Midshipman is a neuroethological model for studying neural control of vocalization. A) Midshipman vocal behavior varies across a range of time scales. Picture of a midshipman fish (top) with schematic side view of the brain, vocal nerve (VN), and sonic swimbladder (SB), the sound-producing organ with vocal muscles (red) attached to the sidewalls. Continuous hums can last from mins to >1 h, as shown in the 1.85 h hum recorded from a captive male (blue trace), and are produced repetitively throughout the night during the summer breeding season (bottom). Close-up of natural hum is adapted from . B) Schematic saggital view of the midshipman brain showing the hindbrain vocal pattern generator that consists of the vocal pre-pacemaker nucleus (VPP), vocal pacemaker nucleus (VPN), and vocal motor nucleus (VMN) (left). Forebrain and midbrain vocal centers drive the hindbrain pattern generator, which provides a precise and synchronous code that controls sonic muscle contraction in a one-to-one manner (compare the series of sound pulses in the natural hum in A to the vocal nerve potentials directly to the right in the bottom trace in B. Extreme temporal precision of motoneuron firing is shown by corresponding traces from an intracellular VMN recording (red) and VN recording (purple; adapted from ). Trace of a long duration VN recording (bottom) adapted from . C) Tissue groups used for transcriptome analysis and their notations. Left: a transverse section at the level of VMN showing bilateral, transneuronal biocytin labeling in VMN and the vocal tract (VoTr) (see ); each VMN innervates the ipsilateral vocal SB muscle. In this study, we surgically separated the midline pair of VMN from the surrounding hindbrain tissue (Rest of H). Right: abbreviations of sample groups according to the tissue and time of collection, used throughout this paper.