Yunlu Zhu

Most fish are inherently unstable and must swim to stabilize posture. How diurnal fish reduce activity at night while maintaining postural control remains unclear. We defined distinct locomotor strategies that larval zebrafish (Danio rerio) use to control posture and navigate the water column in response to light and circadian cues. In the dark, larvae maintain balance by swimming in long bouts with large nose-up rotations, compensating for nose-down drift accrued during prolonged inactivity. Effective postural compensation requires vestibular sensation from the utricle. By contrast, in the light, larvae navigate with short, frequent and variable bouts. While lighting exerts a dominant, masking effect on the locomotor strategies, circadian rhythms modulate swim kinematics. Our results reveal distinct light–dark locomotor strategies and disentangle how ambient light and the internal clock jointly shape balance control and navigation. This work lays the foundation for understanding how external and internal cues interact to govern locomotor activity in freely moving diurnal animals.

Most fish are inherently unstable and must swim to stabilize posture. How diurnal fish reduce activity at night while maintaining postural control remains unclear. We defined distinct locomotor strategies that larval zebrafish ( Danio rerio ) use to control posture and navigate the water column in response to light and circadian cues. In the dark, larvae maintain balance by swimming in long bouts with large nose-up rotations, compensating for nose-down drift accrued during prolonged inactivity. Effective postural compensation requires vestibular sensation from the utricle. By contrast, in the light, larvae navigate with short, frequent, and variable bouts. While lighting exerts a dominant, masking effect on the locomotor strategies, circadian rhythms modulate swim kinematics. Our results reveal distinct light-dark locomotor strategies and disentangle how ambient light and the internal clock jointly shape balance control and navigation. This work lays the foundation for understanding how external and internal cues interact to govern locomotor activity in freely moving diurnal animals.

Fish actively control posture in the pitch axis (nose-up/nose-down) to counter instability and regulate their elevation in the water column. To test the hypothesis that environmental cues shape strategies fish use to control posture, we leveraged a serendipitous finding: larval zebrafish (Danio rerio) lose swim bladder volume and sink mildly after acute loss of lateral line hair cells. Using long-term (48 h) recordings of unrestrained swimming, we discovered that sinking larvae compensated differently depending on light conditions. In the dark, they swim more frequently with an increased nose-up posture. In contrast, larvae in the light do not swim more frequently, but do climb more often. Finally, after lateral line regeneration, larvae returned to normal buoyancy and swam comparably to control siblings. We conclude that larvae can switch postural control strategies depending on the availability of visual information. Our findings complement and extend morphological and kinematic analyses of locomotion. More broadly, by quantifying the variation in strategies our work speaks to the evolutionary substrate for different balance behaviors.

Patients with tauopathies present with profoundly different clinical symptoms ¹ , even within the same disorder ² . A central hypothesis in the field, well-supported by biomarker studies ^3,4 and post-mortem pathology ^5–7 , is that clinical heterogeneity reflects differential degeneration of vulnerable neuronal populations responsible for specific neurological functions. Recent work has revealed mechanisms underlying susceptibility of particular cell types ^8–10 , but relating tau load to disrupted behavior — es- pecially before cell death — requires a targeted circuit-level approach. Here we studied two distinct balance behaviors in larval zebrafish ¹¹ expressing a human 0N/4R-tau allele ¹² in select populations of evolutionarily-conserved and well-characterized brainstem vestibular circuits ^13,14 . We observed that human tau load predicted the severity of circuit-specific deficits in posture and navigation in the ab- sence of cell death. Targeting expression to either mid- or hindbrain balance neurons recapitulated these particular deficits in posture and navigation. By parametrically linking tau load in specific neu- rons to early behavioral deficits, our work moves beyond cell type to close the gap between pathological and neurological conceptions of tauopathy.

The sensation of gravity anchors our perception of the environment and is crucial for navigation. However, the neural circuits that transform gravity into commands for navigation are undefined. We first determined that larval zebrafish ( Danio rerio ) navigate vertically by maintaining a consistent heading across a series of upward climb or downward dive bouts. Gravity-blind mutant fish swim with more variable heading and excessive veering, leading to inefficient vertical navigation. After targeted photoablation of ascending vestibular neurons and spinal projecting midbrain neurons, but not vestibulospinal neurons, vertical navigation was impaired. These data define a sensorimotor circuit that uses evolutionarily-conserved brainstem architecture to transform gravitational signals into persistent heading for vertical navigation. The work lays a foundation to understand how vestibular inputs allow animals to move efficiently through their environment.

Fish actively control posture in the pitch axis (nose-up/nose-down) to counter instability and regulate their elevation in the water column. To test the hypothesis that environmental cues shape strategies fish use to control posture, we leveraged a serendipitous finding: larval zebrafish ( Danio rerio ) lose swim bladder volume and sink mildly after acute loss of lateral line hair cells. Using long-term (48 h) recordings of unrestrained swimming, we discovered that sinking larvae compensated differently depending on light conditions. In the dark, they swim more frequently with an increased nose-up posture. In contrast, larvae in the light do not swim more frequently, but do climb more often. Finally, after lateral line regeneration, larvae returned to normal buoyancy and swam comparably to control siblings. We conclude that larvae can switch postural control strategies depending on the availability of visual information. Our findings complement and extend morphological and kinematic analyses of locomotion. More broadly, by quantifying the variation in strategies our work speaks to the evolutionary substrate for different balance behaviors.

