We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.

Sparse manipulation of neuron excitability during free behavior is critical for identifying neural substrates of behavior. Genetic tools for precise neuronal manipulation exist in the fruit fly, Drosophila melanogaster, but behavioral tools are still lacking to identify potentially subtle phenotypes only detectible using high-throughput and high spatiotemporal resolution. We developed three assay components that can be used modularly to study natural and optogenetically induced behaviors. FlyGate automatically releases flies one at a time into an assay. FlyDetect tracks flies in real time, is robust to severe occlusions, and can be used to track appendages, such as the head. GlobeDisplay is a spherical projection system covering the fly's visual receptive field with a single projector. We demonstrate the utility of these components in an integrated system, FlyPEZ, by comprehensively modeling the input-output function for directional looming-evoked escape takeoffs and describing a millisecond-timescale phenotype from genetic silencing of a single visual projection neuron type.

The striatum shows general topographic organization and regional differences in behavioral functions. How corticostriatal topography differs across cortical areas and cell types to support these distinct functions is unclear. This study contrasted corticostriatal projections from two layer 5 cell types, intratelencephalic (IT-type) and pyramidal tract (PT-type) neurons, using viral vectors expressing fluorescent reporters in Cre-driver mice. Corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing topographic specificity as injection sites move from sensory to motor and frontal areas. Topographic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher topographic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. As predicted by a longstanding model, corticostriatal projections of interconnected cortical areas form parallel circuits in the basal ganglia.

Insects and mammals share similarities of neural organization underlying the perception of odors, taste, vision, sound, and gravity. We observed that insect somatosensation also corresponds to that of mammals. In Drosophila, the projections of all the somatosensory neuron types to the insect's equivalent of the spinal cord segregated into modality-specific layers comparable to those in mammals. Some sensory neurons innervate the ventral brain directly to form modality-specific and topological somatosensory maps. Ascending interneurons with dendrites in matching layers of the nerve cord send axons that converge to respective brain regions. Pathways arising from leg somatosensory neurons encode distinct qualities of leg movement information and play different roles in ground detection. Establishment of the ground pattern and genetic tools for neuronal manipulation should provide the basis for elucidating the mechanisms underlying somatosensation.

The medial entorhinal cortex is part of a neural system for mapping the position of an individual within a physical environment. Grid cells, a key component of this system, fire in a characteristic hexagonal pattern of locations, and are organized in modules that collectively form a population code for the animal's allocentric position. The invariance of the correlation structure of this population code across environments and behavioural states, independent of specific sensory inputs, has pointed to intrinsic, recurrently connected continuous attractor networks (CANs) as a possible substrate of the grid pattern. However, whether grid cell networks show continuous attractor dynamics, and how they interface with inputs from the environment, has remained unclear owing to the small samples of cells obtained so far. Here, using simultaneous recordings from many hundreds of grid cells and subsequent topological data analysis, we show that the joint activity of grid cells from an individual module resides on a toroidal manifold, as expected in a two-dimensional CAN. Positions on the torus correspond to positions of the moving animal in the environment. Individual cells are preferentially active at singular positions on the torus. Their positions are maintained between environments and from wakefulness to sleep, as predicted by CAN models for grid cells but not by alternative feedforward models. This demonstration of network dynamics on a toroidal manifold provides a population-level visualization of CAN dynamics in grid cells.