Neural transcriptome reveals molecular mechanisms for temporal control of vocalization across multiple timescales

BMC Genomics

Andrew H Bass¹  Daniel J Fergus²  Ni Y Feng¹ 

[1] Department of Neurobiology and Behavior, Cornell University, Ithaca, 14853, NY, USA;Current Address: North Carolina Museum of Natural Sciences, Genomics and Microbiology, Raleigh, 27601, NC, USA

关键词: Seasonal reproduction;    Gene expression;    Transcriptome;    Motoneuron;    Vocalization;   

Others  :  1208971
DOI  :  10.1186/s12864-015-1577-2

 received in 2015-01-21, accepted in 2015-04-24,  发布年份 2015

【 摘 要 】

Background

Vocalization is a prominent social behavior among vertebrates, including in the midshipman fish, an established model for elucidating the neural basis of acoustic communication. Courtship vocalizations produced by territorial males are essential for reproductive success, vary over daily and seasonal cycles, and last up to hours per call. Vocalizations rely upon extreme synchrony and millisecond precision in the firing of a homogeneous population of motoneurons, the vocal motor nucleus (VMN). Although studies have identified neural mechanisms driving rapid, precise, and stable neuronal firing over long periods of calling, little is known about underlying genetic/molecular mechanisms.

Results

We used RNA sequencing-based transcriptome analyses to compare patterns of gene expression in VMN to the surrounding hindbrain across three daily and seasonal time points of high and low sound production to identify candidate genes that underlie VMN’s intrinsic and network neuronal properties. Results from gene ontology enrichment, enzyme pathway mapping, and gene category-wide expression levels highlighted the importance of cellular respiration in VMN function, consistent with the high energetic demands of sustained vocal behavior. Functionally important candidate genes upregulated in the VMN, including at time points corresponding to high natural vocal activity, encode ion channels and neurotransmitter receptors, hormone receptors and biosynthetic enzymes, neuromodulators, aerobic respiration enzymes, and antioxidants. Quantitative PCR and RNA-seq expression levels for 28 genes were significantly correlated. Many candidate gene products regulate mechanisms of neuronal excitability, including those previously identified in VMN motoneurons, as well as novel ones that remain to be investigated. Supporting evidence from previous studies in midshipman strongly validate the value of transcriptomic analyses for linking genes to neural characters that drive behavior.

Conclusions

Transcriptome analyses highlighted a suite of molecular mechanisms that regulate vocalization over behaviorally relevant timescales, spanning milliseconds to hours and seasons. To our knowledge, this is the first comprehensive characterization of gene expression in a dedicated vocal motor nucleus. Candidate genes identified here may belong to a conserved genetic toolkit for vocal motoneurons facing similar energetic and neurophysiological demands.

