Since the time of their introduction, optical tweezers (OTs) have grown to be a powerful tool in the hands of biologists. OTs use highly focused laser light to guide, manipulate, or sort target objects, typically in the nanoscale to microscale range. OTs have been particularly useful in making quantitative measurements of forces acting in cellular systems; they can reach inside living cells and be used to study the mechanical properties of the fluids and structures that they contain. As all the measurements are conducted without physically contacting the system under study, they also avoid complications related to contamination and tissue damage. From the manipulation of fluorescent nanodiamonds to chromosomes, cells, and free‐swimming bacteria, OTs have now been extended to challenging biological systems such as the vestibular system in zebrafish. Here, we will give an overview of OTs, the complications that arise in carrying out OTs in vivo , and specific OT methods that have been used to address a range of otherwise inaccessible biological questions.

The vestibular system, which detects gravity and motion, is crucial to survival, but the neural circuits processing vestibular information remain incompletely characterised. In part, this is because the movement needed to stimulate the vestibular system hampers traditional neuroscientific methods. Optical trapping uses focussed light to apply forces to targeted objects, typically ranging from nanometres to a few microns across. In principle, optical trapping of the otoliths (ear stones) could produce fictive vestibular stimuli in a stationary animal. Here we use optical trapping in vivo to manipulate 55-micron otoliths in larval zebrafish. Medial and lateral forces on the otoliths result in complementary corrective tail movements, and lateral forces on either otolith are sufficient to cause a rolling correction in both eyes. This confirms that optical trapping is sufficiently powerful and precise to move large objects in vivo, and sets the stage for the functional mapping of the resulting vestibular processing.

Habituation is a form of learning during which animals stop responding to repetitive stimuli, and deficits in habituation are characteristic of several psychiatric disorders. Due to technical challenges, the brain-wide networks mediating habituation are poorly understood. Here we report brain-wide calcium imaging during larval zebrafish habituation to repeated visual looming stimuli. We show that different functional categories of loom-sensitive neurons are located in characteristic locations throughout the brain, and that both the functional properties of their networks and the resulting behavior can be modulated by stimulus saliency and timing. Using graph theory, we identify a visual circuit that habituates minimally, a moderately habituating midbrain population proposed to mediate the sensorimotor transformation, and downstream circuit elements responsible for higher order representations and the delivery of behavior. Zebrafish larvae carrying a mutation in the fmr1 gene have a systematic shift toward sustained premotor activity in this network, and show slower behavioral habituation.

Information about water flow, detected by lateral line organs, is critical to the behavior and survival of fish and amphibians. While certain aspects of water flow processing have been revealed through electrophysiology, we lack a comprehensive description of the neurons that respond to water flow and the network that they form. Here, we use brain-wide calcium imaging in combination with microfluidic stimulation to map out, at cellular resolution, neuronal responses involved in perceiving and processing water flow information in larval zebrafish. We find a diverse array of neurons responding to head-to-tail (h-t) flow, tail-to-head (t-h) flow, or both. Early in this pathway, in the lateral line ganglia, neurons respond almost exclusively to the simple presence of h-t or t-h flow, but later processing includes neurons responding specifically to flow onset, representing the accumulated displacement of flow during a stimulus, or encoding the speed of the flow. The neurons reporting on these more nuanced details are located across numerous brain regions, including some not previously implicated in water flow processing. A graph theory-based analysis of the brain-wide water flow network shows that a majority of this processing is dedicated to h-t flow detection, and this is reinforced by our finding that details like flow velocity and the total accumulated flow are only encoded for the h-t direction. The results represent the first brain-wide description of processing for this important modality, and provide a departure point for more detailed studies of the flow of information through this network.SIGNIFICANCE STATEMENTIn aquatic animals, the lateral line is important for detecting water flow stimuli, but the brain networks that interpret this information remain mysterious. Here, we have imaged the activity of individual neurons across the entire brains of larval zebrafish, revealing all response types and their brain locations as water flow processing occurs. We find neurons that respond to the simple presence of water flow, and others attuned to the direction, speed, and duration of flow, or the accumulated displacement of water that has passed during the stimulus. With this information, we modeled the underlying network, describing a system that is nuanced in its processing of water flow simulating head-to-tail motion but rudimentary in processing flow in the tail-to-head direction.