Branch architecture of the fly larval abdominal serotonergic neurons

Abstract

In the metazoan central nervous system (CNS), serotonergic neurons send projections throughout the synaptic neuropil. Little is known about the rules that govern these widespread neuromodulatory branching patterns. In this study, we utilize the Drosophila as a model to examine serotonergic branching. Using single cell GFP labeling we show that within each segment of the Drosophila ventral nerve cord (VNC), each of two serotonergic neurons tiles distinct innervation patterns in the contralateral neuropil. In addition, branches extend only a short distance from the target segment. Through ablation-mediated isolation of serotonergic cells, we demonstrate that the distinct areas of innervation are not maintained through competition between neighboring like-serotonergic neurites. Furthermore, the basic branching pattern of serotonergic neurons within the neuropil remains unchanged despite alterations of initial axonal trajectories.

Figure 1. Developmental profile of abdominal CNS serotonergic innervations

Each panel is a dorso-ventral view of a single serotonin-stained A5 abdominal segment, cropped and rotated 90 degrees to provide the frontal view with dorsal up and ventral down. The top left panel is from the end of embryogenesis, stage 17. The scale bar is 10µm and is the same for all panels. The midline is marked with ml and is indicated with a dashed line; the serotonergic axons crossing the midline along the posterior commissure are marked with a pc and the cell bodies are marked with a cb. The larval stages shown are 3 hours after hatching: 3 hr L1; 12 hours after hatching: 12 hr L1; 24 hours after hatching: L2, 72 hours after hatching: L3F and 5 days after hatching or wandering third instars: L3W. Four pupal stages are shown and two adults stages: immediately after hatching: 0 d A, and 77 days after hatching: 77 d A. During embryogenesis, the serotonergic neurons extend main branches (primary branches) that form a circular pattern that are maintained throughout larval development. The primary branches retract during early pupal development, and reform a new pattern during later pupal development, which is maintained throughout adult development.

Figure 2. The medial and lateral serotonergic cells of Drosophila VNC have different innervation patterns

Images of L3F A4 medial (A–D) and A3 lateral (E–H) branch patterns using single cell labeling with GFP (see text). (A,B,E,F) are top views and (C,D,G,H) are a single segment cropped and rotated 90 degrees such that dorsal is at top. The axons crossing the midline in the posterior commissure are indicated at the midline with an arrowhead in each panel. The ipsilateral branches are to the left of each panel and the contralateral branches are to the right. db: dorsal bifurcation, a secondary branch that usually appears from the dorsal lateral point of the medial contralateral primary; ms: secondary branches that run along the edge of the midline; ic: the contralateral projecting secondary branch that originates from the lateral cell ipsilateral branches. The scale bar in H is 10µm and is the same for all panels. Genotypes hs-FLP;UAS-FRT-mcd8-GFP-FRT x ddc-GAL4

Figure 3. Summary of overall branch structure of the abdominal serotonergic neurons

(A) Frontal view with dorsal at top and (B) top view with anterior at top of an A4, A5 or A6 segment. Left columns are cartoon representations and right panels are both stained for serotonin (red) and fasciclin II (blue). Also refer to Fig. 2 and 3 for images of these branches. (A and B) In the cartoon, the medial cell is drawn in red and lateral in green. The fasciclin II bundles are shown in blue and the neuropil in grey. m: medial contralateral branches; i: ipsilateral branches; l: lateral contralateral branches; db: dorsal bifurcation; ms: secondary branches that run along the edge of the midline, just lateral of the dorsal medial fasII bundle; ic: the contralateral projecting secondary branch. (C) Dendrograms of two reconstructed medial cell contralateral branches showing pattern of the secondary branches. The branches are drawn in to show the structure, but are not drawn to scale and do not represent the direction of secondary branch projections. (D) Plot of branch complexity as defined by number of branch points. The x-axis represents the number of segments on a branch and the y-axis the average number of secondary branches of this complexity extending from a medial contralateral primary branch. A secondary branch without any daughter bifurcations is designated 1 by this measure and a branch with one bifurcation a 2.

Figure 4. Genetic ablation of serotonergic cells reveals that the cell boundaries are not maintained through competition

