SUMMARY Optical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but brain motion, which often accompanies behavior, compromises these measurements. Two-photon point-scanning microscopy is especially sensitive to motion artifacts, and two-photon recording of activity has required rigid coupling between the brain and microscope. We developed a two-photon tracking microscope with extremely low-latency (360 μs) feedback implemented in hardware. This microscope can maintain continuous focus on neurons moving with velocities of 3 mm/s and accelerations of 1 m/s2 both in-plane and axially. We recorded calcium dynamics of motor neurons and inter-neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentations and behavioral outputs, and we measured light-induced depolarization of a visual interneuron in a moving animal using a genetically encoded voltage indicator. Our technique can be extended to stabilize recordings in a variety of moving substrates.

Associative learning allows animals to use past experience to predict future events. The circuits underlying memory formation support immediate and sustained changes in function, often in response to a single example. Larval Drosophila is a genetic model for memory formation that can be accessed at molecular, synaptic, cellular, and circuit levels, often simultaneously, but existing behavioral assays for larval learning and memory do not address individual animals, and it has been difficult to form long-lasting memories, especially those requiring synaptic reorganization. We demonstrate a new assay for learning and memory capable of tracking the changing preferences of individual larvae. We use this assay to explore how activation of a pair of reward neurons changes the response to the innately aversive gas carbon dioxide (CO2). We confirm that when coupled to CO2 presentation in appropriate temporal sequence, optogenetic reward reduces avoidance of CO2. We find that learning is switch-like: all-or-none and quantized in two states. Memories can be extinguished by repeated unrewarded exposure to CO2 but are stabilized against extinction by repeated training or overnight consolidation. Finally, we demonstrate long-lasting protein synthesis dependent and independent memory formation.

O ptical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but such recordings are compromised by brain motion that often accompanies behavior. Two-photon point scanning microscopy is especially sensitive to motion artifacts, and to date, twophoton recording of activity has required rigid mechanical coupling between the brain and microscope. To overcome these difficulties, we developed a twophoton tracking microscope with extremely low latency (360 µs) feedback implemented in hardware. We maintained continuous focus on neurons moving with velocities of 3 mm/s and accelerations of 1 m/s 2 both in-plane and axially, allowing high-bandwidth measurements with modest excitation power. We recorded from motor-and inter-neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentation and behavioral outputs. Our technique can be extended to stabilize recordings in a variety of moving substrates. IntroductionTo understand how the brain selects and enacts behavior, it is desirable to record activity in behaving animals.Optical recording of neural activity has become a standard technique in systems neuroscience, but making these measurements in freely behaving animals poses technical challenges [1,2]. Behavior is expressed as motion, and motion of the brain limits the accuracy of optical imaging techniques. Fundamentally, optical measurement of neural activity requires precisely measuring the amount of light emitted by a fluorescent indicator. Movement of a labeled neuron relative to the microscope objective will alter the efficiency with which the indicator is excited and with which fluorescence emissions are collected, as will changes in the position or properties of scattering elements between the objective and the neuron. If these changes or movements accompany the animal's behavior, the result will be a fluorescence signal that varies due to motion, not neural activity.The most common solution to the problem of brain motion is to rigidly couple the brain to the objective. For example, mice [3] and adult flies [4][5][6][7] can be head-fixed to the objective while exploring virtual environments controlled by motion of the animal's legs or wings, or the microscope itself can be mounted on a behaving rodent [8][9][10][11]. Larval zebrafish have been paralyzed and embedded in agar with fictive motion read out through electrical recording of the motor neurons [12]. Whole brain imaging has been accomplished without simultaneous peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/213942 doi: bioRxiv preprint first posted online Nov. 19, 2017; Recording neural activity in unrestrained animals with 3D tracking two photon microscopy measurement of behavior using a variety of techniques in immobilized C. elegans [13][14][15] and with light-sheet microscopy in dissected D. melanogaster larval brains [16]. S...

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Associative learning allows animals to use past experience to predict future events. The circuits underlying memory formation support immediate and sustained changes in function, often in response to a single example. Larval Drosophila is a genetic model for memory formation that can be accessed at the molecular, synaptic, cellular, and circuit levels, often simultaneously, but the standard behavioral assay for learning and memory does not address individual animals. It has also been difficult to form long lasting memories, especially those requiring synaptic reorganization. We demonstrate a new assay for learning and memory capable of tracking the changing preferences of individual larvae. We use this assay to explore how activation of a pair of reward neurons changes the response to the innately aversive gas Carbon Dioxide, CO2. We confirm that when coupled to odor presentation in appropriate temporal sequence, optogenetic reward reduces avoidance of CO2. We find that learning is quantized, all-or-nothing, and can be extinguished by repeated unrewarded exposure to CO2. We find that memories can be stabilized against extinction by repeated training or overnight consolidation. Finally, we demonstrate long-lasting protein synthesis dependent and independent memory formation.