Two new kinds of retinal cells in the eye of a snail, Helix aspersa

JOURNAL OF ULTRASTRUCTURE RESEARCH 50, 216-230(1975)

Two New Kinds of Retinal Cells in the Eye of a Snail, Helix aspersa

JEAN L. BRANDENBURGER

Department of Zoology, University of Cali]ornia, Berkeley, California 94720

Received June 21, 1974

Electron microscopy of tentacular eyes of the common garden snail Helix aspersa, reveals four kinds of retinal cells: supportive, sensory type I, sensory type II, and ganglion. The last two are newly discovered and described in this study. Both kinds of receptor cells bear an array of microvilli distally and an axon basally. These elongated cells interdigitate with pigment-bearing supportive cells. Sensory cell type II differs from type I in possessing stubby microvilli, electron-lucent cytoplasm, and clusters of varying sized, cored and clear vesicles. A type I sensory cell has long villi and large aggregations of 800 A photic vesicles. Ganglion cells are found along the periphery of the retina. They are large, ovoid, and few in number. Each possesses a broad axon basally and many smaller fibers which extend laterally among the axonal fibers of the sensory cells. The neural organization is discussed.

After work ing wi th the eyes of Hel ix aspersa, a p u l m o n a t e sna i l , for over 10

years , I have found two new k inds of cells

in its re t ina . Heretofore, the r e t i n a of this

gas t ropod mo l lu sk has been descr ibed as

possess ing only two types of cells: sensory

a n d suppo r t i ve (9, 25, 26). A th i rd cell

type , gang l ion cell, has been iden t i f i ed as a large e l e c t r o n - l u c e n t cell ne s t l ed a m o n g

the sensory a n d s u p p o r t i v e cells in the ou t e r ha l f of the r e t ina . T h e o ther newly

d iscovered r e t i na l cell is a second type of

sensory cell ( type II), which i n t e r m i n g l e s

wi th sensory cells type I a n d suppo r t i ve cells. Th i s s t u d y charac te r izes these two

new k inds of cells. A p r e l i m i n a r y repor t has

b e e n m a d e (3).

MATERIAL AND METHODS

Adult snails, Helix aspersa, used for this study were either specimens freshly collected from Berkeley gar- dens or those living in a terrarium in the laboratory (for procedure, see 9). Eyes were extirpated from the posterior pair of tentacles and fixed in 2% glutaralde- hyde in 0.1 M cacodylate, pH 7.2, for 1-2 hr at room temperature. Specimens were rinsed briefly, postfixed in chilled 1% osmium tetroxide in the same buffer for 1 hr before dehydration and embedment in Epon. One-micron thick sections were stained with methy- lene blue for light microscopy. Silver-gold thin sections were stained with uranyl acetate and lead citrate and viewed with an RCA 3G electron microscope.

Serial thin sections were made from one eye

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through the central part of the retina at the level of the axonal outflow. This series of over 400 sections covered approximately one-fourth of the retina and allowed me to construct a three-dimensional model of one entire ganglion cell and its processes. The outline of the cell, nucleus, and neurites were drawn on 3 mm thick sheets of transparent plastic with different colored marking pens to distinguish different depths. The sheets of plastic were stacked upon each other using Sjhstrand's method of orientation (27). Each sheet represented five to six thin sections. Thinner sheets and drawings were used to depict regions critical for an understanding of important structures that lie close to one another or are narrow or have an irregular form.

RESULTS

Re t ina

T h e c u p - s h a p e d r e t i na of a ga rden snai l ,

Hel ix aspersa, can be d iv ided in to four regions l i s ted from apica l to basa l : (1)

microvi l la r , (2) p i g m e n t e d , (3) nuc lea r , a n d (4) ne u r a l . Each of the four regions

con t a in s pa r t s of the two p r inc ipa l cell types , sensory ( type I) a n d suppor t ive . T h e

mic rov i l l a r zone consis ts of arrays or rhab - domeres of villi , the p r e s u m e d pho tosen- sory a p p a r a t u s , which ex t end from the ap ica l ends of the sensory cells, a n d s t u b b y microvi l l i of suppo r t i ve cells wedged between the r h a b d o m e r e s . T h e second region is cha rac t e r i zed by two fea tures : p i g m e n t g ranu les in the dis ta l halves of the suppor t -

216

TWO NEW CELLS IN EYE OF SNAIL 217

ive cells and interdigitations of longitudi- nal folds of receptoral and supportive cells which partition each cell into many columns. The next region comprises most of the outer half of the retina. It includes the slender segments of the supportive cells with their elliptical nuclei and the bulbous somas of the sensory ceils with their ovoid nuclei surrounded by large aggregates of 800 • photic vesicles, so designated because they are believed to carry the photo- pigment or precursors thereof to the microvilli (11). The last stratum is a narrow band of neural fibers which arise from the basal ends of the receptoral cells and intermingle with tapering footpieces of the pigmented cells. The neurites penetrate the eye capsule and enter a swelling or neuropile at the head of the optic nerve.

