Research Articles: Neurobiology of Disease Audiogenic seizures in the Fmr1 knockout mouse are induced by Fmr1 deletion in subcortical, vGlut2-expressing excitatory neurons and require deletion in the Inferior Colliculus https://doi.org/10.1523/JNEUROSCI.0886-19.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.0886-19.2019 Received: 24 April 2019 Revised: 6 September 2019 Accepted: 16 October 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2019 the authors 1 Audiogenic seizures in the Fmr1 knockout mouse are induced by Fmr1 deletion in subcortical, 2 vGlut2-expressing excitatory neurons and require deletion in the Inferior Colliculus 3 4 Authors: 5 Darya Gonzalez1, Madison Tomasek1, Seth Hays1, Vinay Sridhar1, Simon Ammanuel1, Chia-wei Chang1, 6 Karen Pawlowski2, Kimberly M. Huber*1, Jay R. Gibson*1. 7 *co-authors 8 1University of Texas Southwestern Medical Center at Dallas, Department of Neuroscience, Dallas, TX 9 75390-9111, USA 10 2University of Texas Southwestern Medical Center at Dallas, Department of Otolaryngology and 11 Biomedical Engineering Program, Dallas, TX 75390-9035, USA 12 13 14 Abbreviated Title: Circuits for audiogenic seizures in Fmr1 knockout (49 char, 50 char max) 15 16 Corresponding Authors: 17 Jay R. Gibson, Kimberly Huber 18 University of Texas, Southwestern Medical Center 19 Department of Neuroscience, Box 9111 20 Dallas, TX 75390-9111 21 Phone: 214-648-5133, Fax: 214-648-1801, Email: [email protected] 22 Phone: 214-648-5134, Fax: 214-648-1801, Email: [email protected] 23 24 Author Contributions 25 JRG, KMH, planned and designed experiments. DG, MT, SH, VS, SA, CWC, KP, and JRG performed 26 experiments and analyzed data. JRG and KMH prepared the manuscript. 1 28 27 pages, 7 figures; Words: title=24 (50), Abstract=247 (250), Introduction=655 (650), Sig. Statement 29 =107 (120), Results=2150, Discussion=1515 (1500). 30 Acknowledgements: We thank Patricia Hahn, Bianca Walker, and Manasa Sarma for technical 31 assistance. We thank the Neuroscience Microscopy Facility for use of confocal microscopes. This facility 32 is supported by the University of Texas, Southwestern Neuroscience department and the Peter O’Donnell 33 Jr. Brain Institute. KP is now affiliated with University of Texas at Dallas, School of Behavioral and 34 Brain Sciences. SH is now affiliated with University of Texas at Dallas, Department of Bioengineering. 35 Funding: NIH funding from NICHD, 1U54HD082008-01 (JRG, KMH) 36 37 38 3 39 Abstract 40 Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading 41 monogenetic cause of autism. One symptom of FXS and autism is sensory hypersensitivity (also called 42 sensory over-responsivity). Perhaps related to this, the audiogenic seizure (AGS) is arguably the most 43 robust behavioral phenotype in the FXS mouse model - the Fmr1 knockout (KO) mouse. Therefore, the 44 AGS may be considered a mouse model of sensory hypersensitivity. Hyperactive circuits are 45 hypothesized to underlie dysfunction in a number of brain regions in FXS patients and Fmr1 KO mice, 46 and the AGS may be a result of this. But the specific cell-types and brain regions underlying AGSs in the 47 Fmr1 KO are unknown. We used conditional deletion or expression of Fmr1 in different cell populations 48 to determine whether Fmr1 deletion in those cells was sufficient or necessary, respectively, for the AGS 49 phenotype in males. Our data indicate that Fmr1 deletion in glutamatergic neurons that express vesicular 50 glutamate transporter 2 (vGlut2) and located in subcortical brain regions is sufficient and necessary to 51 cause AGSs. Furthermore, deletion of Fmr1 in glutamatergic neurons of the inferior colliculus is 52 necessary for AGSs. When we demonstrate necessity, we show that Fmr1 expression in either the larger 53 population of vGlut2-expressing glutamatergic neurons or the smaller population of inferior collicular 54 glutamatergic neurons - in an otherwise Fmr1 KO mouse - eliminates AGSs. Therefore, targeting these 55 neuronal populations in FXS and autism may be part of a therapeutic strategy to alleviate sensory 56 hypersensitivity. 57 58 Significance 59 Sensory hypersensitivity in Fragile X syndrome (FXS) and autism patients significantly interferes with 60 quality of life. Audiogenic seizures (AGSs) are arguably the most robust behavioral phenotype in the 61 FXS mouse model – the Fmr1 knockout - and may be considered a model of sensory hypersensitivity in 62 FXS. We provide the clearest and most precise genetic evidence to date for the cell-types and brain 63 regions involved in causing AGSs in the Fmr1 knockout, and more broadly, for any mouse mutant. 64 Expression of Fmr1 in these same cell-types in an otherwise Fmr1 knockout eliminates AGSs indicating 65 possible cellular targets for alleviating sensory hypersensitivity in FXS and other forms of autism. 