Tiltable objective microscope visualizes discrimination of static and dynamic head movement originates at hair cells

Abstract

Spatio-temporal information on head orientation and movement is fundamental to the sense of balance and motion. Hair cells (HCs) in otolith organs of the vestibular system transduce linear acceleration, including head tilt and vibration. Here, we build a tiltable objective microscope in which an objective lens and specimen tilt together. In vivo Ca2+ imaging of all utricular HCs and ganglion neurons during 360° static tilt and vibration in pitch and roll axes visualizes the direction- and modality-selective topographic responses in larval zebrafish. We find that head vibration is preferentially received by HCs in a specific region whereas static tilt is preferentially transduced by HCs in the outside region. Spatially ordered direction preference in HCs is consistent with hair-bundle polarity, and is preserved in ganglion neurons through topographic innervation. Together, these results demonstrate that the discrimination of different direction and temporal modalities of head orientation and movement originates at HCs.

Biomechanics and neural circuits for vestibular-induced fine postural control in larval zebrafish

“…This suggests that these neurons were slightly inhibited during ipsi-down tilt. To quantify what percentage of neurons were active, we set the threshold to 0.1 for a ΔR/R 0 increase, and neurons with a maximum ΔR/R 0 that was above 0.1 were considered to be active based on the artificial fluorescent intensity changes during the tilt 30 . Based on this criterion, 41% of neurons (136/333) were active only during the ipsi-up tilt, 14% (46/333) were active during both the ipsi-up and -down tilts, and a small proportion of neurons (12/333, 4%) was active only during the ipsi-down tilt (Supplementary Fig.…”

“…We aimed to perform Ca 2+ imaging in TAN neurons during roll tilts, using the imaging system in which an objective lens and a fish were tilted together using a motorized rotation stage 30 (Fig. 3b; Supplementary Fig.…”

“…In this imaging system, two-color ratiometric imaging is used to reduce the motion-related artificial fluorescence intensity change during the tilt. Because this study is the first to use a wide-field version of the tiltable objective microscope (note that a confocal microscopy is used in the original version 30 ), we evaluated the level of artifacts upon roll tilts using green/red Dendra2 (the green Dendra2 was partially photoconverted to red Dendra2 by ultraviolet light such that TAN neurons contained both green and red Dendra2; Supplementary Fig. 2e).…”

“…nMLF neurons are active during ipsilateral-up (contralateral-down) roll tilt nMLF neurons, a cluster of RS neurons that project axons into the spinal cord along the medial longitudinal fasciculus 31 , were the next target of our investigation. We performed Ca 2+ imaging in nMLF neurons at single-cell resolution using a tiltable objective microscope with a spinning disk confocal unit 30 . For this purpose, nMLF neurons were retrogradely labeled with a green chemical fluorescent Ca 2+ indicator, Cal-520 dextran, and a red chemical fluorescent dye, rhodamine dextran.…”

“…In the tiltable-objective microscopes, the images rotated during the tilt. For registration purposes, green and red channel images were counter-rotated 30 . Then, the images were translated in the xy direction using Template Matching and Slice Alignment Plugin.…”