Molecular and Morphological Circuitry of the Octopus Sucker Ganglion

Abstract

The octopus sucker is a profoundly complex sensorimotor structure. Each of the hundreds of suckers that line the octopus arm can move independently or in concert with one another. These suckers also contain an intricate sensory epithelium, enriched with chemotactile receptors. Much of the massive nervous system embedded in the octopus arm mediates control of the suckers. Each arm houses a large axial nerve cord (ANC), which features local enlargements corresponding to each sucker. There is also a sucker ganglion, a peripheral nervous element, situated in the stalk of every sucker. The structure and function of the sucker ganglion remain obscure. We examined the cellular organization and molecular composition of the sucker ganglion in Octopus bimaculoides. The sucker ganglion has an ellipsoid shape and features an unusual organization: the neuropil of the ganglion is distributed as a cap aborally (away from the sucker) and a small pocket orally (toward the sucker), with neuronal cell bodies concentrated in the space between. Using in situ hybridization, we detected positive expression of sensory (PIEZO) and motor (LHX3 and MNX) neuron markers in the sucker ganglion cell bodies. Nerve fibers spread out from the sucker ganglion, targeting the surrounding sucker musculature and the oral roots extending to the ANC. Our results indicate that the sucker ganglion is composed of both sensory and motor elements and suggest that this ganglion is not a simple relay for the ANC, but facilitates local reflexes for each sucker.

Keywords: Octopus bimaculoides; cephalopod; invertebrate; muscular hydrostat; neural circuitry; neuroethology; sensorimotor control.

FIGURE 1

Geography of octopus arm and location of sucker ganglion. (a) Diagram of a transverse cross section of octopus arm. The major components of the arm nervous system are highlighted in blue. Key indicates transverse plane of section. Asterisk denotes acetabulobrachial muscles (ACBM). (b) Transverse arm section with in situ hybridization (ISH) for the neuronal gene complexin (CPLX1). Neuronal cell bodies in the axial nerve cord (ANC) and sucker ganglion (SG) are labeled. Scale bar: 100 µm. (c–f). Longitudinal sections through the octopus arm, as indicated by inset in c. In the longitudinal plane, the coordinate axes correspond to aboral–oral and proximal–distal, as indicated in f. (c) ISH of CPLX1 on a longitudinal section of octopus arm. There is an SG embedded in the ACBM for every sucker. Arrowheads point to the SG. Scale bar: 250 µm. (d, e) The SG is enriched with other pan‐neuronal markers. (d) MUNC18. (e) SYT1. Octopuses have many isoforms of SYT, so SYT1 may not label all neurons. Scale bars: 50 µm. (f) ISH of CPLX1 on a longitudinal section of octopus arm across many suckers. The location of the SG varies based on the location of the sucker. Scale bar: 500 µm. AC, acetabulum; ACBM, acetabulobrachial muscles; ANC, axial nerve cord; IMNC, intramuscular nerve cord; INF, infundibulum; SG, sucker ganglion.

FIGURE 2

Sucker ganglion width changes along the proximal–distal axis. (a, b) Example longitudinal sections through sucker ganglia (SG) used for measurements. Yellow line indicates location of measurement. Scale bars: 50 µm. (a) SG stained for acetylated α‐tubulin (acTUBA, cyan) and F‐actin (magenta). (b) SG stained with hematoxylin and eosin (H&E). (c) SG width decreases down the proximal (P)–distal (D) axis, error bars ± sem. (d) The width of the SG relative to sucker width increases along the P–D axis, error bars ± sem. acTUBA, acetylated α‐tubulin; H&E, hematoxylin and eosin; SG, sucker ganglion.

