Deep three-photon imaging of the brain in intact adult zebrafish.

Behaviors emerge from activity throughout the brain, but noninvasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become noninvasively accessible from embryo to sexually mature adult in a vertebrate model.

In Vivo Measurement of Glycine Receptor Turnover and Synaptic Size Reveals Differences between Functional Classes of Motoneurons in Zebrafish.

The interplay between binding and unbinding of synaptic receptor proteins at synapses plays an important role in determining receptor concentration and synaptic strength, with known links between changes in binding kinetics and synaptic plasticity. The regulation of such kinetics may subserve the specific functional requirements of neurons in intact circuits. However, the majority of studies of synaptic turnover kinetics have been performed in cultured neurons outside the context of normal circuits, and synaptic receptor turnover has not been measured at individual synaptic sites in vivo. We quantified the distribution of glycinergic receptor dynamics using fluorescence recovery after photoconversion of synapses in intact zebrafish and correlated recovery kinetics to synaptic volume in two functionally distinct classes of cells: primary and secondary motoneurons. The rate of fluorescence recovery after photoconversion decreased with synaptic volume in both types of motoneurons, with larger synapses having slower recovery. Primary motoneurons had both larger synapses and associated slower recovery times than secondary motoneurons. Our results suggest that synaptic kinetics are regulated in concert with synaptic sizes and reflect the functional role played by neurons within their circuit.

An olfactory circuit increases the fidelity of visual behavior.

Multimodal integration allows neural circuits to be activated in a behaviorally context-specific manner. In the case of odor plume tracking by Drosophila, an attractive odorant increases the influence of yaw-optic flow on steering behavior in flight, which enhances visual stability reflexes, resulting in straighter flight trajectories within an odor plume. However, it is not well understood whether context-specific changes in optomotor behavior are the result of an increased sensitivity to motion inputs (e.g., through increased visual attention) or direct scaling of motor outputs (i.e., increased steering gain). We address this question by examining the optomotor behavior of Drosophila melanogaster in a tethered flight assay and demonstrate that whereas olfactory cues decrease the gain of the optomotor response to sideslip optic flow, they concomitantly increase the gain of the yaw optomotor response by enhancing the animal's ability to follow transient visual perturbations. Furthermore, ablating the mushroom bodies (MBs) of the fly brain via larval hydroxyurea (HU) treatment results in a loss of olfaction-dependent increase in yaw optomotor fidelity. By expressing either tetanus toxin light chain or diphtheria toxin in gal4-defined neural circuits, we were able to replicate the loss of function observed in the HU treatment within the lines expressing broadly in the mushroom bodies, but not within specific mushroom body lobes. Finally, we were able to genetically separate the yaw responses and sideslip responses in our behavioral assay. Together, our results implicate the MBs in a fast-acting, memory-independent olfactory modification of a visual reflex that is critical for flight control.

Mutation of the Drosophila vesicular GABA transporter disrupts visual figure detection.

The role of gamma amino butyric acid (GABA) release and inhibitory neurotransmission in regulating most behaviors remains unclear. The vesicular GABA transporter (VGAT) is required for the storage of GABA in synaptic vesicles and provides a potentially useful probe for inhibitory circuits. However, specific pharmacologic agents for VGAT are not available, and VGAT knockout mice are embryonically lethal, thus precluding behavioral studies. We have identified the Drosophila ortholog of the vesicular GABA transporter gene (which we refer to as dVGAT), immunocytologically mapped dVGAT protein expression in the larva and adult and characterized a dVGAT(minos) mutant allele. dVGAT is embryonically lethal and we do not detect residual dVGAT expression, suggesting that it is either a strong hypomorph or a null. To investigate the function of VGAT and GABA signaling in adult visual flight behavior, we have selectively rescued the dVGAT mutant during development. We show that reduced GABA release does not compromise the active optomotor control of wide-field pattern motion. Conversely, reduced dVGAT expression disrupts normal object tracking and figure-ground discrimination. These results demonstrate that visual behaviors are segregated by the level of GABA signaling in flies, and more generally establish dVGAT as a model to study the contribution of GABA release to other complex behaviors.

Flies require bilateral sensory input to track odor gradients in flight.

