Tilt In Place Microscopy (TIPM): 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). 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.

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

“…We then rapidly (∼4msec) rotated the galvanometer back to horizontal, and measured the change in fluorescence of a genetically-encoded calcium indicator (GCaMP6s) (Figure 2B). As the duration of the step was orders of magnitude faster than the time constant of GCaMP6s [66], the fluorescence on return to baseline predominantly reflects neuronal activity at the previous eccentric position, and can reliably classify directional sensitivity in comparable projection neurons [54].…”

“…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.…”

“…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][19][20]61]. Here, we used a modern toolkit consisting of: (1) a transgenic line of zebrafish that specifically labels vestibulo-ocular reflex projection neurons [50], (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.…”

“…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).…”

“…Zebrafish are vertebrates that develop externally, are optically transparent [46–49], and perform a vertical vestibulo-ocular reflex by 5 days post-fertilization [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].…”