【 授权许可】

   
2015 Feng et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Bass AH, Gilland EH, Baker R. Evolutionary origins for social vocalization in a vertebrate hindbrain-spinal compartment. Science. 2008; 321:417-21.
• [2]Kingsbury MA, Kelly AM, Schrock SE, Goodson JL. Mammal-like organization of the avian midbrain central gray and a reappraisal of the intercollicular nucleus. PLoS ONE. 2011; 6:e20720.
• [3]Bass AH, Chagnaud BP. Shared developmental and evolutionary origins for neural basis of vocal-acoustic and pectoral-gestural signaling. Proc Natl Acad Sci USA. 2012; 109 Suppl 1:10677-84.
• [4]Elemans C, Spierts I, Müller UK, van Leeuwen JL. Bird song: superfast muscles control dove’s trill. Nature. 2004; 431:146.
• [5]Elemans CPH, Mead AF, Rome LC, Goller F. Superfast Vocal Muscles Control Song Production in Songbirds. PLoS ONE. 2008, 3:e2581.
• [6]Elemans CPH, Mead AF, Jakobsen L, Ratcliffe JM. Superfast Muscles Set Maximum Call Rate in Echolocating Bats. Science. 2011; 333:1885-8.
• [7]Amador A, Perl YS, Mindlin GB, Margoliash D. Elemental gesture dynamics are encoded by song premotor cortical neurons. Nature. 2013; 495:59-64.
• [8]Chagnaud BP, Zee MC, Baker R, Bass AH. Innovations in motoneuron synchrony drive rapid temporal modulations in vertebrate acoustic signaling. J Neurophysiol. 2012; 107:3528-42.
• [9]Goldberg JH, Farries MA, Fee MS. Basal ganglia output to the thalamus: still a paradox. Trends Neurosci. 2013; 36:695-705.
• [10]Yamaguchi A, Kaczmarek LK, Kelley DB. Functional specialization of male and female vocal motoneurons. J Neurosci. 2003; 23:11568-76.
• [11]Lovell PV, Clayton DF, Replogle KL, Mello CV. Birdsong “transcriptomics”: neurochemical specializations of the oscine song system. PLoS ONE. 2008; 3:e3440.
• [12]Lovell PV, Carleton JB, Mello CV. Genomics analysis of potassium channel genes in songbirds reveals molecular specializations of brain circuits for the maintenance and production of learned vocalizations. BMC Genomics. 2013; 14:470.
• [13]Whitney O, Pfenning AR, Howard JT, Blatti CA, Liu F, Ward JM et al.. Core and region-enriched networks of behaviorally regulated genes and the singing genome. Science. 2014; 346:1256780.
• [14]Hilliard AT, Miller JE, Fraley ER, Horvath S, White SA. Molecular microcircuitry underlies functional specification in a basal ganglia circuit dedicated to vocal learning. Neuron. 2012; 73:537-52.
• [15]Warren WC, Clayton DF, Ellegren H, Arnold AP, Hillier LW, Künstner A et al.. The genome of a songbird. Nature. 2010; 464:757-62.
• [16]Fraser BA, Janowitz I, Thairu M, Travis J, Hughes KA. Phenotypic and genomic plasticity of alternative male reproductive tactics in sailfin mollies. Proc R Soc B Biol Sci. 2014; 281:20132310.
• [17]Rittschof CC, Bukhari SA, Sloofman LG, Troy JM, Caetano-Anollés D, Cash-Ahmed A, et al. Neuromolecular responses to social challenge: Common mechanisms across mouse, stickleback fish, and honey bee. Proc Natl Acad Sci USA. 2014;111:17929–34.
• [18]Schunter C, Vollmer SV, Macpherson E, Pascual M. Transcriptome analyses and differential gene expression in a non-model fish species with alternative mating tactics. BMC Genomics. 2014; 15:1-13.
• [19]Sanogo YO, Band M, Blatti C, Sinha S, Bell AM. Transcriptional regulation of brain gene expression in response to a territorial intrusion. Proc R Soc B Biol Sci. 2012;279:4929–38.
• [20]Greenfield DW, Winterbottom R, Collette BB. Review of the Toadfish genera (Teleostei: Batrachoididae). Proc Calif Acad Sci. 2008; 59:665-710.
• [21]Rice AN, Bass AH. Novel vocal repertoire and paired swimbladders of the three-spined toadfish, Batrachomoeus trispinosus: insights into the diversity of the Batrachoididae. J Exp Biol. 2009; 212:1377-91.
• [22]Brantley RK, Bass AH. Alternative male spawning tactics and acoustic signals in the plainfin midshipman fish porichthys notatus Girard (Teleostei, Batrachoididae). Ethology. 1994; 96:213-32.
• [23]McKibben JR, Bass AH. Behavioral assessment of acoustic parameters relevant to signal recognition and preference in a vocal fish. J Acoust Soc Am. 1998; 104:3520-33.
