Efferent neurites to capsular muscles in the eye of a snail, Helix aspersa

Printed in Sweden Copyright © 1974 by Academic Press, Inc. All rights of reproduction in any form reserved

286

J. ULTRASTRUCTURE RESEARCH49, 286--294 (1974)

Efferent Neurites to Capsular Muscles in the Eye of a Snail, Helix aspersa CAROL MORTENSEN and RICHARD M. EAKIN

Department of Zoology, University of California, Berkeley 94720 Received May 30, 1974 Scanning and transmission electron microscopy of the capsule and adjacent tissues of the eye of Helix aspersa revealed strands of tissue interconnecting tentacular wall and optic capsule. These strands are composed of collagen, glial and muscle cells, and bundles of axons. After entering the capsule, the axons synapse with the muscle cells in the capsule. The structure of the capsule and capsular strands is similar to that of the tentacular wall. Tentacle, capsule, and capsular strands appear to be innervated by axons of common origin. The functional significance of the neural pathway here described is discussed. Light-dependent contractions in the eye of Helix aspersa have been reported by Eakin and Brandenburger (2). These pulsations are dependent upon light within the visible spectrum from approximately 440 to 640 nm and occur at the rate of 1 per second. The structural basis for these pulsations was postulated to be muscle cells within the collagenous capsule surrounding the eye. Eakin and Brandenburger noted that the muscle cells of the capsule are innervated by neurites which, they suggested, might be collaterals of axons from the photosensory cells. If correct, these light-initiated pulsations could exert a massaging effect on the retinal sensory cells and facilitate a movement of nutrients and photic vesicles (see 3) to the rhabdomeres at the distal ends of the cells. The present investigation was undertaken to determine the source of innervation of the muscle cells within the capsule of the eye. F r o m preliminary work it is apparent that sensory axons pass into and through the capsule only at the base of the eye, where the optic nerve arises. The axons that innervate the muscle cells of the capsule probably originate in the brain. MATERIALS AND METHODS Adult snails (Helix aspersa), collected from Berkeley gardens, were used in this study. For scanning electron microscopy (SEM), optic tentacles were removed, slit on the side opposite the eye, exposing it, the optic nerve and the tentacular ganglion and nerve. The specimens were then fixed in 2% glutaraldehyde buffered with 0.l M cacodylate, pH 7.2,

I N N E R V A T I O N OF O P T I C C A P S U L E OF A S N A I L

287

for 2.5 hours at room temperature. After thorough rinsing in buffer 5 times over 15 minutes, the specimens were postfixed in 1% osmium tetroxide in 0.1 M cacodylate, pH 7.2, for 1 hour at room temperature. The tissues were rinsed in buffer as before, then incubated in a 1% filtered solution of thiocarbohydrazide or TCH (5) at room temperature for 30 minutes. Samples were thoroughly rinsed in glass distilled water 5 times over 15 minutes and then transferred to 1% osmium tetroxide in distilled water for 30 minutes. Specimens were again rinsed in distilled water as before to deter salt precipitation on tissue surfaces, dehydrated through a graded ethanol series followed by a graded Freon 113 series, then critically point dried. The tissues were mounted on an aluminum stub with silver paint and examined in a Coates and Welter Quickscan 50 field emission scanning electron microscope at 15 kV. To view a specimen with transmission electron microscopy (TEM) after scanning, it was exposed to 100% propylene oxide, embedded in Spurr's low viscosity embedding medium, thin sectioned, and stained with uranyl acetate and lead citrate for examination with an RCA 3G electron microscope. Specimens prepared exclusively for TEM were fixed in 2 % glutaraldehyde in 0.1 M cacodylate, pH 7.2, for 1 hour at room temperature. Tissues were then rinsed briefly in buffer and postfixed in 1% osmium tetroxide in 0.1 M cacodylate, pH 7.2, for 1 hour at room temperature. Specimens were rinsed in buffer, dehydrated through a series of graded ethanols, embedded in Spurr's medium, thin sectioned, stained, and examined.