Measuring ongoing cellular activity is essential to understanding the dynamic functions of biological organisms. The most popular current approach is imaging fluorescence-based genetically encoded Ca ²⁺ indicators (GECIs). While fluorescent probes are useful in many contexts, bioluminescence-based GECIs—probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein—have several distinct advantages. Because bioluminescent (BL) GECIs do not use the bright extrinsic excitation light required for fluorescence, BL GECIs do not photobleach, do not suffer from nonspecific autofluorescent background, and do not cause phototoxicity. Further, BL GECIs can be applied in contexts where directly shining photons on an imaging target is not possible. Despite these advantages, the use of BL GECIs has to date been limited by their small changes in bioluminescence intensity, high baseline signal at resting Ca ²⁺ concentrations, and suboptimal Ca ²⁺ affinities. Here, we describe a new BL GECI, CaBLAM ( Ca ²⁺ B io L uminescence A ctivity M onitor), that displays much higher dynamic range than previous BL GECIs and has a Ca ²⁺ affinity suitable for capturing physiological changes in cytosolic Ca ²⁺ concentration. With these improvements, CaBLAM captures single-cell and subcellular resolution activity at high frame rates in cultured neurons and in vivo , and allows multi-hour recordings in mice and behaving zebrafish. This new advance provides a robust alternative to traditional fluorescent GECIs that can enable or enhance imaging across many experimental conditions.

Balance and movement are impaired in a wide variety of neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and loco-motor kinematics, but without the throughput and scalability necessary to screen candidate genes / potential therapeutics. We present a powerful solution: a Scalable Apparatus to Measure Posture and Locomotion (SAMPL). SAMPL includes extensible imaging hardware and low-cost open-source acquisition software with real-time processing. We first demonstrate that SAMPL’s hardware and acquisition software can acquire data from D. melanogaster , C.elegans , and D. rerio as they move vertically. Next, we leverage SAMPL’s throughput to rapidly (two weeks) gather a new zebrafish dataset. We use SAMPL’s analysis and visualization tools to replicate and extend our current understanding of how zebrafish balance as they navigate through a vertical environment. Next, we discover (1) that key kinematic parameters vary systematically with genetic background, and (2) that such background variation is small relative to the changes that accompany early development. Finally, we simulate SAMPL’s ability to resolve differences in posture or vertical navigation as a function of effect size and data gathered – key data for screens. Taken together, our apparatus, data, and analysis provide a powerful solution for laboratories using small animals to investigate balance and locomotor disorders at scale. More broadly, SAMPL is both an adaptable resource for laboratories looking process video-graphic measures of behavior in real-time, and an exemplar of how to scale hardware to enable the throughput necessary for screening.

Locomotion requires precise control of the strength and speed of muscle contraction and is achieved by recruiting functionally-distinct subtypes of motor neurons (MNs). MNs are essential to movement and differentially susceptible in disease, but little is known about how MNs acquire functional subtype-specific features during development. Using single-cell RNA profiling in embryonic and larval zebrafish, we identify novel and conserved molecular signatures for MN functional subtypes, and identify genes expressed in both early post-mitotic and mature MNs. Assessing MN development in genetic mutants, we define a molecular program essential for MN functional subtype specification. Two evolutionarily-conserved transcription factors, Prdm16 and Mecom, are both functional subtype-specific determinants integral for fast MN development. Loss of prdm16 or mecom causes fast MNs to develop transcriptional profiles and innervation similar to slow MNs. These results reveal the molecular diversity of vertebrate axial MNs and demonstrate that functional subtypes are specified through intrinsic transcriptional codes.

Animals use information about gravity and other destabilizing forces to balance and navigate through their environment. Measuring how brains respond to these forces requires considerable technical knowledge and/or financial resources. We present a simple alternative: Tilt In Place Microscopy (TIPM). TIPM is a low-cost and non-invasive way to measure neural activity following rapid changes in body orientation. Here we used TIPM to study vestibulospinal neurons in larval zebrafish during and immediately after roll tilts. Vestibulospinal neurons responded with reliable increases in activity that varied as a function of ipsilateral tilt amplitude. TIPM differentiated tonic (i.e. sustained tilt) from phasic responses, revealing coarse topography of stimulus sensitivity in the lateral vestibular nucleus. Neuronal variability across repeated sessions was minor relative to trial-to-trial variability, allowing us to use TIPM for longitudinal studies of the same neurons across two developmental timepoints. There, we observed global increases in response strength, and systematic changes in the neural representation of stimulus direction. Our data extend classical characterization of the body tilt representation by vestibulospinal neurons and establish TIPM’s utility to study the neural basis of balance, especially in developing animals. Significance Statement: Vestibular sensation influences everything from navigation to interoception. Here we detail a straight-forward, validated and nearly-universal approach to image how the nervous system senses and responds to body tilts. We use our new method to replicate and expand upon past findings of tilt sensing by a conserved population of spinal-projecting vestibular neurons. The simplicity and broad compatibility of our approach will democratize the study of the brain’s response to destabilization, particularly across development.