Mapping Kenyon cell inputs in Drosophila using dye electroporation

Abstract

Here, we describe a technique for charting the inputs of individual Kenyon cells in the Drosophila brain. In this technique, a single Kenyon cell per brain hemisphere is photo-labeled to visualize its claw-like dendritic terminals; a dye-filled electrode is used to backfill the projection neuron connected to each claw. This process can be repeated in hundreds of brains to build a connectivity matrix. Statistical analyses of such a matrix can reveal connectivity patterns such as random input and biased connectivity. For complete details on the use and execution of this protocol, please refer to Hayashi et al. (2022).¹.

Keywords: Cell Biology; Neuroscience.

Graphical abstract

Figure 1

Pinning the brain (A) Schematic of the brain pinned with the posterior side facing upwards (and the anterior side facing the piece of SYLGARD®), the orientation where the mushroom body calyces are visible (colored red). This is the orientation used in step 2: photo-label a Kenyon cell and step 3: dye-fill projection neurons. (B) Schematic of the brain pinned with the anterior side facing upwards (and the posterior side facing the piece of SYLGARD®), the orientation where the antennal lobes are visible (colored red). This is the orientation used in step 4: score the dye-filled glomeruli.

Figure 2

Photo-labeling a Kenyon cell (A) Locate the mushroom body calyx (outlined in red) and select a Kenyon cell soma (yellow arrow) to photo-label (digital zoom: 2). (B) Zoom in on the Kenyon cell soma, draw a small ROI (cyan box) within the boundaries of the soma, and photo-label the ROI as described in step 2: photo-label a Kenyon cell, sub-step 7 (digital zoom: 32). (C) The soma of the targeted Kenyon cell (yellow arrow) is brighter after photo-conversion (digital zoom: 2). (D) The dendritic arbors and claw-shaped terminals of the photo-labeled Kenyon cell are visible 10 min after photo-conversion (digital zoom: 2). (E) An enlarged view of one of the claws (yellow arrow) formed by the photo-labeled Kenyon cell (digital zoom: 8). (F) The axons of the photo-labeled Kenyon cell (yellow arrow) project in the gamma lobe of the mushroom body (imaged during step 4: score the dye-filled glomeruli) (digital zoom: 1.5). All images are composites, and all scale bars are 10 μm.

Figure 3

Dye-filling projection neurons (A) Align the dye-filled electrode with the mushroom body calyx (digital zoom: 2). (A′) Select a first claw to fill (yellow arrow) (digital zoom: 8). (B) Insert the electrode into the calyx, targeting the claw of interest. Note that the invagination that is created when the electrode is pushed into the calyx will not impact the tissue and the dye-labeling in any meaningful way (digital zoom: 2). (C) Verify that the electrode is located within the claw before electroporating dye into it (digital zoom: 8). (D) The presynaptic bouton (yellow arrow) of the dye-filled projection neuron is visible after dye electroporation and once the electrode is removed (digital zoom: 8). (E) Verify that only one projection neuron was dye-filled after the first electroporation. Only one axon should be visible (yellow arrow). Note that once the dye diffuses throughout the projection neuron, not only will the bouton that was initially filled (white box) be labeled, but the other boutons (in this case only 1 bouton) of that projection neuron will be visible as well (digital zoom: 2). (F) Fill as many claws as possible; in this case, five claws were filled leading to the dye-labeling of five projection neurons (digital zoom: 2). All images are composites, and all scale bars are 10 μm.

Figure 4

Scoring dye-filled glomeruli (A–D) Maximum intensity projections of four different planes in the antennal lobe comprising dye-filled glomeruli (the DC2, VA6, VA1v, VC2 and VC5) and projection neuron somata (1–5), arranged from the most anterior (panel A) to most posterior (panel D) (digital zoom: 1.5). All images are composites, and all scale bars are 10 μm. (E) An example of a connectivity matrix comprised of the sampled input of 200 different Kenyon cell samples. Each row corresponds to a Kenyon cell, and each column corresponds to a glomerulus. Each red dash represents a connection between a Kenyon cell and a given glomerulus, while a yellow dash indicates that a Kenyon cell receives 2 inputs from the same glomerulus.

Figure 5

Examples of successful and unsuccessful outcomes (A) The successful outcome of step 2: photo-label a Kenyon cell, in which only one Kenyon cell is labeled (yellow arrow) (digital zoom: 2). (B) An unsuccessful outcome of step 2, wherein two Kenyon cells are photo-labeled (yellow arrows) (digital zoom: 2). (C) A successful outcome of step 3: dye-fill projection neurons, in which only one projection neuron per claw is filled with dye (yellow arrow) (digital zoom: 8). (D) An unsuccessful outcome of step 3, wherein two projection neurons are filled upon dye electroporation into a single claw (yellow arrows) (digital zoom: 8). (E) An antennal lobe with robustly dye-filled glomeruli that are easy to identify (yellow arrows) (digital zoom: 2). (F) An antennal lobe with lightly labeled glomeruli that are difficult to score (yellow arrows) (digital zoom: 2). All images are composites, and all scale bars are 10 μm.