Longitudinal in vivo imaging of adult Danionella cerebrum using standard confocal microscopy

Abstract

Danionella cerebrum is a new vertebrate model that offers an exciting opportunity to visualize dynamic biological processes in intact adult animals. Key advantages of this model include its small size, life-long optical transparency, genetic amenability, and short generation time. Establishing a reliable method for longitudinal in vivo imaging of adult D. cerebrum, while maintaining viability, will allow for in-depth image-based studies of various processes involved in development, disease onset and progression, wound healing, and aging in an intact live animal. Here, we describe a method for both prolonged and longitudinal confocal live imaging of adult D. cerebrum using custom designed and 3D printed imaging chambers. We created two transgenic D. cerebrum lines to test our imaging system, Tg(mpeg1:dendra2) and Tg(kdrl:mCherry-caax). The first to visualize macrophages and microglia, and the second for spatial registration. Using this approach, we observed differences in immune cell morphology and behavior during homeostasis as well as in response to a stab wound or two-photon-induced brain injury in intact adult fish over the course of several days.

Imaging immunometabolism in situ in live animals

“…Additionally, there are zebrafish mutants available that stay transparent into adulthood [149] . Danionella cerebrum is another emerging fish model that naturally stays transparent throughout life [150] . For this reason, biosensors for single-cell imaging are more likely to be successfully implemented in these organisms.…”

Adult zebrafish as advanced models of human disease

“…To address this issue, Brant M. Weinstein's laboratory has improved protocols for extended imaging of adult zebrafish by using intubation that permits over 20 h of live imaging to capture neutrophils responding to a wound (Castranova et al, 2022). By combining the benefits of both transparency and small organism size, with the complexity of adult brain biology, Pui-Ying Lam has developed methodology for prolonged, longitudinal imaging of the small and life-long optically transparent Danio species Danionella cerebrum (Lam, 2022). Access to live imaging of the adult zebrafish brain often requires surgery but Danionella cerebrum lacks dorsal skull bones, enabling direct access for confocal imaging.…”

Development of sound production inDanionella cerebrum

“…DC has an adult body length of only ∼12 mm and the smallest known vertebrate brain (Britz et al, 2021; Penalva et al, 2018; Schulze et al, 2018). Its small size, together with its life-long transparency has made DC an emerging model system for neuroscience, suitable for whole-brain optical measures of neuronal activity (Schulze et al, 2018; Kadobianskyi et al, 2019; Hoffmann et al, 2023; Lam, 2022; Zada et al, 2023; Lee & Briggman, 2023). Adult DC have a male-specific trait of acoustic signaling, producing click-like sounds by drumming a piece of cartilage against the anterior swim bladder (Schulze et al, 2018; Cook et al, 2024; Vasconcelos et al, 2024).…”