Fruit flies make their living "on the fly" in search of attractive food odors. Flies balance the strength of self-induced bilateral visual motion and bilateral wind cues, but it is unknown whether they also use bilateral olfactory cues to track odors in flight. Tracking an odor gradient requires comparisons across spatially separated chemosensory organs and has been observed in several walking insects, including Drosophila. The olfactory antennae are separated by a fraction of a millimeter, and most sensory neurons project bilaterally and also symmetrically activate the first-order olfactory relay; both properties would seem to constrain the capacity for gradient tracking. Nevertheless, using a modified flight simulator that enables maneuvers in the yaw axis, we found that flies readily steer directly toward a laterally positioned odor plume. This capability is abolished by occluding sensory input to one antenna. Mechanosensory input from the Johnston's organ and olfactory input from the third antennal segment cooperate to direct small-angle yaw turns up the plume gradient. We additionally show that sensory signals from the left antenna contribute disproportionately more to odor tracking than signals from the right, providing further evidence of sensory lateralization in invertebrates.

The neuro-ecology of resource localization in Drosophila: behavioral components of perception and search.

From the moment an adult fruit fly ecloses, its primary objective in life is to disperse and locate the source of an attractive food odor upon which to feed and reproduce. The evolution of flight has greatly enhanced the success of fruit flies specifically and insects more generally. Control of flight by Drosophila melanogaster is unequivocally visual. Strong optomotor reflexes towards translatory and rotational visual flow stabilize forward flight trajectory, altitude and speed. The steering responses to translatory and rotational flow in particular are mediated by computationally separate neural circuits in the fly's visual system, and gaze-stabilizing body saccades are elicited by threshold integration of expanding visual flow. However, visual information is not alone sufficient to enable a fruit fly to recognize and locate an appropriately smelly object due in part to the relatively poor resolution of its compound eyes. Rather, the animal uses an acute sense of smell to actively track odors during flight. Without a finely adapted olfactory system, the fly's remarkable visual capabilities are for naught. The relative importance of vision is apparent in the cross-modal fusion of the two modalities for stable active odor tracking. Olfactory processing in Drosophila is shaped by ecological and functional forces which are inextricably linked. Thus physiologists seeking the functional determinants of olfactory coding as well as ecologists seeking to understand the mechanisms of speciation do well to consider each others' point of view. Here we synthesize a broad perspective that integrates across ultimate and proximate mechanisms of odor tracking in Drosophila.

Context-dependent olfactory enhancement of optomotor flight control in Drosophila.

Sensing and following the chemical plume of food odors is a fundamental challenge faced by many organisms. For flying insects, the task is complicated by wind that distorts the plume and buffets the fly. To maintain an upwind heading, and thus stabilize their orientation in a plume, insects such as flies and moths make use of strong context-specific visual equilibrium reflexes. For example, flying straight requires the regulation of image rotation across the eye, whereas minimizing side-slip and avoiding a collision require regulation of image expansion. In flies, visual rotation stabilizes plume tracking, but rotation and expansion optomotor responses are controlled by separate visual pathways. Are olfactory signals integrated with optomotor responses in a manner dependent upon visual context? We addressed this question by investigating the effect of an attractive food odor on active optomotor flight control. Odorant caused flies both to increase aerodynamic power output and to steer straighter. However, when challenged with wide-field optic flow, odor resulted in enhanced amplitude rotation responses but reduced amplitude expansion responses. For both visual conditions, flies tracked motion signals more closely in odor, an indication of increased saliency. These results suggest a simple search algorithm by which olfactory signals improve the salience of visual stimuli and modify optomotor control in a context-dependent manner, thereby enabling an animal to fly straight up a plume and approach odiferous objects.

The spatial, temporal and contrast properties of expansion and rotation flight optomotor responses in Drosophila.

Fruit flies respond to panoramic retinal patterns of visual expansion with robust steering maneuvers directed away from the focus of expansion to avoid collisions and maintain an upwind flight posture. Panoramic rotation elicits comparatively weak syndirectional steering maneuvers, which also maintain visual stability. Full-field optic flow patterns like expansion and rotation are elicited by distinct flight maneuvers such as body translation during straight flight or body rotation during hovering, respectively. Recent analyses suggest that under some experimental conditions the rotation optomotor response reflects the linear sum of different expansion response components. Are expansion and rotation-mediated visual stabilization responses part of a single optomotor response subserved by a neural circuit that is differentially stimulated by the two flow fields, or rather do the two behavioral responses reflect two distinct control systems? Guided by the principle that the properties of neural circuits are revealed in the behaviors they mediate, we systematically varied the spatial, temporal and contrast properties of expansion and rotation stimuli, and quantified the time course and amplitude of optomotor responses during tethered flight. Our results support the conclusion that expansion and rotation optomotor responses are indeed two separate reflexes, which draw from the same system of elementary motion detectors, but are likely mediated by separate pre-motor circuits having different spatial integration properties, low-pass characteristics and contrast sensitivity.