Genetically enhancing 5-HT synthesis in serotonergic neurons induces spheroid formation along 5-HT neurites. (A) All panels show serotonergic neuropil stained with an antibody against serotonin. Serotonergic cell bodies have been optically sectioned from the pictures in VNC panels. (Scale bar = 10μm). Over-expression of Trh under the control of the Trh-GAL4 driver, which drives expression in serotonergic neurons, results in formation of aberrant swellings (spheroids) along serotonin-IR neurites (arrowheads). From left to right, columns are dorsal neuropil at most caudal portion of L3F VNC (A3-A7), dorsal neuropil of 10 day adult abdominal VNC, OL of 10day adult, magnification of approximate boxed area in third column images. Scale bar in first, second and fourth columns = 10μm, scale bar in third column = 20μm. (B) Schematic of dopaminergic and serotonergic cell body location in fly larval abdominal ventral nerve cord. Open circles indicate 5-HT cell bodies while filled circles represent dopaminergic cell bodies. Boxed region represents approximate regions pictured in (C) and (D). (C) 5-HT immunoreactivity in larval VNC driving UAS-Trh in both the serotonergic and dopaminergic neurons using Trh-GAL4 and th-GAL4, respectively. Dorsolateral dopaminergic neurons are indicated with arrows. Ventromedial dopaminergic neurons are indicated with arrowheads. Asterisks distinguish spheroids from cell bodies. (D) 5-HT immunoreactivity in larval VNC driving UAS-Trh in only the dopaminergic neurons using th-GAL4. Dopaminergic cell bodies are indicated as in (C). Note comparable 5-HT immunoreactivity between dopaminergic and serotonergic cells and lack of 5-HT immunoreactive spheroids in the neuropil. Scale bar in (D) is approximately 10 μm and applies to both images. Abbreviations: VNC = ventral nerve cord, OL = optic lobe, L3F = foraging third instar larva.

Spheroid characterization reveals gene-dose dependence and temporal characteristics of reversible spheroid formation.
(A) Box-whisker plot demonstrating volume distribution of serotonergic varicosities and spheroids in control and Trh over-expressing L3F abdominal CNS. Average volumes ± s.d. are presented to the right of the boxes. P-values based on Student’s t-test comparison to control group. (B) The number of spheroids present in abdominal serotonergic neuropil is dose-dependent upon transgene expression at L3F. ***p < 0.001, Kruskal-Wallis ANOVA. (C) Spheroids are first observed in L3F abdominal neuropil 48 hours after egg lay when UAS-Trh is being maximally driven in serotonergic neurons (Trh-GAL4/Trh-GAL4;UAS-Trh/UAS-Trh). Error bars represent s.d. (D) Larvae of the sensitized genotype (Trh-GAL4/Trh-GAL4;UAS-Trh/+, solid line with circles) are compared to larvae containing a temperature sensitive Tubulin-GAL80 (Trh-GAL4/Trh-GAL4;UAS-Trh/Tub-GAL80^ts, dotted line with squares) to repress Trh over-expression until shifted from 18°C to 30°C. Both groups were held at 18°C for 48 hours after AEL, then shifted to 30°C for 48 hours, then shifted back to 18°C for 48 hours. Asterisks indicate comparisons between age-matched groups from each data set. Crosses indicate comparison of GAL80-ts timepoints to the 96 hour timepoint (^†p < 0.05, **p < 0.01, ^†††, ***p < 0.001, ANOVA). Error bars indicate s.d. B,C,D: n ≥ 8 larval CNS.

Serotonergic spheroids are atypical structures exhibiting differential protein localization
(A) Spheroids in Trh-GAL4;UAS-Trh third instar larval abdominal neuropil stained with antibodies against 5-HT and Trh. (B) Spheroids in Trh-GAL4;UAS-Trh/UAS-h-tau abdominal neuropil stained with antibodies against 5-HT and human tau. Magnified view of boxed region for each shown in (A’) and (B’). Scale bar in (A) = 10μm. Scale bar in (A’) = 1μm. (C and D) Examples of plotted pixel intensity taken along a line placed across the spheroids similar to that pictured in (A’) and (B’), respectively. (E) Protein localization presented as the pixel intensity taken from the core of the spheroid relative to the intensity taken at the periphery. The intensity values obtained from either end of the line were averaged to obtain a single intensity value for the periphery of each spheroid. All markers are compared to Trh using Student’s t-test, two tailed. Representative images of each marker in one spheroid appear above their bar on the graph. Scale bar = 5 μm. Antibodies against Trh, 5-HT (p = 0.002) and dSERT (p = 0.06) all localize to the periphery of the spheroid. UAS-syt-GFP (p = 0.29) and UAS-mCD8-GFP (p = 0.09) also produce GFP immunoreactivity restricted to the periphery of the structure. UAS-GFP (p = 0.0002), UAS-GFP-Atg5 (p = 0.0000002) and UAS-h-Tau (p = 0.0006) are all enriched in the spheroid core based on GFP and tau immunohistochemistry. n=5 spheroids for each marker. Error bars indicate s.d.

Excess serotonin acting in the cytoplasm induces spheroid formation. (A) Genetic and pharmacological manipulations of the sensitized background. Control and experimental groups contain equivalent dosing of GAL4 and UAS constructs (Control 1: Trh-GAL4/+;UAS-Trh/UAS-mCD8-GFP, Control 2: Trh-GAL4/Trh-GAL4;UAS-mCD8-GFP/UAS-Trh, ***,p < 10⁻⁵, number larval VNC shown for each point). (B) Manipulations of 5-HT membrane transporters with and without Tub-GAL80^ts control. White bars represent constitutive expression of Trh-GAL4 from the time Trh expression normally begins. In late expression groups GAL4 activity was suppressed by Tub-GAL80^ts until first larval instar, at which time larvae were transferred to 30°C with or without drug to suppress GAL80 and allow GAL4 activity to proceed. Data points within each group are compared to the constitutively expressing group (black bars). (***, p < 0.0001, Student’s t, two-tailed, number larval VNC shown for each point.) Error bars represent s.d.

Excess serotonin interacts with canonical protein aggregate forming pathways (A) Interactions of the sensitized background with canonical degenerative and aggregate-associated pathways. Control groups are as in Fig. 4. (**, p < 0.007, ***p < 0.0005, Student’s t, two-tailed; number larval VNC shown). Error bars indicate standard deviation from the mean. (B) Representative image demonstrating ubiquitin immunoreactivity in a confocal thin section taken through the approximate center of a spheroid along the Z-axis in Trh-GAL4;Trh-GAL4/UAS-mCD8-GFP;UAS-Trh third instar larval CNS. Scale bar = 5μm.

Fenfluramine-induced swellings in mouse are similar to serotonergic spheroids in the fly.
(A) 5-HT immunoreactivity in saline-injected mouse frontal cortex. Scale bar = 10μm. (B) 5-HT immunoreactivity in fenfluramine-injected mouse frontal cortex. Large swellings are easily visible along serotonergic axons. (C) Swellings along mouse serotonergic axons end in terminal boutons (arrowhead) and occur en passant (arrowhead in (D) along continuous branches (arrows in D). Scale bar in (C) = 10μm. (E) Box-whisker plot demonstrating distribution of serotonergic varicosity and spheroid volume in saline and fenfluramine-injected mouse frontal cortex.