Behaviors emerge from activity throughout the brain, but non-invasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become non-invasively accessible from embryo to sexually mature adult in a vertebrate model.

Cilia are cell surface organelles with key roles in a range of cellular processes, including generation of fluid flow by motile cilia. The axonemes of motile cilia and immotile kinocilia contain 9 peripheral microtubule doublets, a central microtubule pair, and 9 connecting radial spokes. Aberrant radial spoke components RSPH1, 3, 4a and 9 have been linked with primary ciliary dyskinesia (PCD), a disorder characterized by ciliary dysmotility; yet, radial spoke functions remain unclear. Here we show that zebrafish Rsph9 is expressed in cells bearing motile cilia and kinocilia, and localizes to both 9 + 2 and 9 + 0 ciliary axonemes. Using CRISPR mutagenesis, we show that rsph9 is required for motility of presumptive 9 + 2 olfactory cilia and, unexpectedly, 9 + 0 neural cilia. rsph9 is also required for the structural integrity of 9 + 2 and 9 + 0 ciliary axonemes. rsph9 mutant larvae exhibit reduced initiation of the acoustic startle response consistent with hearing impairment, suggesting a novel role for Rsph9 in the kinocilia of the inner ear and/or lateral line neuromasts. These data identify novel roles for Rsph9 in 9 + 0 motile cilia and in sensory kinocilia, and establish a useful zebrafish PCD model.

Acoustic behavior is widespread across vertebrates, including among fishes. We report robust acoustic displays during aggressive interactions for a laboratory colony of Danionella dracula, a miniature and transparent species of teleost fish closely related to zebrafish (Danio rerio), which are hypothesized to be sonic based on the presence of a hypertrophied muscle associated with the male swim bladder. Males produce bursts of pulsatile sounds and a distinct postural display–extension of a hypertrophied lower jaw, a morphological trait not present in other Danionella species—during aggressive, but not courtship interactions. Females show no evidence of sound production or jaw extension in such contexts. Novel pairs of size-matched or -mismatched males were combined in resident-intruder assays where sound production and jaw extension could be linked to individuals. In both dyad contexts, resident males produced significantly more sound pulses than intruders. During heightened sonic activity, the majority of highest sound producers also showed increased jaw extension. Residents extended their jaw more than intruders in size-matched, but not -mismatched contexts. Larger males in size-mismatched dyads produced more sounds and jaw extensions compared to their smaller counterparts, and sounds and jaw extensions increased with increasing absolute body size. These studies establish D. dracula as a sonic species that modulates putatively acoustic and postural displays during aggressive interactions based on residency and body size, providing a foundation for further investigating the role of multimodal displays in a new model clade for neurogenomic and neuroimaging studies of aggression, courtship, and other social interactions.

No abstract

Although optical microscopy has allowed us to study the entire brain in early developmental stages, access to the brains of live, adult vertebrates has been limited. Danionella, a genus of miniature, transparent fish closely related to zebrafish has been introduced as a neuroscience model to study the entire adult vertebrate brain. However, the extent of optically accessible depth in these animals has not been quantitatively characterized. Here, we show that two- and three-photon microscopy can be used to access the entire depth of the adult wild type Danionella dracula brain without any modifications to the animal other than mechanical stabilization. Three-photon microscopy provides high signal to background ratio and optical sectioning through the deepest part of the brain. While vasculature can be observed with two-photon microscopy, the deeper regions have low contrast. We show that multiphoton microscopy is ideal for readily penetrating the entire adult brain within the geometry of these animals' head structures and without the need for pigment removal. With multiphoton microscopy enabling optical access to the entire adult brain and a repertoire of methods that allow observation of the larval brain, Danionella provides a model system for readily studying the entire brain over the lifetime of a vertebrate.