Tilt in Place Microscopy: a Simple, Low-Cost Solution to Image Neural Responses to Body Rotations

Abstract

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), a low-cost and noninvasive 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 time points. 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 the utility of TIPM to study the neural basis of balance, especially in developing animals.SIGNIFICANCE STATEMENTVestibular sensation influences everything from navigation to interoception. Here, we detail a straightforward, 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 on 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 response of the brain to destabilization, particularly across development.

Neuronal birthdate reveals topography in a vestibular brainstem circuit for gaze stabilization

“…Instead, retrograde tracing studies across chick, zebrafish, frog, and mouse had collectively identified coarse axonal projections – e.g., to spinal or ocular motor circuits – as their primary organizational axis 18–20,62 . Here, we used a modern toolkit consisting of: (1) a transgenic line of zebrafish that specifically labels vestibulo-ocular reflex projection neurons 28 , (2) in vivo birthdating 56 early in development, and (3) spatial mapping of tilt responses 54 . These tools allowed us to extend the “axonal projection” model of vestibular brainstem organization to functional channels within an individual vestibular nucleus.…”

“…All experiments were performed on 5 dpf larvae (Transgenic lines). Stimulation paradigms were presented as previously reported in 54 and are described again below. Larvae were paralyzed using bath application of 0.8 mg/mL pancuronium bromide (Sigma-Aldrich P1918, St. Louis, MO) for 8-10 minutes.…”

“…Projection neurons responsible for the vertical vestibulo-ocular reflex are directionally selective and respond primarily to either nose-up or nose-down head/body tilts (Figure 2A) [64][65][66] . To categorize the directional selectivity of individual projection neurons, we used Tilt In Place Microscopy (TIPM) with a two-photon microscope 54 (Figure 2B). Briefly, we used a galvanometer to rotate to, and hold larvae at an eccentric angle (either 19°nose-up or nose-down).…”

“…We then rapidly (~4msec) rotated the galvanometer back to horizontal and measured the change in fluorescence of a genetically-encoded calcium indicator (UAS:GCaMP6s). As the duration of the step was orders of magnitude faster than the time constant of GCaMP6s 67 , the fluorescence on return to horizontal predominantly reflects neuronal activity at the previous eccentric position, and can reliably classify directional sensitivity in comparable projection neurons 54 . We categorized the subtype identity (nose-up/nose-down) of 471 projection neurons from 22 larvae according to the direction(s) that evoked excitation.…”

“…Zebrafish are vertebrates that develop externally, are optically transparent [46][47][48][49] , and perform a vertical vestibulo-ocular reflex by 5 days postfertilization 27,28,50 . Larvae possess both the semicircular canal and otolithic vestibular end-organs 27,51,52 , stimulation of which elicits reliable responses from central projection neurons 48,53,54 . Prior work established that vertical (eyes-up and eyes-down) motor neurons are spatially and developmentally organized 8 and suggested that similar principles might organize the vestibular periphery 53,55 .…”