Visual place learning in Drosophila melanogaster

Abstract

The ability of insects to learn and navigate to specific locations in the environment has fascinated naturalists for decades. The impressive navigational abilities of ants, bees, wasps and other insects demonstrate that insects are capable of visual place learning, but little is known about the underlying neural circuits that mediate these behaviours. Drosophila melanogaster (common fruit fly) is a powerful model organism for dissecting the neural circuitry underlying complex behaviours, from sensory perception to learning and memory. Drosophila can identify and remember visual features such as size, colour and contour orientation. However, the extent to which they use vision to recall specific locations remains unclear. Here we describe a visual place learning platform and demonstrate that Drosophila are capable of forming and retaining visual place memories to guide selective navigation. By targeted genetic silencing of small subsets of cells in the Drosophila brain, we show that neurons in the ellipsoid body, but not in the mushroom bodies, are necessary for visual place learning. Together, these studies reveal distinct neuroanatomical substrates for spatial versus non-spatial learning, and establish Drosophila as a powerful model for the study of spatial memories.

Figure 1. Drosophila trained in a thermal-visual arena exhibit place learning

(a) Illustration of the arena. The floor is composed of 64 thermoelectric modules, the panorama is provided by a 24×192 LED display, and flies are recorded using a CMOS camera under infrared (IR) illumination. (b) Thermal imaging view of the arena’s floor showing the uniformly warm surface with a single cool tile; also shown is the heated ring barrier. Bottom panel: temperature readings across the arena (black line). (c) Trajectories of 4 representative flies from trials #1,2 and 10 are shown below a diagrammatic representation of the visual panorama denoting the location of the cool tile in the previous trial (dashed square), and the current location of the cool tile (blue square). In this coupled condition the position of the cool tile relative to the visual panorama remains constant even as its absolute position changes between trials.

Figure 2. Flies use visual cues to improve in place learning task

Flies were trained with a coupled visual panorama (red, n=33 experiments, 495 flies), with an uncoupled (gray, n=21 experiments, 315 flies) or dark (black n=23 experiments, 345 flies) visual surround. (a) When trained in the coupled condition (red) flies reduce the time to find the cool tile by nearly half, whereas flies trained with an uncoupled panorama (gray) or in the dark (black) show little or no improvement in time to locate the target. The improvement exhibited with the coupled visual panorama is due to flies taking (b) shorter, (c) more direct paths to the target (d) rather than simply increasing walking velocity. See Methods for details of calculations. Values are mean ± SEM.

Figure 3. Following training flies exhibit a persistent search bias in the absence of the cool tile and retain this memory for several hours

Flies are tested in a probe trial (#11) where the visual display is relocated but no cool tile is present. (a) Trajectories from four representative flies, each plotted for 60 s after leaving their starting quadrant. Flies start in the top-left quadrant (Q1, Start); the dashed square denotes the “expected” location of the cool tile (Q2, Target). (b) Flies preferentially search in the quadrant where they have been trained to find the cool spot (Q2), even when the cool spot is absent; values are mean ± SEM, n=33 experiments, 495 flies. (c) Probe learning index is significantly greater than zero (indicating learning) when flies are trained with a coupled visual panorama (red, p<0.0001, n=33), but not when trained with an uncoupled (grey, p=0.28, n=21) or dark (black, p=0.39, n=23) visual panorama. (d) To test place memory retention, flies were tested in a probe trial at several time intervals following training (n≥5). Flies retain visual place memories for at least 2 hours after training. Box plots indicate the median value (solid black line), 25 and 75 percentiles (box), and the data range (dashed whiskers). For details of calculations and additional statistics, see Methods.

Figure 4. Subsets of ellipsoid-body ring neurons are required for place learning

(a–c) GAL4 driver lines targeting subsets of cells in the mushroom body or (d–f) the ellipsoid body; shown are the expression patterns for each driver using a GFP reporter. (g) White boxes denote spatial learning prior to Kir2.1 induction; grey boxes indicate performance following Kir2.1 expression. Silencing ellipsoid body neurons projecting to R1 and R4 (R15B07) or R1 (R28D01) severely impairs place learning (p<.001, red lines) while silencing mushroom body neurons (a–c), or a separate subset of eb neurons projecting to R4 alone (R38H02), leaves place learning intact. Box plots are as described in Fig 3, n≥8 experiments. (h) Schematic representation of ellipsoid body ring neuron anatomy. (i–m) Flies with impaired place learning (expressing Kir2.1) show normal (i) walking velocity, (j) heat aversion, (k) optomotor response, (l) visual pattern discrimination during tethered flight, and (m) olfactory learning. For i–k (n≥8 experiments), values are mean ± SEM. For l (n≥6 flies) & m (n=8 experiments), values are mean ± SD. See Methods for details of calculations and additional statistical analysis.