Visual projection neurons in the Drosophila lobula link feature detection to distinct behavioral programs
- Janelia Research Campus, Howard Hughes Medical Institute, United States
Figures
Figure 1 with 1 supplement
Introduction to lobula columnar (LC) neurons.
Schematics (A–C) and confocal images (D–K) show the lobula and adjacent parts of the visual system. (A,D,G,H) Horizontal sections. (B,E,I,J) Anterior views. (C,F,K) Cross-section views of the …
Figure 1—figure supplement 1
Examples of additional LC cell types and similar neurons.
These cell types were not selected for further analyses in this study since they did not project to glomerular target regions in the ventrolateral central brain, covered only part of the lobula as …
Figure 2 with 1 supplement
Expression patterns of LC neuron split-GAL4 lines.
Split-GAL4 driven expression of 20xUAS-CsChrimson-mVenus (insertion in attP18; visualized using anti-GFP antibody labeling; green) and a neuropil marker (anti-Brp, magenta) are shown. Genotypes are …
Figure 2—figure supplement 1
VNC expression patterns of the corresponding brains shown in Figure 2.
Imaging parameters and brightness or contrast adjustments were identical for each brain/VNC pair. Scale bar represents 50 µm. Original confocal stacks are available from www.janelia.org/split-GAL4.
Figure 3 with 2 supplements
LC neuron terminals in the central brain are organized into distinct neuropil structures.
(A) Illustration of the projection patterns of 12 LC cell types that project to major optic glomeruli in the PVLP (or in the PVLP/PLP boundary region). Image is a substack maximum intensity …
Figure 3—figure supplement 1
A second type of columnar VPN projects to the LC22 target region.
Main image and inset show two views along approximately orthogonal axes of MCFO labeled LC22 (green) and LPLC4 (magenta) cells generated using a GAL4 driver line (R11C10) with expression in both …
Figure 3—figure supplement 2
Presynaptic marker expression in individual LC cell types.
Single confocal sections are shown. Images were rotated to show similar views. The anti-Brp reference pattern (grey) and the presynaptic marker (syt-smHA, magenta) are shown. The white appearance of …
Figure 4 with 1 supplement
Multicolor stochastic labeling reveals differences in the arrangement of the terminal arbors of different LC types within their target glomerulus.
(A–C) LC16. (A) Position of the dendrites of three LC16 cells in the lobula in a layer cross-section view. Each cell occupies a distinct position along the long (DV) and short (AP) axes of the …
Figure 4—figure supplement 1
Terminal arbor arrangements of additional LC cell types.
(A–C) LC6, (D–F) LC11, (G–I) LC13, (J–L) LC12, (M–O) LC18, (P–R) LPLC2. Substantial overlap of the arbors of co-labeled single cells in their target regions was also observed for the remaining LC …
Figure 5 with 2 supplements
Layer specific arborizations of LC neurons in the lobula.
(A) Anti-Brp neuropil marker shows bands of different intensity in the lobula that can serve as approximate markers of layer boundaries. The image is a maximum intensity projection through 10 …
Figure 5—figure supplement 1
Layer positions of arbors of known cell types.
Lobula terminals of MCFO labeled T5, Tm9, Tm4, Tm3 and Tm20 cells (Fischbach and Dittrich, 1989) are shown. Anti-Brp pattern is in grey. Layer boundaries are marked with white lines. Golgi studies (F…
Figure 5—figure supplement 2
Potential presynaptic sites of LC neurons in the lobula.
In addition to the target glomerulus in the central brain (see Figure 3 and Figure 3—figure supplement 2), split-GAL4 driven pJFRC51-3XUAS-IVS-syt::smHA in su(Hw)attP1 expression also resulted in …
Figure 6 with 1 supplement
Column spread of LC neurons and other cell types in the lobula in cross-section views.
Cells were labeled using MCFO. Cross-section views of the lobula were generated using Vaa3D. The AP and DV axes of the lobula are indicated. Anti-Brp reference marker is shown in grey. LC neuron …
Figure 6—figure supplement 1
Layer cross-section views of lobula arbors of the LC neuron types not shown in Figure 6.
Cross-section views of LC cells of the indicated types were generated as described in the Figure 6 legend. Asterisks in the LC10b and LC21 panels mark two MCFO labeled cells (LC10c and LC11, …
Figure 7 with 2 supplements
Single cell shapes of LC neurons.
