrows indicate the rostro-caudal location of peak responses within L1.

Supplementary Figure 11 A schematic model of 5-HT3AR+ cell action within thalamocortical circuits.

5-HT3AR cells within layer (L) 1 of primary auditory cortex (A1) are driven by direct, topographically-organized inputs from the ventral medial geniculate body (MGBv). These cells are also co-activated by neuromodulators such as acetylcholine (ACh) via nAChRs. The nAChR recruitment of these cells is developmentally regulated by Lynx1, a protein that reduces nAChR function31,32. The axons of non-late-spiking (non-LS) 5-HT3AR cells descend to L4, preferentially targeting intracolumnar PV-cell somata. This suppresses MGBv-evoked spiking in PV cells, creating temporal windows of enhanced thalamic drive to L4 pyramidal cells that may gate plasticity mechanisms.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11

Life Sciences Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1: MGB and neuromodulatory inputs converge on superficial 5-HT3AR+ interneurons.
Fig. 2: 5-HT3AR+ interneurons target intracolumnar PV interneurons in L4.
Fig. 3: 5-HT3AR+ interneurons gate a window of disinhibition by differentially targeting PV and pyramidal cells.
Fig. 4: 5-HT3AR+ cell silencing prevents tonotopic plasticity.
Fig. 5: Nicotinic recruitment of 5-HT3AR+ cells controls critical-period timing.
Fig. 6: VIP+ cells contribute to PV cell suppression, but are not necessary for critical-period plasticity.
Fig. 7: A cortical L1 map tunes auditory cortex.
Supplementary Figure 1: MGB projects to cortical layer 1.
Supplementary Figure 2: 5-HT3AR+ interneurons receive direct MGB inputs.
Supplementary Figure 3: 5-HT3AR+ interneurons differentially affect MGB-evoked EPSPs in PV and pyramidal cells.
Supplementary Figure 4: 5-HT3AR+ interneurons provide spatially and temporally precise inhibition.
Supplementary Figure 5: Silencing of 5-HT3AR+ cells abolishes a critical period for auditory thalamocortical plasticity.
Supplementary Figure 6: Developmental changes in genes encoding 5-HT3AR and nAChRs within 5-HT3AR+ interneurons.
Supplementary Figure 7: Lynx1 expression in cortical interneurons.
Supplementary Figure 8: Nicotine induces a barrage of IPSCs in PV and pyramidal cells from Lynx1-knockout mice.
Supplementary Figure 9: Lynx1-knockout mice exhibit normal peripheral hearing thresholds.
Supplementary Figure 10: A topographic organization of 5-HT3AR+ interneurons.
Supplementary Figure 11: A schematic model of 5-HT3AR+ cell action within thalamocortical circuits.