Figure 1.
Schematic depiction of holothurian nervous system.
A) The semi-transparent Leptosynapta clarki is readily found just beneath the sediment during the springtime at False Bay, San Juan Island, WA. Major structures of the echinoderm nervous system are visible through the skin: buccal tentacles (T), circumoral nerve ring (NR), radial nerve cords (RNC) and the dorsal papillae (P). B) FMRFamide-like immunoreactivity was found in processes extending from the nerve ring (NR) into the tentacles (T) (tentacle nerves: TN), in the esophageal region (E) and throughout the body with projections and interneurons (IN) from the radial nerve cords (RNC). C) Histamine-like immunoreactivity was identified in processes extending from the NR and in abundant neurons in the tentacles (T), as well as in processes from the RNCs to sensory-like structures in the papillae (P).
Figure 2.
FMRFamide-like immunoreactivity (green) is seen throughout the buccal region of Leptosynapta clarki juveniles.
Note that some of the staining in the nerve ring is background based on comparisons to control samples (no primary antibody). Red indicates muscle fibers visualized by phalloidin (actin filaments) stain. Nuclei are labeled with Draq-5 and are colored in blue. A) Composite panel of FMRFamide-like immunoreactivity surrounding the circumoral nerve ring (NR) and projecting into each tentacle as two radial nerve extensions (tentacle nerves: TN); scale bar 100 µm B) Composite panel of two tentacles at higher magnification shows the furcation of these extensions from the mid tentacle region (MT) culminating in putative sensory cells visible at the tip of tentacle (TT); scale bar 60 µm C) Higher magnification of one tentacle clearly shows the extension of one putative sensory cell beyond the muscle fiber (red) and into epithelial tissue; scale bar 50 µm (D) further magnification of these cells reveals projections characteristic of sensory cells; scale bar 10 µm.
Figure 3.
Mouth region and tentacles of adult Leptosynapta.
A) Dissection of adult tentacle reveals an abundance of FMRFamide-like immunoreactive radial nerve extensions throughout the tentacle and a concentration of putative sensory cells in the esophageal region (circle); scale bar 25 µm B) DIC image corresponding to A. Arrows point to cross-sections of two buccal tentacles. C) Dense aggregation of processes and cell bodies are visible in the esophageal region and the anterior region of the radial nerve cord (RNC) where it meets the circumoral nerve ring (NR). Arrows point to cell bodies; scale bar 50 µm D) FMRFamide-like immunoreactivity in tentacles of adult. Arrows point to sensory cells (SC) in the periphery of the tentacle and their processes projecting to the NR via the TN; scale bar 50 µm. Blue: Nuclear stain using Draq-5, Green: Anti-FMRFamide antibody.
Figure 4.
FMRFamide-like immunoreactivity (FMRF-IR) is also found throughout the mid-body and posterior region of adult Leptosynapta clarki.
A) Close association with interneurons and muscles in proximity of the radial nerve cord (RNC). This section shows both radial (RM) and longitudinal muscles (LM). scale bar 25 µm B) putative interneurons are found intermittently along the RNC and appear associated with axons in the RNC. RM are visible in the background; scale bar 25 µm C,D) high magnification images of a FMRF-IR process associated with a muscle fibre (arrows). No cell bodies are visible in these views; scale bars 25 µm and 10 µm respectively E) FMRF-IR network of axons in proximity of radial nerve cord is also associated with individual cell bodies (CB). In this image, bleed-through fluorescence from dispersed muscle fibers (M) is visible; scale bar 50 µm. F) close-up of network also seen in E. Cell bodies are clearly visible in body wall; scale bar 50 µm. G) at higher magnification these cell bodies do not appear to have the characteristic projections of those seen in the buccal region or other parts of the mid-body. scale bar 25 µm. Blue: Nuclear stain using Draq-5, Green: Anti-FMRFamide antibody.
Figure 5.
Histaminergic-like immunoreactivity found in the buccal tentacle region of juvenile Leptosynapta clarki.
Cell bodies are clearly labeled with histamine and are morphologically similar to FMRFamide-like neurons in the tentacles (see Fig. 3). A) individual cells with typical, sensory-like projections are visible at the epithelial surface (b) putative neurons without projections are found in deeper tissue layers (a). In this preparation only few processes are visible. scale bar 50 µm. B) corresponding individual DIC optical section of Fig. 5A shows the position of the two distinct cell types in the tentacle. scale bar 50 µm. C) Higher magnification of the sensory-like tentacle neurons; scale bar 25 µm. D) Close-up of putative sensory neuron with characteristic projection into distal tentacle region. scale bar 10 µm. Blue: Nuclear stain using Draq-5, Green: Anti-histamine antibody.
Figure 6.
Histaminergic-like immunoreactivity (HA-IR) seen throughout the mid-body region of Leptosynapta clarki juveniles and adults.
A) high concentration of HA-IR neurons in the center of the body wall papillae (c). These neurons project to the radial nerve cord (RNC) (b) and connect in dense bundles (a) (see also D for cross section of RNC); scale bar 100 µm. B) Higher magnification view of RNC and two body wall papillae; ); scale bar 50 µm. Neurons in the papillae have distinct sensory protrusions that reach to the outside of the epithelial layer (C and E; scale bars 50 µm). D) cross section of RNC with projections from body wall papillae sensory neurons to RNC; HA-IR is combined with DIC image which shows the association of histaminergic neurons with latitudinal muscle fibers; scale bar 50 µm. F:G) Sensory neurons of body wall papillae form distinct glomeruli-like structure indicating sites of dense synaptic connections (E). This glomeruli-like structure is visible in G and H; scale bar 25 µm.
Figure 7.
Leptosynapta clarki adults respond to histamine injection (200 µM) with a drastic increase in anterior to posterior directed peristaltic contractions.
Thick lines in the middle of the boxplots show the median number of anterior to posterior contractions per minute and the whiskers delineate the interquartile range. Control and sham specimen (filtered seawater, 200 µM) rarely displayed anterior to posterior peristalsis, while histamine injected animals consistently responded with rapid increases in the frequency of anterior to posterior peristalsis (F(1,8) = 46.8874, P = 0.001).