Optic capsule

RESULTS

The capsule which surrounds the eye of the snail H. aspersa contains muscle cells embedded in a matrix of collagenous fibers. These cells are long and fusiform with their long axes and thick and thin myofilaments oriented parallel to the circumference of the eye. Axons in the capsule may be identified by the presence of small clear synaptic vesicles (500 A diameter), large granular and agranular synaptic vesicles (800-1 000 ~ diameter), and neurotubules. Single naked nerve fibers are found in close apposition to muscle cells (Fig. 1). Stretches of the plasma membranes of neurite and muscle fiber are parallel, and the intercellular gaps of these presumed neuromuscular junctions have a constant width of 200 A. Axons accompanied by other neurites (Fig. 2) as well as glial processes (Fig. 3) are also observed to make contact with muscle cells. Glial cells (gliointerstitial cells, 7) are identified by the presence of electron-dense granules which are 200-600 nm in diameter, oval or round, and membrane bounded. Alpha particles of glycogen are also present. Glial cells may make contact with muscle cells in conjunction with axons (Fig. 4) or alone. Axons and glia run in bundles between the muscle cells in the collageneous matrix (Fig. 5) and on the surface of the capsule.

Capsular strands Scanning electron microscopy of tentacles, dissected to expose the eye, optic nerve, and tentacular ganglion and nerve (Fig. 6), reveals strands of tissue interconnecting

288

MORTENSENAND EAKIN

tentacular wall and capsule of the eye (arrow, Fig. 7). Strands also lie On the surface of the capsule (arrow, Fig. 8). Transmission electron microscopy shows that these strands contain axonal bundles similar to those found within the capsule of the eye (Figs. 9 and 10). Details of axonal profiles within these bundles are unclear owing to the method of preparation of the specimens for SEM. Specimens prepared exclusively for T E M demonstrate that these strands contain glial and muscle cells, bundles of axons, and neuromuscular junctions (Figs. 11 and 12).

DISCUSSION

Innervation of the capsule We have shown by SEM that small strands of tissue connect the tentacular wall to the eye (Figs. 6-8) and by T E M that these bridges contain bundles of nerve fibers together with glia, muscle cells, and collagen fibers (Figs. 9 and 10). The use of the thiocarbohydrazide technique of Kelley et al. (5), which allows SEM and then T E M of the same specimen, enabled us to be certain of the identification and composition of the strands connecting the capsule to the tentacular wall. As noted in the introduction, the present study was made to identify the neural pathways to the capsular muscles of the eye of Helix aspersa which were believed by Eakin and Brandenburger (2) to effect the light-stimulated pulsations of that organ. They further postulated that collaterals from the axons of the photoreceptoral cells enter the capsule of, the eye by way of the neural outflow at the basal pole of the retina. Functional contacts of these collaterals with the capsular musculature would complete a neural pathway from receptor to effector. The present findings, however, do not support this hypothesis; they suggest, instead, that motor impulses are carried to the capsular muscles by bundles of nerve fibers which travel from the brain to the capsule of the eye through the tentacular wall.

Neuromuscular junctions Contacts between neurites and muscle cells were observed in the wall of the optic tentacle, the capsule of the eye, and the fibrous cords between eye and tentacle. We FIG. 1. Contact between a single axon (A) and a muscle cell (M) in capsule. ASV, agranular synaptic vesicle; CSV, small clear synaptic vesicle; GSV, granular synaptic vesicle, x 23 000. FIG. 2. Dense accumulation of small clear synaptic vesicles (CSV) in an axon (A) making contact with a muscle cell (M) in capsule. AB, axonal bundle, x 23 000. F~G. 3. Axon (A) and glial cell (G) containing electron-dense granules (EDG) in contact with muscle (M) in capsule, x 23 000. FIG. 4. Axon (A) and glial cell (G) in close apposition to muscle cell (M) in collagen0us matrix (CO) of capsule, x 23 000. FIG. 5. Bundle of axons (A) at periphery of capsule. CO, collagen; G, glia; GL, glycogen; GSV, granular synaptic vesicle; N, neurotubule. × 23 000.

o:.