Maximum intensity projection images of MCFO labeled single cells were manually segmented to exclude other labeled cells or background signal and converted to inverted grayscale images. Cells are …
Figure 7—figure supplement 1
Layer patterns of single cells of 22 LC neuron types.
Images are reoriented views (generated using Vaa3D) of MCFO labeled LC neurons. Anti-Brp neuropil marker is in grey. For each cell type, two different views, along the AP and along the DV axes of …
Figure 7—figure supplement 2
Single cell labeling of LPLC arbors in lobula and lobula plate.
(A) LPLC1. (B) LPLC2. (C) LPLC4. Numbers indicate the four LP layers (Fischbach and Dittrich, 1989). Similar to lobula layers, the LP layers can be identified by changes in the intensity of anti-Brp …
Figure 8 with 2 supplements
Optogenetic activation of LC neurons induces distinct behavioral responses that differ between LC cell types.
(A) A representative video image of group of freely walking flies in the circular arena assay. (B) Representative video images of a freely behaving fly on a small glass platform in the single-fly …
Figure 8—figure supplement 1
Quantification of locomotor behaviors and determination of behavioral penetrance.
(A) A representative tracking of a fly with LC16 activation in the arena assay. Arrows indicate the start and end positions. Fly’s trajectory is denoted in red (during stimulation, 1 s) or in white …
Figure 8—figure supplement 2
Variability of LC16 backward walking behavior across trials and across flies.
In the circular arena experiments, each fly was repeatedly tested in a series of trials (see Materials and methods). For most analyses, we pooled data across all flies and trials (as shown in the …
Figure 9 with 2 supplements
Experiments with additional LC6 and LC16 driver lines confirm the activation phenotypes of these cell types.
(A,B) Behavioral penetrance for different controls and multiple split-GAL4 driver lines for (A) jumping (flies that jumped within 200 ms of stimulation onset) with LC6 controls based on the OL0077B …
Figure 9—figure supplement 1
Expression patterns of multiple split-GAL4 driver lines for LC6 and LC16.
Brain (A,B) and VNC (C,D) expression patterns of split-GAL4 drivers for LC6 (A,C) and LC16 (B,D). Split-GAL4 driven expression of 20xUAS-CsChrimson-mVenus (insertion in attP18; visualized using …
Figure 9—figure supplement 2
Quantification of LC16 activation induced locomotor behaviors and determination of behavioral penetrance.
Jitter plots show the distribution of mean velocity and angular speed during optogenetic stimulation of multiple LC16 split-GAL4 driver lines and control lines. Color codes are the same as in Figure …
Figure 10 with 2 supplements
Reaching behavior resulting from activation of LC10 subtypes.
(A) Representative video images of a fly exhibiting reaching behavior in the single-fly assay. Time stamps indicate milliseconds (ms) after the start of reaching. (B) Comparison of lobula layer …
Figure 10—figure supplement 1
Expression patterns of LC10 split-GAL4 driver lines.
Brain (A) and VNC (B) expression patterns using 20xUAS-CsChrimson-mVenus in attP18 visualized with an anti-GFP antibody (green) and a neuropil marker (anti-Brp, magenta) are shown. While some driver …
Figure 10—figure supplement 2
Stochastic single cell labeling reveals LC10 subtype expression patterns of the LC10 split-GAL4 driver lines.
(A) Examples (10 per driver line) of MCFO-labeled LC10 cells from five different lines. Subtype classification is indicated for each cell (white lowercase letters). Anti-Brp pattern (used to …
Figure 11 with 1 supplement
LC6 and LC16 activation behaviors resemble avoidance responses evoked by visual looming.
(A) LC6 and LC16 project to adjacent, non-overlapping target glomeruli. The image was generated using a 3D image rendering software (FluoRender) (Wan et al., 2012) on aligned confocal images. (B) …
Figure 11—figure supplement 1
Behavioral consequences of silencing LC6 and LC16 by Kir2.1 expression.
Split-GAL4 driver lines OL0077B (LC6, A–B) or OL0046B (LC16, C) were crossed to flies with pJFRC49-10XUAS-IVS-eGFPKir2.1 in a DL strain background. pBDPGAL4U crossed to the same effector or DL flies …
Figure 12 with 1 supplement
LC16 and LC6, but not LC11, respond to visual looming stimuli with robust calcium increases.
(A) Visual stimuli evoked calcium responses of LC neurons were imaged in head-fixed flies. (B) The axon terminals of LC cells bundle to form cell-type specific glomeruli (subset shown in C). We …
Figure 12—figure supplement 1
LC16 and LC6 are tuned to slower looming speeds.
