Whole-brain recordings give us a global perspective of the brain in action. In this study, we describe a method using light field microscopy to record near-whole brain calcium and voltage activity at high speed in behaving adult flies. We first obtained global activity maps for various stimuli and behaviors. Notably, we found that brain activity increased on a global scale when the fly walked but not when it groomed. This global increase with walking was particularly strong in dopamine neurons. Second, we extracted maps of spatially distinct sources of activity as well as their time series using principal component analysis and independent component analysis. The characteristic shapes in the maps matched the anatomy of subneuropil regions and, in some cases, a specific neuron type. Brain structures that responded to light and odor were consistent with previous reports, confirming the new technique’s validity. We also observed previously uncharacterized behavior-related activity as well as patterns of spontaneous voltage activity.

Highlights d Custom 3D image registration method to analyze neuronal population imaging data d Fly dopamine neuron population encodes innate valence of odors or tastes d DAN compartment activity is modulated by different physiological states d DAN compartment activity correlates with movement conveying behavioral state

SummaryNeuromodulation permits flexibility of synapses, neural circuits and ultimately behavior. One neuromodulator, dopamine, has been studied extensively in its role as reward signal during learning and memory across animal species. Newer evidence suggests that dopaminergic neurons (DANs) can modulate sensory perception acutely, thereby allowing an animal to adapt its behavior and decision-making to its internal and behavioral state. In addition, some data indicate that DANs are heterogeneous and convey different types of information as a population. We have investigated DAN population activity and how it could encode relevant information about sensory stimuli and state by taking advantage of the confined anatomy of DANs innervating the mushroom body (MB) of the fly Drosophila melanogaster. Using in vivo calcium imaging and a custom 3D image registration method, we find that the activity of the population of MB DANs is predictive of the innate valence of an odor as well as the metabolic and mating state of the animal. Furthermore, DAN population activity is strongly correlated with walking or running, consistent with a role of dopamine in conveying behavioral state to the MB. Together our data and analysis suggest that distinct DAN population activities encode innate odor valence, movement and physiological state in a MB-compartment specific manner. We propose that dopamine shapes innate odor perception through combinatorial population coding of sensory valence, physiological and behavioral context.

17Whole brain recordings give us a global perspective of the brain in action. We describe here a method 18 using light field microscopy to record both whole brain calcium and voltage activity at high speed in 19 behaving adult flies. We show first that global activity maps can be obtained for various stimuli and 20 behaviors. Notably, we found that brain activity increased on a global scale when the fly walked but not 21 when it groomed. Second, maps of spatially distinct sources of activity as well as their time series can be 22 extracted using principal component analysis and independent component analysis. Their characteristic 23 shapes matched the anatomy of sub-neuropil regions and in some cases a specific neuron type. Brain 24 structures that responded to light and odor were consistent with previous reports, confirming the 25 validity of the new technique. We also observed previously uncharacterized behavior-related activity, as 26 well as patterns of spontaneous activity in the central complex. 27 28 29 peer-reviewed)

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly's head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly's ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts.The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.