Investigating the causes of stimulus-evoked changes in cone reflectance using a combined adaptive optics SLO-OCT system

In vivo functional imaging of human photoreceptors is an emerging field, with compelling potential applications in basic science, translational research, and clinical management of ophthalmic disease. Measurements of light-evoked changes in the photoreceptors has been successfully demonstrated using adaptive optics (AO) coherent flood illumination (CFI), AO scanning light ophthalmoscopy (SLO), AO optical coherence tomography (OCT), and full-field OCT with digital AO (dAO). While the optical principles and data processing of these systems differ greatly, and while these differences manifest in the resulting measurements, we believe that the underlying physiological processes involved in each of those techniques are likely the same. AO CFI and AOSLO systems are more widely used than OCT systems. However, those systems produce only two-dimensional images and so, less can be said about the anatomical and physiological origins of the observed signal. OCT signal, on the other hand, provides 3D imaging but at a cost of high volume of data, making it impractical to clinical purposes. In light of this, we employed a combined AO OCT SLO system with point for point correspondence between the OCT and SLO images to measure functional responses simultaneously with both and investigate SLO retinal functional biomarkers based on OCT response. The resulting SLO images reveal reflectance changes in the cones which are consistent with those previously reported using AO CFI and AO SLO. The resulting OCT volumes show phase changes in the cone outer segment (OS) consistent with those previously reported by us and others. We recapitulate a model of the cone OS previously proposed to explain AO-CFI reflectance changes, and show how this model can be used to predict the signal in AO SLO. The limitations of the model is also discussed in this manuscript.

Dopaminergic mechanism underlying reward-encoding of punishment omission during reversal learning in Drosophila

Animals form and update learned associations between otherwise neutral sensory cues and aversive outcomes (i.e., punishment) to predict and avoid danger in changing environments. When a cue later occurs without punishment, this unexpected omission of aversive outcome is encoded as reward via activation of reward-encoding dopaminergic neurons. How such activation occurs remains unknown. Using real-time in vivo functional imaging, optogenetics, behavioral analysis and synaptic reconstruction from electron microscopy data, we identify the neural circuit mechanism through which Drosophila reward-encoding dopaminergic neurons are activated when an olfactory cue is unexpectedly no longer paired with electric shock punishment. Reduced activation of punishment-encoding dopaminergic neurons relieves depression of olfactory synaptic inputs to cholinergic neurons. Synaptic excitation by these cholinergic neurons of reward-encoding dopaminergic neurons increases their odor response, thus decreasing aversiveness of the odor. These studies reveal how an excitatory cholinergic relay from punishment- to reward-encoding dopaminergic neurons encodes the absence of punishment as reward, revealing a general circuit motif for updating aversive memories that could be present in mammals.

Suppression of GABAergic neurons through D2-like receptor secures efficient conditioning in Drosophila aversive olfactory learning

The GABAergic system serves as a vital negative modulator in cognitive functions, such as learning and memory, while the mechanisms governing this inhibitory system remain to be elucidated. In Drosophila, the GABAergic anterior paired lateral (APL) neurons mediate a negative feedback essential for odor discrimination; however, their activity is suppressed by learning via unknown mechanisms. In aversive olfactory learning, a group of dopaminergic (DA) neurons is activated on electric shock (ES) and modulates the Kenyon cells (KCs) in the mushroom body, the center of olfactory learning. Here we find that the same group of DA neurons also form functional synaptic connections with the APL neurons, thereby emitting a suppressive signal to the latter through Drosophila dopamine 2-like receptor (DD2R). Knockdown of either DD2R or its downstream molecules in the APL neurons results in impaired olfactory learning at the behavioral level. Results obtained from in vivo functional imaging experiments indicate that this DD2R-dependent DA-to-APL suppression occurs during odor-ES conditioning and discharges the GABAergic inhibition on the KCs specific to the conditioned odor. Moreover, the decrease in odor response of the APL neurons persists to the postconditioning phase, and this change is also absent in DD2R knockdown flies. Taken together, our findings show that DA-to-GABA suppression is essential for restraining the GABAergic inhibition during conditioning, as well as for inducing synaptic modification in this learning circuit. Such circuit mechanisms may play conserved roles in associative learning across species.

Whole-brain calcium imaging during physiological vestibular stimulation in larval zebrafish

During in vivo functional imaging, animals are head-fixed and thus deprived from vestibular inputs, which severely hampers the design of naturalistic virtual environments. To overcome this limitation, we developed a miniaturized ultra-stable light-sheet microscope that can be dynamically rotated during imaging along with a head-restrained zebrafish larva. We demonstrate that this system enables whole-brain functional imaging at single-cell resolution under controlled vestibular stimulation. We recorded for the first time the dynamic whole-brain response of a vertebrate to physiological vestibular stimulation. This development largely expands the potential of virtual-reality systems to explore complex multisensory-motor integration in 3D.