FIGURE 3

Internal neural organization of the sucker ganglion. (a–c) The axes for a sucker: proximal–distal, external–internal, and aboral–oral. (a) An en face, horizontal cartoon of the suckers showing that the suckers are arranged in two offset rows. In the horizontal plane, each sucker has a proximal (P)–distal (D) axis, and an internal side (InS)–external side (ExS) axis. The P–D axis corresponds to the P–D axis for the arm as whole (i.e., from head to arm tip). The InS corresponds to the side of the sucker that hugs the midline of the arm. The ExS corresponds to the side of the sucker that is situated on the outer edge of the arm. (b) A transverse cartoon of a sucker. In the transverse plane, each sucker has an aboral–oral axis and an ExS–InS axis. The aboral side is the side further from the sucker; the oral side is the side closer to the sucker. (c) A cartoon of a transverse section through the sucker ganglion (SG). Like the whole sucker, the SG has an aboral–oral axis and an ExS–InS axis in the transverse plane. (d–g) Organization of the SG demonstrated by acetylated α‐tubulin (acTUBA, cyan) immunostaining. Horizontal virtual slices produced by fusing multiple 1‐µm confocal sections from aboral (d) to oral (g). See Video S1 for a 3D rendering of this data set. (d) Most aborally is a neuropil (NP) cap and an internal belt (in.belt) that is composed of circumferential fibers and large cell bodies. (e) The in.belt is situated aboral to the cell body (CB) region. (f) Oral to the CB territory is an outer belt (out.belt), composed of circumferentially running fiber bundles. (g) On the oral side of the SG is a pocket of oral NP (oNP). Inset highlights the slight asymmetry of the horizontal sections through the SG. (h–k) Longitudinal sections from external (h) to internal (k) through the SG prepared for in situ hybridization with CPLX1 demonstrating the location of neuronal cell bodies. Neurons are located in the in.belt and are dense throughout the full extent of CB region. Scale bars: 50 µm. acTUBA, acetylated α‐tubulin; CB, cell bodies; D, distal; ExS, external side; in.belt, internal belt; InS, internal side; NP cap, neuropil cap; oNP, oral neuropil; out.belt, outer belt; P, proximal.

FIGURE 4

Connective tissue splits the sucker ganglion. (a, b) Picrosirius Red staining of a longitudinal section through the sucker ganglion (SG). Red denotes the presence of connective tissue. (a) Brightfield image depicting the full extent of the SG. Black arrowheads point to connective tissue plate (CTP) between the cell body (CB) region and neuropil (NP) cap. (b) Polarized light image of the same field, highlighting the red labeling of connective tissue. White arrowheads point to CTP. Scale bars: 50 µm. CB, cell bodies; CTP, connective tissue plate; NP cap, neuropil cap.

FIGURE 5

Gene expression patterns in the cell body territory of the sucker ganglion. (a–c) The sucker ganglion (SG) is enriched with sensory and motor neuron markers. (a) In situ hybridization (ISH) for the mechanosensory ion channel marker PIEZO. Inset demonstrates PIEZO labeling in the inner belt (in.belt). (b) ISH for LIM homeobox 3 transcription factor (LHX3), a motor neuron marker. (c) ISH for motor neuron and pancreas homeobox transcription factor (MNX). (d–f) Presysnaptic markers for cholinergic neurons label a subset of neurons in the cell body region. (d) ISH for choline acetyl transferase (CHAT). (e) ISH for vesicular acetylcholine transporter (VACHT). (f) ISH for solute carrier family 5 member 7 (SLC5A7). Scale bars: 50 µm. (g–j) The molecular profile of the SG differs from that of the ANC. No positive labeling was detected in the SG for (g) NK6 homeobox transcription factor (NXK6), a motor neuron marker, or (i) dorsal root ganglia homeobox transcription factor (DRGX), despite positive labeling in the ANC (h, NKX6) or ACBM (j, DRGX). Scale bars for g–i: 50 µm. Scale bar for j: 20 µm. (k–n) Markers of inhibitory neurotransmission, (k) glutamate acid decarboxylase (GAD) and (m) vesicular inhibitory amino acid transporter (VIAAT), do not show detectable labeling in the SG, although the ANC (l, GAD) and ACBM (n, VIAAT) demonstrate cellular labeling readily. Scale bars for k–m: 50 µm. Scale bar for n: 20 µm. All sections longitudinal. ACBM, acetabulobrachial muscles; ANC, axial nerve cord; CB, cell bodies; in.belt, inner belt; NP cap, neuropil cap.

FIGURE 6

Cells in the local musculature feed back to the sucker ganglion. (a) Maximum projection of the sucker ganglion (SG) and surrounding musculature in the horizontal plane stained for acetylated α‐tubulin (acTUBA, cyan). Arrowheads point to a cell body in the musculature feeding back to SG. Scale bar: 100 µm. (b–e) Close‐up images of cells in musculature stained with acTUBA. (b, c) Cells exhibit a complex morphology. Yellow arrowheads point to processes that extend into the musculature; white arrowheads indicate cell bodies. Arrows point in the direction of the SG. Scale bars: 25 µm. (d) Some cells are located in clusters. White arrowheads indicate cell bodies. Arrow points in the direction of SG. Scale bar: 50 µm. (e) Some cells are located close to the SG. White arrowheads indicate cell bodies. Arrow points in the direction of the SG. Scale bar: 25 µm. ACBM, acetabulobrachial muscles; acTUBA, acetylated α‐tubulin; in.belt, inner belt; SG, sucker ganglion.

