Spontaneous spiking activity in a focally recorded cilia cluster and the lack thereof in adjacent skin.

Spontaneous spiking activity in a focally recorded cilia cluster and the lack thereof in adjacent skin.</p

SEM images of papillae surfaces.

A: Three clusters of cilia on a lobe. B: Each cluster is surrounded by numerous tubercles, some of which in this image are ruptured (dashed ellipse). C: There are about 100 cilia in each cluster, with all showing the dilated heads of paddle cilia. D: Paddle cilia-like structures (arrows) are observed within the several broken tubercles of B, above. Scale bars: A, 25 μm; B, 10 μm; C, 3 μm; and D, 2 μm.</p

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

Efferent spikes in papillae from LOVN stimulation.

Shown are 7 of 30 records where two papillae were recorded simultaneously during single LOVN stimuli. Following the stimulus artifacts in mid-traces, single spikes (arrows) appeared in papillae at variable latencies. The spikes in the papillar pair had nearly identical latencies. Spikes sometimes failed in both pairs (record 7), or more often, either failed to appear in the papilla of the upper trace pairs (e.g., record 5) or failed to fully invade (record 6).</p

Chemoceptive responses of papillae to betaine perfusion (0.1M trimethylglycine).

A. The record shows the latency from activity induced by perfusion onset to activity induced by betaine stimulation. B. Betaine stimulation (arrowhead) induces spike activity. Shown are 8 recordings of different papillae from 3 different animals. The arrowhead indicates betaine contact with papillae. C. In recordings of B, comparing activity 3 seconds before and after papillar contact, betaine increased spike frequency from an average baseline of 4.04 ± 0.68 (median: 4.00 Hz, standard error: 0.678) to 14.04 ± 1.1 Hz (median: 13.33 Hz, standard error: 1.01) with significant difference (* p < 0.008, Wilcoxon Signed-Rank test).</p

Anti-tubulin and phalloidin staining of papillae in whole-mounted tissues.

Cilia clusters on the lobules are green (anti-tubulin). Muscle is red (phalloidin). A-C: Antibody and phalloidin penetrated only the superficial level of the tissues, probably through the clusters, revealing the clusters of sensory cilia in the lobules (arrowheads) and the adjacent muscle fibers (arrows). Finer cilia surround the central bundles. Muscle fibers lie immediately beneath the cilia clusters. D: A confocal section of C showing off the glomerular tangles below the cilia (arrowhead) and muscle fibers (arrows). Scale bars A, 200 μm; B-D, 50 μm.</p

The oral veil (OV) with papillae, tentacle, and their innervation.

A: Pleurobranchaea foraging (La Jolla Shores, CA.; photo by Tracy Clark). B: Papillae (pp) on the oral veil. T, the tentacle portion of the oral veil C: A single papilla. Papillae are multi-lobed and vary in size, with larger papillae near the midline of the oral veil. D: Branching of the two sensory nerves, the large oral veil nerve (LOVN) and the tentacle nerve (TN) innervating both the tentacle (T) and adjacent oral veil. The nerves overlap in their innervation of the OV and bring sensory information to the cerebropleural ganglion (CPlG).</p

The SeN spreads under the skin of the papillae between neighboring cilia clusters.

A: A horizontal section cut through the skin shows the TH-li cilia clusters (arrowheads). B: A horizontal section cut a little deeper reveals the subepithelial layer with a distinct and very dense network of TH-li neurites and neuronal cell bodies (arrows). C: A cross section of the papillae. The subepithelial TH-li neuronal network (arrows) spreads under the skin of the papillae, interconnecting the neighboring clusters of cilia (arrowheads). D: Nervelets (arrows) coming from cilia clusters (arrowheads) with lateral paths toward other clusters. Scale bars: A, C, and D, 50 μm; B, 100μm.</p

LOVN afferent spikes originate in the subepithelial network.

Orthodromic action potentials originating in papillae were not identifiable in the sensory LOVN. A. A gentle water jet from a Pasteur pipet was applied twice (arrows) at the midline of the isolated oral veil, likely stimulating multiple papillae mechanically. Recordings of a single papilla (upper trace) and the LOVN (lower trace) show typical loosely correlated activities. B. An expanded record of A, showing difficulty in identifying common spikes. Recording sites were separated by ~2 cm of nerve. The record also shows the relatively longer duration spikes of the ciliary clusters relative to nerve. Other parts of the expanded records are available in Supplementary Information. C. Stimulating a single papilla with a suction electrode (0.2 V, 4 msec duration, single stimuli at intervals of 4–10 sec) over 55 trials failed to drive correlated spikes in the LOVN with similar latencies but drove a few larger spikes at long latencies (triangles). Larger trace: Overlays of the 55 trials. Spiking post-stimulus was significant (Wilcoxon Signed Rank test, using right-tailed Z distribution, p = 0.018). Latencies of these large-amplitude spikes were quite variable, as expected if synaptically activated. Smaller dark trace: The average of the 55 trials in which, if present, smaller, orthodromically driven spikes should have additively emerged from the noise but did not.</p

Sections through oral veil papillae and tentacle stained with anti-tubulin AB (green) and phalloidin (red), showing nerve and muscle structures, respectively.

A: Horizontal section of a papilla at relatively low power. Arrowheads indicate papillar lobules with cilia clusters. B, C: Extensively branching nerves (arrows) approach clusters of cilia (arrowheads, which are also labeled by anti-tubulin AB. D: Higher magnification shows that each cilia cluster (arrowheads) is innervated by a small nerve branch (arrows). Scale bars: A, 500 μm; B, C: 100 μm; D, 50 μm.</p