• [24]Ibara RM, Penny LT, Ebeling AW, van Dykhuizen G, Cailliet G. The mating call of the plainfin midshipman fish, Porichthys notatus. In: Predators and prey in fishes. Springer Netherlands, Dordrecht; 1983: p.205-12. [Developments in Environmental Biology of Fishes]
• [25]Rubow TK, Bass AH. Reproductive and diurnal rhythms regulate vocal motor plasticity in a teleost fish. J Exp Biol. 2009; 212:3252-62.
• [26]McIver EL, Marchaterre MA, Rice AN, Bass AH. Novel underwater soundscape: acoustic repertoire of plainfin midshipman fish. J Exp Biol. 2014; 217:2377-89.
• [27]Bass AH, Marchaterre MA, Baker R. Vocal-acoustic pathways in a teleost fish. J Neurosci. 1994; 14:4025-39.
• [28]Goodson JL, Bass AH. Vocal–acoustic circuitry and descending vocal pathways in teleost fish: Convergence with terrestrial vertebrates reveals conserved traits. J Comp Neurol. 2002; 448:298-322.
• [29]Kittelberger JM, Bass AH. Vocal motor and auditory connectivity of the midbrain periaqueductal gray in a teleost fish. J Comp Neurol. 2013; 521:791-812.
• [30]Chagnaud BP, Baker R, Bass AH. Vocalization frequency and duration are coded in separate hindbrain nuclei. Nat Commun. 2011; 2:346-11.
• [31]Bass AH. Central pattern generator for vocalization: is there a vertebrate morphotype? Curr Opin Neurobiol. 2014; 28:94-100.
• [32]Bass AH, Baker R. Sexual dimorphisms in the vocal control system of a teleost fish: Morphology of physiologically identified neurons. J Neurobiol. 1990; 21:1155-68.
• [33]Remage-Healey L, Bass AH. A rapid neuromodulatory role for steroid hormones in the control of reproductive behavior. Brain Res. 2006; 1126:27-35.
• [34]Remage-Healey L, Bass AH. Rapid, hierarchical modulation of vocal patterning by steroid hormones. J Neurosci. 2004; 24:5892-900.
• [35]Remage-Healey L, Bass AH. Estradiol interacts with an opioidergic network to achieve rapid modulation of a vocal pattern generator. J Comp Physiol A. 2009; 196:137-46.
• [36]Remage-Healey L, Bass AH. Plasticity in brain sexuality is revealed by the rapid actions of steroid hormones. J Neurosci. 2007; 27:1114-22.
• [37]Goodson JL, Bass AH. Rhythmic midbrain-evoked vocalization is inhibited by vasoactive intestinal polypeptide in the teleost Porichthys notatus. Brain Res. 2000; 865:107-11.
• [38]Goodson JL, Bass AH. Forebrain peptides modulate sexually polymorphic vocal circuitry. Nature. 2000; 403:769-72.
• [39]Feng NY, Bass AH. Melatonin action in a midbrain vocal-acoustic network. J Exp Biol. 2014; 217:1046-57.
• [40]Sturdy CB, Wild JM, Mooney R. Respiratory and telencephalic modulation of vocal motor neurons in the zebra finch. J Neurosci. 2003; 23:1072-86.
• [41]Rübsamen R, Betz M. Control of echolocation pulses by neurons of the nucleus ambiguus in the rufous horseshoe bat, Rhinolophus rouxi. I. Single unit recordings in the ventral motor nucleus of the laryngeal nerves in spontaneously vocalizing bats. J Comp Physiol A. 1986; 159:675-87.
• [42]Riede T, Goller F. Peripheral mechanisms for vocal production in birds - differences and similarities to human speech and singing. Brain Lang. 2010; 115:69-80.
• [43]Hage SR, Jürgens U. On the role of the pontine brainstem in vocal pattern generation: a telemetric single-unit recording study in the squirrel monkey. J Neurosci. 2006; 26:7105-15.
• [44]Hage SR, Gavrilov N, Salomon F, Stein AM. Temporal vocal features suggest different call-pattern generating mechanisms in mice and bats. BMC Neurosci. 2013; 14:99.
• [45]Bass A, Andersen K. Inter- and intrasexual dimorphisms in the vocal control system of a teleost fish: motor axon number and size. Brain Behav Evol. 1991; 37:204-14.
• [46]Fergus DJ, Bass AH. Localization and divergent profiles of estrogen receptors and aromatase in the vocal and auditory networks of a fish with alternative mating tactics. J Comp Neurol. 2013; 521:2850-69.
• [47]Bass AH, Remage-Healey L. Central pattern generators for social vocalization: Androgen-dependent neurophysiological mechanisms. Horm Behav. 2008; 53:659-72.