Stimulus evoked calcium responses to different loom speeds. The responses are mean ∆F/F during a time window in which the response peaks (2 s before and after the looming stimulus stops expanding). …
Figure 13 with 1 supplement
Behavioral responses to unilateral LC neuron stimulation differ from bilateral activation behaviors and are directional.
(A) Schematic illustration of a genetic method for stochastic labeling and activation of LC neurons. A ‘stop-cassette’ reporter (pJFRC300-20XUAS-FRT>-dSTOP-FRT>-CsChrimson-mVenus in attP18) was used …
Figure 13—figure supplement 1
Turning behavior of LC10 flies with bilateral labeling that is stronger in one brain hemisphere (trial count = 212, fly count = 18).
Data for flies with uniform bilateral expression or no labeling are the same as in the main Figure and included for comparison.
Videos
Video 1
Composite of aligned images showing a series of sections through the ventrolateral central brain with the target regions of 18 LC (or LPLC) neurons visualized by presynaptic marker expression.
Original images, composite image assembly and color scheme are as in Figure 3. The anti-Brp reference pattern (grey) of the template brain used for alignment is shown both together with the labeled …
Video 2
Three-dimensional rendering of the target regions of LC (or LPLC) neurons in the ventrolateral central brain shown in Figure 3 and Video 1.
Image assembly and color scheme are as described in Figure 3. Anti-Brp reference pattern is in grey. Terminals of LC10 cell types in the AOTu are not included. The movie was generated using Vaa3D.
Video 3
An example of an LC neuron activation phenotype (backward walking and turning) in the arena assay.
A representative video showing groups of freely walking flies tested in the circular arena. The video is shown at 0.4x actual speed and the red indicators at the corners indicate the timing of …
Video 4
Examples of distinct LC neuron activation phenotypes in the arena assay.
One representative fly for each phenotype is shown before, during and after optogenetic stimulation (1 s each, 3 s in total). Flies’ centers of mass are tracked and their trajectories are …
Video 5
Examples of distinct LC neuron activation phenotypes in the arena assay.
10 representative flies for each phenotype are shown for the duration of optogenetic stimulation (1 s). Flies’ centers of mass are tracked for the duration of stimulation and their trajectories are …
Video 6
Examples of distinct CsChrimson activation phenotypes in the single-fly assay.
10 representative flies of each genotype are shown during the 50 ms optogenetic stimulation and for the following 450 ms. Jumping and reaching phenotypes are shown in the side view whereas forward …
Video 7
Examples of phenotypes upon bilateral and unilateral activation of LC16 in the stochastic activation experiment.
A representative fly for bilateral and unilateral LC16 activation is shown before, during and after optogenetic stimulation (1 s each, 3 s in total). Flies’ centers of mass are tracked and their …
Additional files
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Supplementary file 1
Four tables with information on LC neuron anatomy, results of behavioral experiments and experimental genotypes.
(A) Summary of the anatomical properties of the 22 LC neuron types described in detail in this study. Cell numbers and lateral arbor spreads are listed as mean ± SD. Arbors sizes in visual column units were estimated from the measurements of lateral arbor spread. Further details are provided in the Materials and methods. (B) Details of the behavioral experiments in Figures 8–13. This table includes information on split-GAL4 hemidrivers (the AD and DBD halves), the behavioral penetrance for each of the five examined phenotypes, and trial and fly counts, from both the arena and single-fly assays. While the raw data counts within each assay are the same, a small number of trials could not be scored by either manual annotation or automatic tracking; as a result, there are some small differences in the number of quantified data points for the two scoring methods. Use of fly culture media different from standard cornmeal molasses food with supplemental retinal is indicated as follows: ret-: standard cornmeal molasses food without supplemental retinal. Vit-: Vitamin A-deficient food based on the grape juice recipe (see Materials and methods) with supplemental retinal. Vit-, ret-: Vitamin A-deficient food without supplemental retinal. norpA indicates flies that are rendered blind by a null mutation in the norpA gene, norpA[36]. (C) Split-GAL4 driver lines for the LC neuron types described in this study. Split-GAL4 line names in bold indicate drivers used in behavioral experiments. (D) Details of the fly lines used to generate the data for the anatomy Figures and Videos 1 and 2.
- https://doi.org/10.7554/eLife.21022.042