(A–D) Single GFP labeled cells generated in the same way as in figure 1 are shown for comparison. (A) A medial A5 serotonergic neuron is labeled with GFP. The medial A5 serotonergic neuron extends its contralateral axonal projections throughout the 5^th abdominal segment and halfway through the 6^th abdominal segment (halfway between the A5 and A6 commissure). For quantifying the distance of projections, we define the full innervation of a segment as 1 segment away. Therefore, this cell sends its projections 1.5 segments away. (B) Top view of GFP alone from (A) is shown to highlight the anterior-posterior extensions. (C) Frontal view of a medial A5 serotonergic neuron labeled with GFP. (D) Frontal view of a lateral A3 serotonergic neuron labeled with GFP. The dorsal secondary branch (db) and lateral cell contralateral projection (ic) are also indicated. (E–H) Reaper-hid (rpr-hid) expression is limited to early serotonergic development by driving UAS-rpr hid with the eg-GAL4 promoter. Expression of rpr-hid early in serotonergic development causes apoptosis of a random subset of serotonergic neurons during embryogenesis, leaving a variable number of serotonergic neurons intact. (E) The top view image of a VNC stained with serotonin (red) and BP102 (green) shows a typical outcome of rpr-hid expression. BP102 is a general neuropil marker and is used to demark the segmental boundaries by staining the commissural axons. Serotonergic neurons cross the midline along the posterior commissure of each segment (denoted by the white arrowheads). In this VNC only a single medial A5 remains. The larva before dissection was morphologically and behaviorally grossly normal. The contralateral projection of this serotonergic neuron extends in the posterior direction and spans its segment and 3/4 into the adjacent posterior segment, which is calculated from the overlaid BP102 staining (1.75 segments). (F) Top view of serotonin staining alone from (E) is shown. (G) Frontal view of a single isolated medial A5 stained with serotonin. The medial pattern is maintained and is grossly indistinguishable from the medial pattern seen in medial cells labeled with GFP that have all neighboring cells intact (C). (H) Frontal view of a single isolated lateral A3 stained with serotonin. The lateral pattern is maintained and is grossly indistinguishable from the lateral pattern seen in lateral cells labeled with GFP that have all neighboring cells intact (D). (I) The average farthest posterior intersegmental distance that a medial cell sends its contralateral axons is shown for various segments, with comparisons between GFP labeled and rpr-hid isolated cells during L3F and L3W. Isolated serotonergic cells (rpr-hid) do not send their contralateral axonal extensions any farther than serotonergic cells with all neighboring cells intact (GFP-labeled cell). In L3W rpr-hid larvae, serotonergic cells have grown in isolation for five days with little/no increase in axonal extension in comparison to GFP labeled neurons in L3W. Isolated serotonergic cells (rpr-hid) do not send their contralateral axonal extensions any farther than serotonergic cells with all neighboring cells intact (GFP-labeled cell). Data are mean ±SD. Number of samples for each measure is indicated in the graph. Genotypes: (A–D) hs-FLP;UAS-FRT-mcd8-GFP-FRT x ddc-GAL4, (E–H) UAS-rpr-hid x eg-GAL4

Figure 5. The primary branch pattern is maintained despite altered axon routing or number

Effects of overexpression of drl (A,B,C,D,E), robo2 (F,G,H,I,J) and hb (K,L,M,N,O). The midline is indicated with a dashed line and the dorsal secondary branch (db) and lateral cell ipsilateral projection (ic) are also indicated. (A,F,K) are top views of serotonin stained segments with eg-GAL4 used as a driver. (B,G,L) shows a top view of segments stained for surviving serotonergic neurons with serotonin (red) and BP102 (B,G) and GFP (L). (C,D,H,I,M,N) are serotonin staining along of surviving neurons are side views from posterior except for (N) which is a top view. (A–E) drl expression driven by eg-GAL4 early in serotonergic development misdirects crossing axons to the anterior commissure of the adjacent posterior segment instead of the posterior commissure of its own segment. After crossing in the adjacent segment, the axon grows back to the correct segment (A), which is denoted by the white arrowheads. (B–D) drl expression in combination with rpr-hid generates isolated serotonergic cells, which reveals that the medial (C) and lateral patterns (D) are preserved even when the axons enter the contralateral side from an adjacent segment. (B) is a top view showing serotonin in red and BP102 in green. (E) Schematic representation of the medial and lateral pattern seen in serotonergic cells expressing drl. This pattern is identical to GFP labeled cells (Figure 2), except the axon crosses the midline along the adjacent anterior commissure, which is denoted by a dotted line as it crosses the midline (vertical blue dotted line). (F–J) robo2 expression driven by eg-GAL4 early in serotonergic development blocks axons from crossing the midline (F). (G–I) robo2 expression in combination with rpr-hid creates single isolated serotonergic cells whose branches do not cross the midline, but instead project into the ipsilateral neuropil. (G) Shows a single isolated medial A4 stained with serotonin (red) and BP102 (green). (H) Shows a frontal view of a single medial cell. (I) Shows a frontal view of a single lateral cell. (J) Schematic of the medial and lateral pattern seen in serotonergic cells. This pattern is the mirror image of the normal medial and lateral patterns. (K–N) Hunchback (hb) expression driven by eg-GAL4 early in serotonergic development causes the creation of multiple cells with circular medial projections. Three to five medially projecting serotonergic neurons can be seen per side of each segment (arrowheads) (K). There is a loss of cells with lateral projections. (L–N) Hb and rpr-hid combined expression can produce isolated multiples of cells with medial circular projections. The axons from these cells fasciculate along the same circular medial branch pattern and cannot be individually resolved under light microscopy. (N) Dorsal view of (M). (O) Schematic representation of serotonergic cells expressing hb. Genotypes: (A) UAS-drl x eg-GAL4 (B–D) UAS-rpr-hid x UAS-drl x eg-gal4; (F) UAS-robo2 x eg-GAL4 (G–I) UAS-rpr-hid x UAS-robo2 x eg-GAL4 (K) UAS-hb;UAS-hb x eg-GAL4 (L–N) UAS-rpr-hid x UAS-hb;UAS-hb x eg-GAL4