Recording cilia activity in ctenophores: effects of nitric oxide and low molecular weight transmitters

Cilia are the major effectors in Ctenophores, but very little is known about their transmitter control and integration. Here, we present a simple protocol to monitor and quantify cilia activity and provide evidence for polysynaptic control of cilia coordination in ctenophores. We also screened the effects of several classical bilaterian neurotransmitters (acetylcholine, dopamine, L-DOPA, serotonin, octopamine, histamine, gamma-aminobutyric acid (GABA), L-aspartate, L-glutamate, glycine), neuropeptide (FMRFamide), and nitric oxide (NO) on cilia beating in Pleurobrachia bachei and Bolinopsis infundibulum. NO and FMRFamide produced noticeable inhibitory effects on cilia activity, whereas other tested transmitters were ineffective. These findings further suggest that ctenophore-specific neuropeptides could be major candidates for signal molecules controlling cilia activity in representatives of this early-branching metazoan lineage

A peripheral subepithelial network for chemotactile processing in the predatory sea slug Pleurobranchaea californica.

Many soft-bodied animals have extensive peripheral nervous systems (PNS) with significant sensory roles. One such, the sea slug Pleurobranchaea californica, uses PNS computations in its chemotactile oral veil (OV) in prey tracking, averaging olfactory stimuli across the OV to target likely source direction, or "stimulus place". This suggests a peripheral subepithelial network (SeN) interconnecting sensory sites to compute the directional average. We pursued anatomy and connectivity of previously described ciliated putative sensory cells on OV papillae. Scanning electron microscopy (SEM) confirmed paddle-shaped cilia in clusters. Anti-tubulin and phalloidin staining showed connections to branching nervelets and muscle fibers for contraction and expansion of papillae. Ciliary cell processes could not be traced into nerves, consistent with sensory transmission to CNS via secondary afferents. Anti-tyrosine hydroxylase-stained ciliated cells in clusters and revealed an at least partially dopaminergic subepithelial network interconnecting clusters near and distant, connections consistent with PNS averaging of multiple stimulated loci. Other, unidentified, SeN neurotransmitters are likely. Confirming chemotactile functions, perfusible suction electrodes recorded ciliary spiking excited by both mechanical and appetitive chemical stimuli. Stimuli induced sensory nerve spiking like that encoding stimulus place. Sensory nerve spikes and cilia cluster spikes were not identifiable as generated by the same neurons. Ciliary clusters likely drive the sensory nerve spikes via SeN, mediating appetitive and stimulus place codes to CNS. These observations may facilitate future analyses of the PNS in odor discrimination and memory, and also suggest such SeNs as potential evolutionary precursors of CNS place-coding circuitry in the segmented, skeletonized protostomes and deuterostomes

Mechanoceptive responses of papillae at perfusion onset.

A: “On” responses from papillae at onset of ASW flow. The records are from 24 recordings of different papillae in three different animals. Immediately upon the step change in pressure in the suction electrode with ASW perfusion, brief bursts of spikes occurred. Responses quickly adapted while flow was maintained. Recordings from skin adjacent to papillae showed little spontaneous and no induced mechanosensitive activity (not shown). Perfusion offset (downward arrowhead) was 5 seconds after onset (upward arrowhead) for most (19 out of 24) recordings, and for 5 recordings it was 2 seconds after onset. B: Papillar spike frequencies for 1 second time-windows before and after onset of perfusion, and before perfusion offset (box plots show distributions of the firing frequencies, including mean, interquartile distribution, and outliers). Averaged pre-perfusion baseline firing frequency was 1.625 ± 0.31 Hz (median: 1.0 Hz, standard error: 0.311), increasing to 11.30 ± 0.90 Hz (median: 12.0 Hz, standard error: 0.897) at perfusion onset. Spike activity rapidly adapted to lower firing frequency (4.92 ± 0.75 Hz; median: 3.5 Hz, standard error: 0.752) measured at 1 second before perfusion offset. These effects were significant (Wilcoxon Signed-Rank tests p < 0.00002 for perfusion onset response vs. pre-perfusion (*) and for perfusion onset vs. adaptation (**), and slightly less significant p < 0.0003 for pre-perfusion vs. adaptation).</p

TH-li in branching nerves and sensory clusters.

TH-li staining of cilia clusters and nerves is green; DAPI staining of cell nuclei in the epithelium is blue. A, B: Thick branching TH-li nerves (arrows) approach the clusters of cilia (arrowheads) described earlier. Note individual TH-li neuronal cell bodies spread around nerve branches and lateral branchlets near cilia clusters’ bases. C, D: Several TH-li cell bodies (arrows) each produces a single cilia-like projection to the surface. These presumed receptor cells lie at the base of the cilia cluster (arrowhead). Neuropil-like areas are visible below clusters. An axon of a cell body of these TH-li cells at the left arrow in C branches laterally. Scale bars: A and B, 100 μm; C and D, 50 μm.</p