FIGURE 7

Asymmetric gene expression of FMRF in the sucker ganglion. (a) Transverse section of the axial nerve cord with in situ hybridization (ISH) for FMRF. FMRF expression is found abundantly throughout the cell body layer. Inset indicates the location of the panel image. (b) Longitudinal section through the sucker ganglion (SG) with ISH for FMRF. Arrowhead point to FMRF labeling of a small subset of cells. (c) Horizontal section through the SG with FMRF ISH. FMRF labeling is restricted to the external side of the SG, as indicated by arrowheads. ANC, axial nerve cord; CB, cell bodies; CBL, cell body layer; ExS, external side of sucker; InS, internal side of sucker; in.belt, inner belt; NP, neuropil; NP cap, neuropil cap. Scale bars: 50 µm.

FIGURE 8

Distribution of sucker ganglion nerve fibers. (a) Maximum projection of a whole mount of a sucker stained for acetylated α‐tubulin (acTUBA, cyan). Many fascicles enter and exit the sucker ganglion (SG). Scale bar: 250 µm. (b) Distribution of fascicle diameters. Diameters range from 6 to 45 µm, with the peak in the 11–20 µm range. (c) Transverse section through the SG stained for acTUBA (cyan) and F‐actin (magenta). Location of nerve fiber exits is denoted with asterisk. Scale bar: 50 µm. (d) Distribution of fiber exits around the SG (n = 3 ganglia, 2 stained for acTUBA, 1 stained for SMI‐31) plotted in the transverse plane. There is a gap corresponding to the neuropil (NP) cap (***) and two gaps corresponding to a protrusion of the cell body region (**). Color key by location of root in the aboral–oral and external–internal planes. (e–g) The largest nerve fibers exit aborally. (e) Horizontal maximum projection of the aboral portion of the SG stained for acTUBA. Red arrow heads indicate large nerve bundles. (f) Horizontal maximum projection of the oral portion of the sucker ganglion stained with acTUBA. No large nerve bundles are present. Scale bars: 100 µm. (g) Distribution of fiber exits around the SG (n = 1 ganglion, stained for acTUBA) plotted in the transverse plane. Color key corresponds to size of fascicles. The largest fiber bundles exit aborally, corresponding to the portion between the NP cap (***) and cell body protrusion (**). (h) Example of SG nerves (blue) segmented out of a transverse whole mount slice stained for acTUBA. Red traces depict the axial nerve cord oral roots. (i) Schematic of the average trajectory, which is a vector created from the nerve exit point to the center of the processes. (j) Distribution of average trajectories in the transverse plane (n = 1 ganglion, stained for acTUBA). Color key corresponds to nerve root location in the aboral–oral, external–internal plane. Asterisks depict aboral gap (***) and lateral gaps (**) as in d and g. Nerve targets correspond to nerve exit. Key illustrates the transverse orientation. (k) Distribution of average trajectories in the horizontal plane (n = 1 ganglion, stained for acTUBA). Color key corresponds to nerve target location in the horizontal plane. There is a bias away from the proximal–distal axis. Key illustrates the horizontal orientation. acTUBA, acetylated $\mathit{\alpha }$‐tubulin; CB, cell bodies; ExS, external side; InS, internal side; NP cap, neuropil cap; SG, sucker ganglion.

FIGURE 9

Summary of sucker ganglion internal complexity and nerve fiber connections. (a) Cartoon depicting internal organization of the sucker ganglion (SG). The inner belt (red; in.belt), outer belt (orange; out.belt), and nerves (blue) connecting to the SG are highlighted. (b) Schematic depicting nerve connections. Oral nerves from the axial nerve cord are green. Nerves from the SG that target the oral nerves are pink. Nerves from the SG that target local musculature are blue. AC, acetabulum; ACBM, acetabular brachial muscles; CB, cell bodies; CTP, connective tissue plate; in.belt, inner belt; INF, infundibulum; NP cap, neuropil cap; oNP, oral neuropil; out.belt, outer belt; SG, sucker ganglion.