Elias, L. J. | bioRxiv Science

bioRxiv2025

JACUZI-SD: An automated, high-throughput, minimally stressful approach to sleep depriving larval zebrafish.

While sleep deprivation broadly disrupts health and well-being, the neural and molecular mechanisms that signal increased sleep pressure remain poorly understood. A key obstacle to progress is the fact that traditional methods for inducing sleep deprivation (SD) in animal models often introduce confounding stress or disrupt circadian rhythms. Here, we present JACUZI-SD (Jetting Automated Currents Under Zebrafish to Induce Sleep Deprivation), a fully automated, high-throughput system designed to induce sleep deprivation in larval zebrafish with minimal stress. By delivering randomized water pulses via a custom milli-fluidic device integrated with a 96-well plate and controlled by an Arduino, JACUZI-SD promotes wakefulness during the natural dark cycle without the stress associated with existing SD methods. Our results demonstrate that JACUZI-SD reduces total sleep time by 41-64% and elicits a robust rebound sleep characterized by increased sleep bout length following deprivation. Importantly, this method avoids activating the hypothalamic-pituitary-interrenal (HPI) stress axis, as evidenced by reduced stress marker expression compared to other deprivation methods. Additionally, the system reliably activates established sleep pressure pathways, including the upregulation of galanin in the neurosecretory preoptic area, while also revealing biologically relevant inter-individual variability in homeostatic rebound responses. JACUZI-SD provides a powerful, minimally invasive platform for dissecting the neural and molecular underpinnings of sleep homeostasis in vertebrates.

neuroscience↗

bioRxiv2022

Over-expression of thyroid hormone receptor β2 in zebrafish changes the distribution of cone spectral signals

Zebrafish retinal cone signals shift in spectral shape through larval, juvenile, and adult development as expression patterns of eight cone-opsin genes change. An algorithm extracting signal amplitudes for the component cone spectral types is developed and tested on two thyroxin receptor {beta}2 (tr{beta}2) gain-of-function lines crx:mYFP-2A-tr{beta}2 and gnat2:mYFP-2A-tr{beta}2, allowing correlation between opsin signaling and opsin immunoreactivity in lines with different developmental timing and cell-type expression of this red-opsin-promoting transgene. Both adult transgenics became complete, or nearly complete, red-cone dichromats, with disproportionately large LWS1 opsin amplitudes as compared to controls, where LWS1 and LWS2 amplitudes were about equal, and significant signals from SWS1, SWS2, and Rh2 opsins were detected. But in transgenic larvae and juveniles of both lines it was LWS2 amplitudes that increased, with LWS1 cone signals rarely encountered. In gnat2:mYFP-2A-tr{beta}2 embryos at 5 days post fertilization (dpf), red-opsin immunoreactive cone density doubled, but red-opsin amplitudes (LWS2) increased < 10%, and green, blue and UV opsin signals were unchanged, despite co-expressed red opsins, and the finding that an sws1 UV-opsin reporter gene was shut down by the gnat2:mYFP-2A-tr{beta}2 transgene. By contrast both LWS2 red-cone amplitudes and the density of red-cone immunoreactivity more than doubled in 5 dpf crx:mYFP-2A-tr{beta}2 embryos, while UV-cone amplitudes were reduced 90%. Embryonic cones with tr{beta}2 gain-of-function transgenes were morphologically distinct from control red, blue or UV cones, with wider inner segments and shorter axons than red cones, suggesting cone spectral specification, opsin immunoreactivity and shape are influenced by the abundance and developmental timing of tr{beta}2 expression. Significance StatementAs different combinations of eight cone-opsin mRNAs are successively expressed during zebrafish development and maturation, the composite cone-ERG spectral signal shifts. Amplitudes of each of the eight resulting cone signals are inferred computationally from the composite signal, both in controls and in two thyroxin-receptor {beta}2 (tr{beta}2) gain-of-function transgenics, crx:mYFP-2A-tr{beta}2 and gnat2:mYFP-2A-tr{beta}2, tr{beta}2 being a transcription factor required for expression of the red-cone opsins LWS1 and LWS2. Adult transgenics become red cone dichromats with excess LWS1 amplitudes, but larvae and juveniles evoke excess LWS2 amplitudes. Controls retain 5 to 6 cone signals of changing composition throughout development. The progression of transgene-induced amplitude alterations is slower in gnat2:mYFP-2A-tr{beta}2, with supernormal red-opsin antigenicity not immediately correlating with red-cone signaling.

neuroscience↗