Figure 7.
5-HT regulates THC and alters adoptive transfer outcomes
(A) Injection of 5-HT into crayfish leads to maximal THC increases by 20 min post-injection. Twenty microliters of hemolymph was collected from crayfish prior to experimentation in order to determine a pre-injection THC. After allowing crayfish to rest for 24 h, they were injected with saline or 5-HT and bled again at a specified time point (x axis) following the injection to determine a post-injection THC. The % change was determined as: (post-injection THC – pre-injection THC/pre-injection THC) ∗ 100. Grey bars represent the average percent change in THC of animals injected with saline, and black bars represent average % change in THC of animals injected with 5-HT. Comparisons between groups were made with one-way ANOVA analysis followed by Tukey’s multiple comparison tests. Error bars indicate the standard error of the mean (SEM). Asterisks indicate significant difference from saline control (independent samples t-test, 10 min, p = 0.018; 20 min, p = 0.021). For the 5-HT group (black bars), for the four time points (10, 20, 40, and 60 min), sample size n = 11, 9, 10, and 8 animals, respectively. For the saline group (grey bars), n = 8, 9, 9, and 8, respectively.
(B,Inset) Recipient crayfish were soaked in 5-HT for 24 h after adoptive transfer of hemocytes from donor crayfish. The total number of niche cells increases, despite the potential immune response to the introduction of foreign cells. This effect of 5-HT has also been observed without adoptively transferred cells (Benton et al., 2011). The niches of 5-HT-treated crayfish increase by an average of 65.5% cells, which represents a significant increase compared with the control crayfish maintained in pond water (independent samples t-test, p = 0.001). Error bars indicate SEM; n = 11 niches per group.
(B) After the adoptive transfer of EdU-labeled donor hemocytes, recipient crayfish were soaked in pond water alone (control) or pond water + 5-HT (10−9 M) (5-HT soak). 40% (control) and 50% (5-HT-soaked) of niches contained EdU-labeled cells; therefore, the number of recipient niches is not substantially altered by increased levels of 5-HT. However, the numbers of donor hemocytes that are incorporated into niches increases after 5-HT incubation of recipients, and the types of labeled cells observed are also altered. In 5-HT-treated recipients, 83% of labeled niche cells (15) contained EdU nuclear labeling and were elliptical with no (or sparse) granules; these are similar in size and shape to resident niche cells. In pond water controls, only one cell contained nuclear label (bars with diagonal lines) and had these morphological features. 5-HT treatment of recipients tends to have the opposite influence on “other EdU-labeled cells” (cytoplasmic labeling with or without nuclear labeling). 5-HT treatment resulted in a reduction in the mean numbers of these “other EdU+” cells (black bars). t-tests comparing the data for each of these labeling types revealed a significant difference in “EdU nuclear label” (p = 0.017) between the 5-HT soak and control groups, but no statistical difference in the “other EdU + cells” (p = 0.68). Error bars represent SEM; n = 12 niches per group.