Itia A. Favre-Bulle

Scattering of Sculpted Light in Intact Brain Tissue, with implications for Optogenetics

Optogenetics uses light to control and observe the activity of neurons, often using a focused laser beam. As brain tissue is a scattering medium, beams are distorted and spread with propagation through neural tissue, and the beam’s degradation has important implications in optogenetic experiments. To address this, we present an analysis of scattering and loss of intensity of focused laser beams at different depths within the brains of zebrafish larvae. Our experimental set-up uses a 488 nm laser and a spatial light modulator to focus a diffraction-limited spot of light within the brain. We use a combination of experimental measurements of back-scattered light in live larvae and computational modelling of the scattering to determine the spatial distribution of light. Modelling is performed using the Monte Carlo method, supported by generalised Lorenz–Mie theory in the single-scattering approximation. Scattering in areas rich in cell bodies is compared to that of regions of neuropil to identify the distinct and dramatic contributions that cell nuclei make to scattering. We demonstrate the feasibility of illuminating individual neurons, even in nucleus-rich areas, at depths beyond 100 μm using a spatial light modulator in combination with a standard laser and microscope optics.

Optical trapping of otoliths drives vestibular behaviours in larval zebrafish

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.The neural circuits of the vestibular system, which detects gravity and motion, remain incompletely characterised. Here the authors use an optical trap to manipulate otoliths (ear stones) in zebrafish larvae, and elicit corrective tail movements and eye rolling, thus establishing a method for mapping vestibular processing.

Hypothalamic Projections to the Optic Tectum in Larval Zebrafish

The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH) to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC)/stratum griseum periventriculare (SPV), and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil.

Ultrasensitive rotating photonic probes for complex biological systems

We use rotational photonic tweezers to access local viscoelastic properties of complex fluids over a wide frequency range. This is done by monitoring both passive rotational Brownian motion and also actively driven transient rotation between two angular trapping states of a birefringent microsphere. These enable measurement of high- and low-frequency properties, respectively. Complex fluids arise frequently in microscopic biological systems, typically with length scales at the cellular level. Thus, high spatial resolution as provided by rotational photonic tweezers is important. We measure the properties of tear film on a contact lens and demonstrate variations in these properties between two subjects over time. We also show excellent agreement between our theoretical model and experimental results. We believe that this is the first time that active microrheology using rotating tweezers has been used for biologically relevant questions. Our method demonstrates potential for future applications to determine the spatial-temporal properties of biologically relevant and complex fluids that are only available in very small volumes.

Luminance Changes Drive Directional Startle through a Thalamic Pathway

Looming visual stimuli result in escape responses that are conserved from insects to humans. Despite their importance for survival, the circuits mediating visual startle have only recently been explored in vertebrates. Here we show that the zebrafish thalamus is a luminance detector critical to visual escape. Thalamic projection neurons deliver dim-specific information to the optic tectum, and ablations of these projections disrupt normal tectal responses to looms. Without this information, larvae are less likely to escape from dark looming stimuli and lose the ability to escape away from the source of the loom. Remarkably, when paired with an isoluminant loom stimulus to the opposite eye, dimming is sufficient to increase startle probability and to reverse the direction of the escape so that it is toward the loom. We suggest that bilateral comparisons of luminance, relayed from the thalamus to the tectum, facilitate escape responses and are essential for their directionality.

Cellular resolution imaging of vestibular processing across the larval zebrafish brain

The vestibular system, which reports on motion and gravity, is essential to postural control, balance, and egocentric representations of movement and space. The motion needed to stimulate the vestibular system complicates studying its circuitry, so we previously developed a method for fictive vestibular stimulation in zebrafish, using optical trapping to apply physical forces to the otoliths. Here, we combine this fictive stimulation with whole-brain calcium imaging at cellular resolution, delivering a comprehensive map of the brain regions and cellular responses involved in basic vestibular processing. We find these responses to be broadly distributed across the brain, with unique profiles of cellular responses and topography in each brain region. The most widespread and abundant responses involve excitation that is rate coded to the stimulus strength. Other responses, localized to the telencephalon and habenulae, show excitation that is only weakly rate coded and that is sensitive to weak stimuli. Finally, numerous brain regions contain neurons that are inhibited by vestibular stimuli, and these inhibited neurons are often tightly localised spatially within their regions. By exerting separate control over the left and right otoliths, we explore the laterality of brain-wide vestibular processing, distinguishing between neurons with unilateral and bilateral vestibular sensitivity, and revealing patterns by which conflicting vestibular signals from the two ears can be mutually cancelling. Our results show a broader and more extensive network of vestibular responsive neurons than has previously been described in larval zebrafish, and provides a framework for more targeted studies of the underlying functional circuits.

Investigation of Optical Properties of Otoliths with Optical Trapping

Investigating the functioning of biological system often ask to, not only image the different processes and elements involved, but also manipulate them to find information that could not be found visually, such as forces of motion, strength of bondings, elasticity, viscosity to cite a few.

Scattering in Zebrafish Brain for Optogenetics

In the previous chapter, I focussed on the theory and modelling of light scattering in brain tissue. In this chapter, I present the measurements of backscattered light in-vivo in the zebrafish brain and compare those with my Monte Carlo method and model.

Optical Systems to Decode Brain Activity

Different methods have been developed over the past decades for studies of brain function. These methods aim to decode brain activity in terms of the communication that takes place among neurons, and to identify the patterns of neural activity that ultimately produce behaviour.

Optical Manipulation of Otoliths In-Vivo

Despite the light scattering that occurs in biological tissues, in-vivo optical trapping is possible, and has been demonstrated for targets such as red blood cells [1] and nanoparticles [2]. Those studies show that OT can trap and manipulate small objects in free flowing channels in relatively shallow tissue (50 \(\upmu \)m) without any correction to the incoming beam.