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Research Articles: Development/Plasticity/Repair Neurod1 is essential for the primary tonotopic organization and related auditory information processing in the midbrain Iva Macovaa,d, Kateryna Pysanenkob, Tetyana Chumakb, Martina Dvorakovaa,d, Romana Bohuslavovaa, Josef Sykab, Bernd Fritzschc and Gabriela Pavlinkovaa aInstitute of Biotechnology CAS, Vestec, Czechia bInstitute of Experimental Medicine CAS, Prague, Czechia cDepartment of Biology, University of Iowa, Iowa City, IA, USA dFaculty of Science, Charles University, Prague, Czechia https://doi.org/10.1523/JNEUROSCI.2557-18.2018 Received: 10 October 2018 Revised: 17 November 2018 Accepted: 5 December 2018 Published: 12 December 2018 Author contributions: I.M., K.P., T.C., M.D., R.B., and B.F. performed research; I.M., K.P., T.C., M.D., R.B., B.F., and G.P. analyzed data; I.M. and K.P. wrote the first draft of the paper; R.B., J.S., B.F., and G.P. designed research; J.S., B.F., and G.P. edited the paper; B.F. and G.P. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. We thank Dr. K. Kandler for their helpful comments on an earlier version of this MS, Dr. D. Šuta for his help in the processing of single unit data, and A. Pavlinek for editing the MS. This research was supported by the Czech Science Foundation (No. 17-04719S to GP); by BIOCEVCZ.1.05/1.1.00/02.0109 from the ERDF; by the institutional support of the Czech Academy of Sciences RVO: 86652036; by Charles University (GA UK No. 324615 to IM and GAUK No. 780216 to MD) and by NIH (R01 AG060504 to BF). Correspondence should be addressed to To whom correspondence should be addressed. Email: [email protected] or [email protected] Cite as: J. Neurosci 2018; 10.1523/JNEUROSCI.2557-18.2018 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. 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Copyright © 2018 the authors 1 Neurod1 is essential for the primary tonotopic organization and related 2 auditory information processing in the midbrain 3 Authors: Iva Macovaa,d,1, Kateryna Pysanenkob,1, Tetyana Chumakb, Martina Dvorakovaa,d, 4 Romana Bohuslavovaa, Josef Sykab,2, Bernd Fritzschc,2,3, Gabriela Pavlinkovaa,2,3 5 6 Affiliations: 7 aInstitute of Biotechnology CAS, Vestec, Czechia 8 bInstitute of Experimental Medicine CAS, Prague, Czechia 9 cDepartment of Biology, University of Iowa, Iowa City, IA, USA 10 dFaculty of Science, Charles University, Prague, Czechia 11 1I.M. and K.P. contributed equally to this work. 12 2contributed to the conceptualization, evaluation and writing of this report. 13 3To whom correspondence should be addressed. 14 Email: [email protected] or [email protected] 15 16 17 Conflict of interest: The authors declare no competing financial interests. 18 Acknowledgments: We thank Dr. K. Kandler for their helpful comments on an earlier version of 19 this MS, Dr. D. Šuta for his help in the processing of single unit data, and A. Pavlinek for editing 20 the MS. 21 Funding: This research was supported by the Czech Science Foundation (No. 17-04719S to 22 GP); by BIOCEVCZ.1.05/1.1.00/02.0109 from the ERDF; by the institutional support of the 23 Czech Academy of Sciences RVO: 86652036; by Charles University (GA UK No. 324615 to IM 24 and GAUK No. 780216 to MD) and by NIH (R01 AG060504 to BF). 25 1 26 Abstract 27 Hearing depends on extracting frequency, intensity and temporal properties from sound to 28 generate an auditory map for acoustical signal processing. How physiology intersects with 29 molecular specification to fine tune the developing properties of the auditory system that enable 30 these aspects remains unclear. We made a novel conditional deletion model that eliminates the 31 transcription factor NEUROD1 exclusively in the ear. These mice (both sexes) develop a 32 truncated frequency range with no neuroanatomically recognizable mapping of spiral ganglion 33 neurons onto distinct locations in the cochlea nor a cochleotopic map presenting topographically 34 discrete projections to the cochlear nuclei. The disorganized primary cochleotopic map alters 35 tuning properties of the inferior colliculus units, which display abnormal frequency, intensity, 36 and temporal sound coding. At the behavioral level, animals show alterations in the acoustic 37 startle response, consistent with altered neuroanatomical and physiological properties. We 38 demonstrate that absence of the primary afferent topology during embryonic development leads 39 to dysfunctional tonotopy of the auditory system. Such effects have never been investigated in 40 other sensory systems due to the lack of models. 