4 66 Introduction 67 68 Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading 69 monogenetic cause of autism (Bassell and Warren, 2008). It is caused by loss of function mutations in 70 FMR1 which encodes an RNA binding protein, FMRP. Many of the impairments in FXS are reproduced 71 in the FXS mouse model, the Fmr1 knockout (KO) mouse (Bakker, 1994). 72 Sensory hypersensitivity (or sensory over-responsivity) and abnormal sensory processing occur in 70- 73 90% of FXS and autistic patients, and these traits can significantly disrupt behavior (Musumeci et al., 74 1994, Miller et al., 1999, Rojas et al., 2001, Baranek et al., 2008, Ben-Sasson et al., 2009, Hagerman et 75 al., 2009). FXS patients display increased physiological auditory responses as observed by the event- 76 related potential (ERP) amplitude and reduced habituation of the ERP in response to repeated sounds 77 (Castren et al., 2003, Van der Molen et al., 2011, Ethridge et al., 2016). These ERP changes correlate 78 with sensory hypersensitivity and communication deficits in FXS patients, suggesting that 79 hyperexcitability of auditory pathways contribute to these symptoms (Ethridge et al., 2016). Fmr1 KO 80 mice also have an enhanced auditory ERP in the form of reduced habituation and enhanced sound-evoked 81 firing of auditory cortical neurons (Rotschafer and Razak, 2013). The mice also have audiogenic 82 seizures (AGSs) (Musumeci et al., 2000, Chen and Toth, 2001). 83 As a result of these and other observations, it is hypothesized that brain circuits are hyperexcitable in FXS 84 (Contractor et al., 2015). In support of this idea, individuals with FXS have an increased incidence of 85 epilepsy (Musumeci et al., 1999, Sabaratnam et al., 2001, Berry-Kravis et al., 2010). Indeed, in the Fmr1 86 KO mouse, circuit hyperexcitability and potential underlying mechanisms have been well demonstrated in 87 neocortex and hippocampus (Chuang et al., 2005, Galvez and Greenough, 2005, Goncalves et al., 2013, 88 Cea-Del Rio and Huntsman, 2014, Zhang et al., 2014a, Contractor et al., 2015). However, establishing a 89 link between a specific hyperexcitable circuit and altered behavior in the Fmr1 KO mouse has been 90 elusive. 91 We consider the AGS in the Fmr1 KO mice to be a model of sensory stimulus hypersensitivity in FXS. 92 AGSs occur in other autism mouse models, such as with Syngap1 and Ube3a deletion (Jiang et al., 1998, 93 Clement et al., 2012). The AGS is arguably the most robust behavioral phenotype in the Fmr1 KO mouse 94 and has been reproduced in 49 original research articles since 2000 and in multiple strain backgrounds 95 (Table I). But interpretations from all these studies are limited by lack of knowledge of the circuits or 96 cell-types in which Fmr1 functions to cause the AGS. 5 97 From studies of seizure-prone rat and mouse strains, AGSs likely originate from hyperexcitable circuits in 98 the brain stem (Faingold, 2002, 2004, Ribak, 2015). In the Fmr1 KO, studies using c-fos expression to 99 mark active neurons during the AGS implicate cells in the midbrain and pons (Chen and Toth, 2001), but 100 this experimental approach cannot determine if FMRP deletion in these active neurons causes the AGS or 101 if they are simply indirectly activated. 102 By crossing mice with conditional deletion or expression of Fmr1 with cell type and/or brain region 103 specific Cre lines, we determined the locus in which Fmr1 deletion causes AGSs. Fmr1 is expressed in 104 many cell types throughout the brain: neurons, astrocytes, oligodendrocytes, and endothelial cells (Zhang 105 et al., 2014b). Our results indicate that Fmr1 deletion in subcortical glutamatergic neurons that express 106 vesicular glutamate transporter 2 (vGlut2) underlies AGSs. Fmr1 deletion in glutamatergic neurons in the 107 inferior colliculus is necessary for the phenotype, which represents the most precise genetic localization 108 to date for causing AGS in mice. This latter finding pertaining to the inferior colliculus implicates a 109 potentially hyperexcitable, localized circuit underlying a behavioral phenotype in the Fmr1 KO mouse. 110 Finally, selective Fmr1 expression in glutamatergic neurons in an otherwise Fmr1 KO mouse eliminates 111 AGSs suggesting that targeting these neurons may be part of a strategy to alleviate sensory 112 hypersensitivity in FXS and in autism. 113 114 Materials and Methods 115 116 Mice 117 For conditional Fmr1 deletion, experimental mice were produced by crossing a sire expressing Cre- 118 recombinase (Cre) in a specific neuronal population to an Fmr1loxP/+ dam (Mientjes et al., 2006). We refer 119 to the loxP allele as “conditional off”, or cOFF, and the genotype as Fmr1cOFF/+.