• [48]Forlano PM, Sisneros JA, Rohmann KN, Bass AH. Neuroendocrine control of seasonal plasticity in the auditory and vocal systems of fish. Front Neuroendocrinol. 2015;37:129–45.
• [49]Fergus DJ, Feng NY, Bass AH. Gene expression patterns in auditory hair cell epithelium: insights into the molecular basis of seasonal plasticity in frequency sensitivity [abstract]. Washington DC: Society for Neuroscience, 183.02/SS28;2014.
• [50]Balakrishnan CN, Mukai M, Gonser RA, Wingfield JC, London SE, Tuttle EM et al.. Brain transcriptome sequencing and assembly of three songbird model systems for the study of social behavior. PeerJ. 2014; 2:e396.
• [51]Parra G, Bradnam K, Korf I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics. 2007; 23:1061-7.
• [52]Parra G, Bradnam K, Ning Z, Keane T, Korf I. Assessing the gene space in draft genomes. Nucleic Acids Res. 2009; 37:289-97.
• [53]Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ et al.. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res. 2008; 36:3420-35.
• [54]Huth TJ, Place SP. De novo assembly and characterization of tissue specific transcriptomes in the emerald notothen, Trematomus bernacchii. BMC Genomics. 2013; 14:805.
• [55]Ballman KV, Grill DE, Oberg AL, Therneau TM. Faster cyclic loess: normalizing RNA arrays via linear models. Bioinformatics. 2004; 20:2778-86.
• [56]Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010; 11:R25.
• [57]Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26:139-40.
• [58]Brantley RK, Bass AH. Cholinergic neurons in the brain of a teleost fish (Porichthys notatus) located with a monoclonal antibody to choline acetyltransferase. J Comp Neurol. 1988; 275:87-105.
• [59]Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M, Tanabe M. Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res. 2014; 42(Database issue):D199-205.
• [60]Wu G, Fang Y-Z, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. J Nutr. 2004; 134:489-92.
• [61]Delage E, Puyaubert J, Zachowski A, Ruelland E. Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: Convergences and divergences among eukaryotic kingdoms. Prog Lipid Res. 2013; 52:1-14.
• [62]Ophir AG, SCHRADER SB, Gillooly JF. Energetic cost of calling: general constraints and species-specific differences. J Evol Biol. 2010; 23:1564-9.
• [63]Gillooly JF, Ophir AG. The energetic basis of acoustic communication. Proc R Soc B Biol Sci. 2010; 277:1325-31.
• [64]Walsh PJ, Mommsen TP, Bass AH. Biochemical and molecular aspects of singing in batrachoidid fishes. In Biochemistry and Molecular Biology of Fishes. Volume 4. Metabolic Biochemistry. Edited by Hochachka PW, Mommsen TP; Amsterdam: Elsevier; 1995:279–289.
• [65]Rome LC. Design and function of superfast muscles: new insights into the physiology of skeletal muscle. Annu Rev Physiol. 2006; 68:193-221.
• [66]Bartos M, Vida I, Jonas P. Synaptic mechanisms of synchronized gamma oscillations in inhibitory interneuron networks. Nat Rev Neurosci. 2007; 8:45-56.
• [67]Pereda AE. Electrical synapses and their functional interactions with chemical synapses. Nat Rev Neurosci. 2014; 15:250-63.
• [68]Bass AH, Marchaterre MA. Sound-generating (sonic) motor system in a teleost fish (Porichthys notatus): Sexual polymorphisms and general synaptology of sonic motor nucleus. J Comp Neurol. 1989; 286:154-69.
• [69]Judge SIV, Bever CT. Potassium channel blockers in multiple sclerosis: neuronal Kv channels and effects of symptomatic treatment. Pharmacol Ther. 2006; 111:224-59.
• [70]Gutman GA, Chandy KG, Adelman JP, Aiyar J, Bayliss DA, Clapham DE et al.. International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels. Pharmacol Rev. 2003; 55:583-6.
• [71]Ding S, Matta SG, Zhou F-M. Kv3-like potassium channels are required for sustained high-frequency firing in basal ganglia output neurons. J Neurophysiol. 2011; 105:554-70.
• [72]Richardson FC, Kaczmarek LK. Modification of delayed rectifier potassium currents by the Kv9.1 potassium channel subunit. Hear Res. 2000; 147:21-30.
