Initially acquired memory dissipates rapidly if not consolidated. Such memory decay is thought to result either from the inherently labile nature of newly acquired memories or from interference by subsequently attained information. Here we report that a small G protein Rac-dependent forgetting mechanism contributes to both passive memory decay and interference-induced forgetting in Drosophila. Inhibition of Rac activity leads to slower decay of early memory, extending it from a few hours to more than one day, and to blockade of interference-induced forgetting. Conversely, elevated Rac activity in mushroom body neurons accelerates memory decay. This forgetting mechanism does not affect memory acquisition and is independent of Rutabaga adenylyl cyclase-mediated memory formation mechanisms. Endogenous Rac activation is evoked on different time scales during gradual memory loss in passive decay and during acute memory removal in reversal learning. We suggest that Rac's role in actin cytoskeleton remodeling may contribute to memory erasure.

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

The Drosophila brain contains a relatively simple circuit for forming Pavlovian associations, yet it achieves many operations common across memory systems. Recent advances have established a clear framework for Drosophila learning and revealed the following key operations: a) pattern separation, whereby dense combinatorial representations of odors are preprocessed to generate highly specific, nonoverlapping odor patterns used for learning; b) convergence, in which sensory information is funneled to a small set of output neurons that guide behavioral actions; c) plasticity, where changing the mapping of sensory input to behavioral output requires a strong reinforcement signal, which is also modulated by internal state and environmental context; and d) modularization, in which a memory consists of multiple parallel traces, which are distinct in stability and flexibility and exist in anatomically well-defined modules within the network. Cross-module interactions allow for higher-order effects where past experience influences future learning. Many of these operations have parallels with processes of memory formation and action selection in more complex brains.

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, 'Voltron', that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 minutes of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.Animal behavior is produced by patterns of neuronal activity that span a wide range of spatial and temporal scales. To understand how neural circuits mediate behavior thus requires high-speed recording from ensembles of neurons for long periods of time.Although the activity of large numbers of neurons can now be routinely recorded using genetically encoded calcium indicators (GECIs) (1), the slow kinetics of calcium signals complicate the measurement of action potentials, and sub-threshold voltage signals are missed entirely (1-3). Voltage imaging using genetically encoded voltage indicators (GEVIs) can overcome these challenges, enabling imaging of fast spikes and subthreshold dynamics in genetically defined neurons (4, 5). The high imaging speed and excitation intensity required for voltage imaging, combined with the smaller volume of the cellular membrane, place increased demands on voltage indicators relative to GECIs.Extant GEVIs rely on fluorescence from either microbial rhodopsins (6-8) or fluorescent proteins (FPs) (9-13). These fluorophores lack the brightness and photostability to allow in vivo voltage imaging from large fields of view over timescales of many behavioral events, precluding the millisecond-timescale interrogation of neural circuits. Recent development of improved rhodamine dyes such as the Janelia Fluor ® (JF) dyes enable their use in complex biological experiments due to their high brightness and photostability (14), compatibility with self-labeling protein tags (15, 16), and the ability to traverse the blood-brain barrier for in vivo delivery (17). Here we describe a 'chemigenetic' GEVI scaffold-termed 'Voltron'-which incorporates these synthetic fluorophore dyes. Voltron provides an increased photon yield that enables in vivo imaging of neuronal spiking and sub-threshold voltage signals in model organisms with order-of-magnitude improvement in the number of neurons imaged simultaneously over substantially longer durations.Our design for a chemigenetic voltage indicator combines a voltage-sensitive microbial rhodopsin domain (6, 7, 11) with a self-labeling protein tag domain (F...