290

MORTENSEN AND EAKIN

interpret these contacts as neuromuscular junctions because of the following features. I n all three places the contacts are characterized by parallel, straight o r curved, plasmalemmas of neurite and muscle cell. The intercellular gap is about 200 A wide and contains lightly stained, finely granular material. The lack of morphological sPecializations of pre- or postjunctional membranes in optic tentacles, penis retractor muscle, rectum, and foot of Helix has been reported by a number of workers (see 8), and it is likely a common characteristic of neuromuscular junctions in this animal. Three distinctive types of vesicles are present in axons in close apposition to the smooth muscle cells of the capsule, tentacle, and fibrous strands. Clusters of small clear vesicles lie adjacent to the prejunctional membrane (see Figs. 1 and 2), and large granular and agranular vesicles are also presentwithin the axoplasm (Figs. 1 and 4). The significance of the different types of vesicles at neuromuscular junctions is uncertain owing to a lack of physiological and pharmacological information. Because of the close relation of the small clear synaptic vesicles to the axonal membrane at neuromuscular junctions it may be assumed that these vesicles are actively involved in neurotransmission. It is presently believed that morphologically different vesicles near neuromuscular junctions in gastropods contain chemically different transmitters Or precursors thereof. Furthermore, it is thought that adrenergic transmitter substances, such as norepinephrine or dopamine, are stored in the dense, granular vesicles. The chemical nature of the transmitter in the axons containing predominantly agranular vesicles is unknown. The reader is referred to Rogers (9) for a further discussion of the possible chemical nature of the transmitter substances in the neural vesicles of mollusks. Functional significance The similar organization of neural, muscular, and connective tissues in tentacular wall, optic capsule, and capsular bridges (8; this paper) indicates their involvement in movements of the optic tentacle and in the accommodation of the eye. Contractions of muscle cells within the tentacle probably produce certain motions of the optic tentacle, such as bending or shifts in position when the tentacle is fully extended; contractions of muscle cells within the capsule may cause localized changes in the curvature of the eye; and contractions of muscle cells in the capsular strands might also implement alterations in the shape of the eye as well as change the position of Fio. 6. TCH prepared specimen. E, eye; ON, optic nerve; TG, tentacular ganglion; TN, tentacular nerve; TW, tentacular wall. x 90. FIG. 7. Higher magnification of area outlined in Fig. 6. Arrow indicates strand of tissue from tentacular wall to capsule of eye: x 180. FIG. 8. TCH prepared specimen. Arrow indicates strand of tissue on surface of capsule. E, eye; ON optic nerve; TG, tentacular ganglion; TW, tentacular wall. x 360. FIG. 9. Thin section through strand at arrow in Fig. 8. AB, axonal bundle, x 12 600.