• [73]Stocker M, Hellwig M, Kerschensteiner D. Subunit assembly and domain analysis of electrically silent K+ channel alpha-subunits of the rat Kv9 subfamily. J Neurochem. 1999; 72:1725-34.
• [74]Brown D. M-currents: an update. Trends Neurosci. 1988; 11:294-9.
• [75]Grove CL, Szabo TM, McIntosh JM, Do SC, Waldeck RF, Faber DS. Fast synaptic transmission in the goldfish CNS mediated by multiple nicotinic receptors. J Physiol. 2011; 589:575-95.
• [76]McMahon LL, Yoon KW, Chiappinelli VA. Electrophysiological evidence for presynaptic nicotinic receptors in the avian ventral lateral geniculate nucleus. J Neurophysiol. 1994; 71:826-9.
• [77]Bellingham MC, Berger AJ. Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors. J Neurophysiol. 1996;76:3758-70.
• [78]Miles GB, Hartley R, Todd AJ, Brownstone RM. Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion. Proc Natl Acad Sci USA. 2007; 104:2448-53.
• [79]Zagoraiou L, Akay T, Martin JF, Brownstone RM, Jessell TM, Miles GB. A cluster of cholinergic premotor interneurons modulates mouse locomotor activity. Neuron. 2009; 64:645-62.
• [80]Hellstr MJ, Oliveira ALR, Meister BR, Cullheim S. Large cholinergic nerve terminals on subsets of motoneurons and their relation to muscarinic receptor type 2. J Comp Neurol. 2003; 460:476-86.
• [81]Muennich EAL, Fyffe REW. Focal aggregation of voltage-gated, Kv2.1 subunit-containing, potassium channels at synaptic sites in rat spinal motoneurones. J Physiol. 2004; 554:673-85.
• [82]Brown DA, Passmore GM. Neural KCNQ (Kv7) channels. Br J Pharmacol. 2009; 156:1185-95.
• [83]Caulfield MP, Birdsall NJ. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev. 1998; 50:279-90.
• [84]Luetje CW, Piattoni M, Patrick J. Mapping of ligand-binding sites of neuronal nicotinic acetylcholine-receptors using chimeric alpha-subunits. Mol Pharmacol. 1993; 44:657-66.
• [85]McIntosh JM, Dowell C, Watkins M, Garrett JE, Yoshikami D, Olivera BM. Alpha-conotoxin GIC from Conus geographus, a novel peptide antagonist of nicotinic acetylcholine receptors. J Biol Chem. 2002; 277:33610-5.
• [86]Chambers MS, Atack JR, Broughton HB, Collinson N, Cook S, Dawson GR et al.. Identification of a novel, selective gaba aα5 receptor inverse agonist which enhances cognition. J Med Chem. 2003; 46:2227-40.
• [87]Remage-Healey L, Bass AH. From social behavior to neural circuitry: Steroid hormones rapidly modulate advertisement calling via a vocal pattern generator. Horm Behav. 2006; 50:432-41.
• [88]Forlano PM, Deitcher DL, Myers DA, Bass AH. Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source. J Neurosci. 2001; 21:8943-55.
• [89]Labrie F, Luu-The V, Lin SX, Simard J, Labrie C, El-Alfy M et al.. Intracrinology: role of the family of 17 beta-hydroxysteroid dehydrogenases in human physiology and disease. J Mol Endocrinol. 2000; 25:1-16.
• [90]Kusakabe M, Nakamura I, Young G. 11beta-hydroxysteroid dehydrogenase complementary deoxyribonucleic acid in rainbow trout: cloning, sites of expression, and seasonal changes in gonads. Endocrinology. 2003; 144:2534-45.
• [91]Genova RM, Marchaterre MA, Knapp R, Fergus D, Bass AH. Glucocorticoid and androgen signaling pathways diverge between advertisement calling and non-calling fish. Horm Behav. 2012; 62:426-32.
• [92]Forlano PM, Kim SD, Krzyminska ZM, Sisneros JA. Catecholaminergic connectivity to the inner ear, central auditory, and vocal motor circuitry in the plainfin midshipman fish Porichthys Notatus. J Comp Neurol. 2014; 522:2887-927.
• [93]Stacey N. Hormones, pheromones and reproductive behavior. Fish Physiol Biochem. 2003; 28:229-35.
• [94]Forlano PM, Deitcher DL, Bass AH. Distribution of estrogen receptor alpha mRNA in the brain and inner ear of a vocal fish with comparisons to sites of aromatase expression. J Comp Neurol. 2005; 483:91-113.
• [95]Davies KJ. Oxidative stress, antioxidant defenses, and damage removal, repair, and replacement systems. IUBMB Life. 2000; 50:279-89.