41 42 Significance statement 43 All sensory systems form a topographical map of neuronal projections from peripheral sensory 44 organs to the brain. Neuronal projections in the auditory pathway are cochleotopically organized, 45 providing a tonotopic map of sound frequencies. Primary sensory maps typically arise by 46 molecular cues, requiring physiological refinements. Past work has demonstrated physiologic 47 plasticity in many senses without ever molecularly undoing the specific mapping of an entire 48 primary sensory projection. We genetically manipulated primary auditory neurons to generate a 2 49 scrambled cochleotopic projection. Eliminating tonotopic representation to auditory nuclei 50 demonstrates the inability of physiological processes to restore a tonotopic presentation of sound 51 in the midbrain. Our data provide the first insights into the limits of physiology-mediated 52 brainstem plasticity during the development of the auditory system. 53 54 Keywords: auditory pathway, inferior colliculus, inner ear, Neurod1 mutation, sensory 55 topographical map, plasticity 56 57 INTRODUCTION 58 Sensory systems develop topographic maps of sensory inputs in nervous systems: the 59 somatosensory system projects topographical maps of the skin to the brainstem, midbrain, and 60 cortex (Penfield and Boldrey, 1937; Renier et al., 2017), the olfactory system projects odor 61 properties to a given olfactory glomerulus (Mombaerts, 1999; Gogos et al., 2000), the retina 62 develops a topographical map in the midbrain (Sperry, 1963; Goodhill, 2007; Huberman et al., 63 2008), and vestibular, lateral line and electroreceptive organs project to specific nuclei of the 64 hindbrain and midbrain to establish computational maps (Krahe and Maler, 2014; Chagnaud et 65 al., 2017). In the auditory system, cochlear sensory hair cells are connected to the brain by spiral 66 ganglion neurons that are organized within the cochlea in an orderly fashion according to 67 frequency, so called tonotopic organization, with high frequencies at the base and low 68 frequencies at the apex (Rubel and Fritzsch, 2002; Muniak et al., 2016). This tonotopic (or 69 cochleotopic) organization is maintained throughout the auditory pathways (Kandler et al., 70 2009). The formation of a tonotopic map requires the precise projection of the spiral ganglion 71 neuron afferents of the cochlea onto the first auditory nuclei of the hindbrain, the cochlear nuclei. 3 72 In contrast to the better-characterized visual system or olfactory system, only some molecular 73 mechanisms are known to lead to the cochleotopic mapping of spiral ganglion afferents onto the 74 cochlear nuclei (Cramer and Gabriele, 2014; Goodrich, 2016; Yang et al., 2017) but it is 75 unknown how much the initial cochleotopic map is physiologically refined (Marrs and Spirou, 76 2012). Developing second order maps are highly plastic (Hubel et al., 1977; Renier et al., 2017) 77 and convergence of multiple sensory organs can plastically reshape primary sensory maps as a 78 compromise between activity and molecular cues (Constantine-Paton and Law, 1978; Elliott et 79 al., 2015). Indeed, the auditory system is well known for having a high level of plastic changes 80 throughout life (Syka, 2002; Eggermont, 2017) but how embryonic development affects and 81 possibly limits these later plastic changes remains unclear due to the lack of models (Kral et al., 82 2016) beyond simply removing parts of the cochlea (Harrison, 2016). 83 In the formation of the primary tonotopic map, several factors apparently define the 84 precision of some spiral ganglion neuron projections (Cramer and Gabriele, 2014; Lu et al., 85 2014; Goodrich, 2016; Yang et al., 2017). The basic Helix-Loop-Helix (bHLH) gene Neurod1 86 (Liu et al., 2000) was shown to be essential for inner ear neuronal development as well as normal 87 growth of the cochlea. Subsequent work on mutants null for Neurod1 showed retention of some 88 sensory neurons using specific neuronal tracing techniques (Kim et al., 2001). NEUROD1 cross- 89 regulates other transcription factors in neurons and hair cells, leading to the transformation of 90 some neurons to intraganglionic hair cells, and transformation of some outer hair cells to inner 91 hair cells (Jahan et al., 2010b). In addition, deletion of Neurod1 leads to gross projection 92 mapping errors of the few remaining neurons (Jahan et al., 2010a) that go beyond those 93 described in other primary sensory system (Huberman et al., 2008). In previously generated 94 mutants, Neurod1 deletion occurs both in the ear and the central auditory nuclei, which limits 4 95 spiral ganglion neuronal viability and hampers physiological assessment of the wiring defects 96 (Gurung and Fritzsch, 2004; Fritzsch et al., 2006; Jahan et al., 2010a). 97 We therefore generated a novel mutant with a conditional deletion of Neurod1 only in the 98 ear to spare many spiral ganglion neurons and to retain Neurod1 expression in the auditory 99 nuclei and auditory midbrain. We show here how a shortened and nearly overlapping 100 cochleotopic projection from spiral neurons to the cochlear nucleus is expanded across the entire 101 inferior colliculus, affecting the frequency, intensity, and temporal processing of the central 102 auditory system of adult mice at the physiological and behavioral level.