INNERVATION OF OPTIC CAPSULE OF A SNAIL

291

292

MORTENSEN AND EAKIN

the eye within the optic tentacle. The innervation of the capsule of the eye by motor fibers from the tentacular wall suggests a coordination of the movements of the optic tentacle and the changes in shape and position of the eye. Newell (6) has claimed that the eye of a prosobranch mollusk, Littorina littorea, is able to accommodate for form vision by changing the curvature of its cornea through the action of radially arranged corneal muscles. He records that when "the front surface of the eye is stimulated by touching it with the point of a needle its radius of curvature becomes shorter." And he concludes: "There is good evidence that when in air sharp images of distant objects can be focused on the retina. Moreover, it seems likely that with accommodation near objects could also be brought into focus." With regard to stylommatophora n pulmonates, such as Helix, Newell believes that "eyes borne at the tips of tentacles must be held rigidly in position to maintain a constant orientation of eyes to the body to implement guided movements o f the animal in relation to objects in its environment." Willem (10) claimed that Helix pomatia can preceive small objects at close range (1-2 ram) and vaguely distinguish larger ones up to a centimeter. Similar observations were recorded by Buddenbrock (1) on several species of Helix. Eakin (unpublished) has observed on occasion that H. aspersa avoided a piece of metal held close to one of the optic tentacles. Thus, there are indications of form vision in Helix. It is not known whether accommodation occurs in Helix aspersa. Because the tentacles are often moving about the animal's head, however, some kind of accommodation seems necessary to keep images in focus on the retina. The probable mechanism of accommodation, if present, is change in shape of the eye by the musculature of optic capsule and capsular strands. It is possible that an innervation of capsule and optic tentacle by motor fibers which travel common pathways from the brain could coordinate changes in position and shape of the eyes with movements of the tentacles. It is known (4) that in higher nonsegmented invertebrates, incompatible movements are prevented by mutually inhibitory circuits in the brain, where the motor impulses for these movements are generated, rather than by proprioceptive arcs. The fact that the capsule of the eye and capsular strands are innervated by axons which travel with the neurites innervating the tentacular wall suggests that these motor fibers originate in a similar part of the brain and that they may have mutually excitatory and inhibitory central circuits. The neuromuscular system in the optic capsule and capsular strands, here described, may be involved in the light-dependent pulsations of the eye of Helix aspersa deFIG. 10. Thin section through strand at arrow in Fig. 7. AB, axonal bundle; GC, glial cell. x 12 000. Fro. 11. Strand of tissue on surface of optic capsule. AB, axonai bundle; C, capsule of eye; EDG, electron-dense granule; GC, glial cell; M, muscle. × 9 100. FIG. 12. Higher magnification of neuromuscular junction in Fig. 11. A, axon; M, muscle, x 23 000.

294

MORTENSEN AND EAKIN

scribed by Eakin and Brandenburger (2). It is possible that the m o t o r fibers to the capsular muscles may directly or indirectly synapse with neurites from the photosensory cells of the retina, and thus constitute the efferent segment of a reflex arc. Collaterals of afferent axons in the optic nerve to the capsular muscles, as conjectured by Eakin and Brandenburger, have not been found. Further study is needed to determine the neurological control of the pulsations of the eyes of H. aspersa. This investigation was supported by grant GM 10292 of the USPHS. The authors appreciate the use of the scanning electron microscope in the Electron Microscope Laboratory of the University of California at Berkeley which was provided by NSF grant GB 38359.

REFERENCES 1. BUDDENBROCK,W. v., Zool. Jahrb. 37, 313 (1920). 2. EAKIN, R. M. and BRANDENBURGZR,J. L., in ARCENEAUX,C. J. (Ed.), 30th Ann. Proc. Electron Microsc. Soc. Amer., Baton Rouge, Louisiana, p. 46 (1972). 3. EAKIN, R. M., BRANDENBURG~R,J. L., MORTENSEN,C. and KING, D., 8th lnt. Congr. Electron Microsc., Canberra 1974, in press. 4. HORRIDG~,G. A., Interneurones, p. 101. Freeman, San Francisco, 1968. 5. KELLEY, R. O., DZKKER, R. A. F. and BLUEMINK,J. G., J. Ultrastruc. Res., 45, 254 (1973). 6. NEWELL, G. E., Proc. Zool. Soc. London 144, 75 (1965). 7. NICAISZ,G., lnt. Rev. Cytol. 34, 251 (1973). 8. ROGERS, D. C., Z. Zellforsch. Mikrosk. Anat. 89, 80 (1968). 9. - ibid. 99, 315 (1969). 10. WILLEt~,V., Arch. Biol. ~2, 57 (1892).