The miniscule calcium carbonate crystals in your ear canals have a big job: They shift as your body changes position, letting your brain know your body’s location in, and linear acceleration through, space. Key to survival, these crystals (called otoliths or “ear stones”) are part of the body’s vestibular system, which has eluded understanding in large part because the movement needed to stimulate it has been a barrier. “It’s difficult to study activity in a moving brain,” says Ethan Scott, associate professor in the School of Biomedical Sciences at Australia’s University of Queensland.
But in a major innovation to overcome this barrier, Scott and his colleagues targeted the tiny crystals with optical trapping.1,2 Observing the effects with fluorescence microscopy, they not only clarified how the brain detects gravity and movement, they also won the 2018 Australian Museum Eureka Prize for Excellence in Interdisciplinary Scientific Research (see figure).
They used infrared (IR) laser light to move otoliths in immobile zebrafish, thereby generating a sense of movement in the brain and eliciting behaviors observed using their customized imaging system. “By tricking an animal into thinking it is moving while the brain remains stationary, we can now use advanced microscopy to study the cells and circuits across the brain responsible for motion processing, for the first time,” Scott says.
During optical trapping (OT) of ear stones in the saccule (sac) and utricle (ut)—two organs of the inner ear involved in sensing gravity and movement—in a zebrafish larva, researchers performed volumetric selective planar illumination microscopy (SPIM) of an area 300 μm deep into the brain. With the help of 2D galvo mirrors and an electrically tunable lens coordinating control, the imaging technique scans two 488 nm sheets of light through the z-axis, one from the front of the zebrafish and the other from the side (a); associate professor Ethan Scott, Dr. Itia A Favre-Bulle, and professor Halina Rubinsztein-Dunlop are part of the Optical Physics in Neuroscience team at the University of Queensland that conducted the work, which won the 2018 Australian Museum Eureka Prize for Excellence in Interdisciplinary Scientific Research.
The team’s microscopy approach was enabled by protein engineering and fluorescence imaging advances—namely genetically encoded calcium indicators (GECIs) and selective planar illumination microscopy (SPIM). Their system provided two scanning light sheets and a fluorescence emission channel, along with a camera to image responses in the animal. To achieve optical trapping, a pair of 1064 nm beams, steered individually using gimbal-mounted mirrors, applied medial and lateral forces to two 55 µm otoliths in each live fish. The researchers used galvanometric mirrors to generate scanning light sheets and detected fluorescence emissions using the same objective they used to deliver the trapping beams.
Adjusting the imaging focal plane by way of an electrically tunable lens that, when synchronized with the galvo mirrors, allowed them to perform volumetric imaging without moving either the zebrafish or the imaging objective. Superior stability enabled them to consistently apply the trapping forces, which require sub-micron precision. Knowing that evidence of movement perception would show up in the fishes’ tails and eyes, the researchers imaged the tails using a low-power objective beneath the specimens, and the eyes using a fluorescence imaging camera. The results were consistent with previous work showing that optical trapping in this setup drives compensatory and proportional movements in both the tail and eyes.
A cellular-resolution whole-brain map
The researchers next performed brain-wide calcium imaging during the optical trapping to identify which brain regions participate in vestibular perception and processing. Their results described the responses of individual neurons to various stimulus strengths and identified two functional neuron classes that proved to be excitable by—and one that is inhibited by—vestibular stimuli. They were able to identify 10 distinct brain regions that produced consistent vestibular responses. For each one, they generated a functional profile showing registered anatomical locations and their response types, as well as laterality of the vestibular neurons within them. They also reported the cellular responses elicited by the induced stimuli oriented in various directions.
The team’s work apparently is the first to perform calcium imaging on vestibular processing, and the first to map this processing brain-wide and at cellular resolution. However, the research makes clear that there is much more to learn, and thus opens the door to further investigation. “Across all of these observations,” they wrote, “the breadth and richness of the responses suggests a more extensive system for vestibular processing across larval zebrafish brain than has previously been appreciated.”
1. I. A. Favre-Bulle, G. Vanwalleghem, M. A. Taylor, H. Rubinsztein-Dunlop, and E. K. Scott., Biorxiv. (2018); https://doi.org/10.1101/302752.
2. I. A. Favre-Bulle, A. B. Stingoe, H. Rubinsztein-Dunlop, and E. K. Scott., Nat. Commun., 8, 630 (2017).
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