• [96]Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006; 443:787-95.
• [97]Cozzolino M, Pesaresi MG, Gerbino V, Grosskreutz J, Carrì MT. Amyotrophic Lateral Sclerosis: New insights into underlying molecular mechanisms and opportunities for therapeutic intervention. Antioxid Redox Signal. 2012; 17:1277-330.
• [98]Hayes JD, Pulford DJ. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance part I. Crit Rev Biochem Mol. 1995;30:445–520.
• [99]Hermosura MC, Nayakanti H, Dorovkov MV, Calderon FR, Ryazanov AG, Haymer DS et al.. A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders. Proc Natl Acad Sci USA. 2005; 102:11510-5.
• [100]Bandyopadhyay U, Cotney J, Nagy M, Oh S, Leng J, Mahajan M et al.. RNA-seq profiling of spinal cord motor neurons from a presymptomatic sod1 als mouse. PLoS ONE. 2013; 8:e53575.
• [101]Arnold AP, Nottebohm F, Pfaff DW. Hormone concentrating cells in vocal control and other areas of the brain of the zebra finch (Poephila guttata). J Comp Neurol. 1976; 165:487-511.
• [102]Bass AH, Horvath BJ, Brothers EB. Nonsequential developmental trajectories lead to dimorphic vocal circuitry for males with alternative reproductive tactics. J Neurobiol. 1996; 30:493-504.
• [103]DeVoogd TJ, Pyskaty DJ, Nottebohm F. Lateral asymmetries and testosterone-induced changes in the gross morphology of the hypoglossal nucleus in adult canaries. J Comp Neurol. 1991; 307:65-76.
• [104]Clower RP, Nixdorf BE, DeVoogd TJ. Synaptic plasticity in the hypoglossal nucleus of female canaries: structural correlates of season, hemisphere, and testosterone treatment. Behav Neural Biol. 1989; 52:63-77.
• [105]Bass AH, Forlano PM. Neuroendocrine mechanisms of alternative reproductive tactics: the chemical language of reproductive and social plasticity. Cambridge University Press, Cambridge; 2008.
• [106]Gahr M, Garcia-Segura LM. Testosterone-dependent increase of gap-junctions in HVC neurons of adult female canaries. Brain Res. 1996; 712:69-73.
• [107]Matsumoto A, Arnold AP, Zampighi GA, Micevych PE. Androgenic regulation of gap junctions between motoneurons in the rat spinal cord. J Neurosci. 1988; 8:4177-83.
• [108]Perez J, Tranque PA, Naftolin F, Garcia-Segura LM. Gap junctions in the hypothalamic arcuate neurons of ovariectomized and estradiol-treated rats. Neurosci Lett. 1990; 108:17-21.
• [109]Rome LC, Syme DA, Hollingworth S, Lindstedt SL, Baylor SM. The whistle and the rattle: the design of sound producing muscles. Proc Natl Acad Sci USA. 1996; 93:8095-100.
• [110]Sisneros JA, Forlano PM, Knapp R, Bass AH. Seasonal variation of steroid hormone levels in an intertidal-nesting fish, the vocal plainfin midshipman. Gen Comp Endocrinol. 2004; 136:101-16.
• [111]Zhong S, Joung J-G, Zheng Y, Chen Y-R, Liu B, Shao Y et al.. High-throughput illumina strand-specific RNA sequencing library preparation. Cold Spring Harb Protoc. 2011; 2011:940-9.
• [112]Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30:btu170-2120.
• [113]Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J et al.. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013; 8:1494-512.
• [114]Li B, Fillmore N, Bai Y, Collins M, Thomson JA, Stewart R, et al. Evaluation of de novo transcriptome assemblies from RNA-Seq data. Genome Biol. 2014;15:553.
• [115]Schmieder R, Edwards R. Quality control and preprocessing of metagenomic datasets. Bioinformatics. 2011; 27:863-4.
• [116]Glusman G, Caballero J, Robinson M, Kutlu B, Hood L. Optimal scaling of digital transcriptomes. PLoS ONE. 2013; 8:e77885.
• [117]Robinson MD, Smyth GK. Moderated statistical tests for assessing differences in tag abundance. Bioinformatics. 2007; 23:2881-7.
• [118]R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria; 2014.
• [119]Mardia KV, Kent JT, Bibby JM. Multivariate Analysis. New York: Academic Press; 1979.
• [120]Forlano PM, Marchaterre M, Deitcher DL, Bass AH. Distribution of androgen receptor mRNA expression in vocal, auditory, and neuroendocrine circuits in a teleost fish. J Comp Neurol. 2010; 518:493-512.