In addition to the two retinal cells just described, there are two newly discovered cells: the ganglion cell and a second kind of' sensory cell, designated sensory cell type II.

Ganglion Ceils

General structure. Ganglion cells are distinguished by their electron-lucent cytoplasm, large size, and ovoid shape (Fig. 1). They measure between 11-16 tim in diameter and are found along the periphery of the retina in the nuclear and neural regions. I estimate that there are about 12 ganglion cells per eye as determined by two different procedures: counting these cells in serial sections through one quarter of an eye, and enumerating probable ganglionic axons in cross sections of an optic nerve proximal to the swelling. The nucleus of a ganglion is a large, spherical, centrally situated body measuring 8 ~m in diameter and possessing scattered globular chromatin. An undulat- ing nuclear envelope forms inpocketings (arrows, Figs. 1-2). Polysomes, granules, microtubules, and 45-60 A filaments may be seen near nuclear pores (NP, Fig. 4) in a section grazing one of the nuclear indentations. Most of the large organelles, such as mitochondria and Golgi bodies, are absent

from a lighter zone of cytoplasm or halo immediately surrounding the nucleus (Figs. 1-2).

The most conspicuous components of the soma of a ganglion cell are clusters of large, irregular vesicles, varying from 500 to 1200

in diameter (Fig. 2). Some have dense cores; others are granular; still others are clear. The majority of them are 1000 • with dense cores, with or without a halo. A few spiny or coated vesicles are found (arrows, Fig. 5). In contrast, the 800 ]k clear photic vesicles, which occur in sensory cells of type I, are remarkably uniform in size and shape (Fig. 3). The origin of the cored vesicles is by dehiscence from the electron- dense Golgi cisternae (Fig. 5). Golgi centers are numerous; 13 were counted in a repre- sentative section through one ganglion cell. Other cytoplasmic organelles and inclusions are: mitochondria, rough and smooth endoplasmic reticulum, lysosomes, mul- tivesicular bodies, microtubules, ribosomes, and clusters of small 300 A granules. The last named elements (GR, Fig. 4) seem to be beta granules of glycogen based upon their size, morphology and characteristic weak staining with lead.

Neurites. Neural fibers of the ganglion cells are of two types: a single broad axon (Fig. 1) and numerous slender neurites (Fig. 6-11). The plastic model which I constructed of one ganglion cell was most helpful in understanding the relationships between neural processes. All neurites are connected to the cell soma at its basal end. Each ganglion cell is like an octopus with one broad arm (axon) passing into the optic nerve and several smaller arms (dendrites) extending into the neuropile of the retina to mingle with receptor cell neurites. I have classified the dendrites into two kinds: (1) long, branching fibers; (2) short, undivided spines.

Dendrites. Six to eight long dendritic processes are associated with a ganglion cell. Each measures 5-15 tim in length and 0.5 to 1 tim in diameter. Two different fibers from the same cell are shown in Figs.

218 JEAN L. BRANDENBURGER

FIG. 1. Ganglion cell (GC) in retina of H. aspersa with large nucleus (N) and broad axon (A). CA, capsule; L, lysosomes; PC, pigmented cells; SC I, sensory cell type I; V, vesicles. Arrows, indentations of nuclear envelope.

x 9 600.


Fro. 2. Part of ganglion cell (GC) with clusters of cored vesicles (DV). ER, endoplasmic reticulum; G, Golgi cisternae; L, lysosomes; M, mitochondria; N, nucleus. Arrows, indentations of nuclear envelope. × 22 000.

Fla. 3. Paracrystalline array of photic vesicles in sensory cell type I. × 28 000. Fro. 4. Cytoplasmic region of ganglion cell grazing one of the nuclear indentations with nuclear pores (NP). F,

filaments; GR, glycogen granules; MT, microtubule; P, polysomes; R, ribosomes. × 27 000. FIG. 5. Golgi cisternae (G) filled with electron-dense material in ganglion cell. CV, clear vesicle; DV,

dense-cored vesicles; ER, endoplasmic reticulum; M, mitochondrion. Arrows, spiny vesicles. × 19 000.

6 a n d 9. O n e (in F ig . 6) l eaves b a s a l l y f rom t h e cel l (GC) by a n a r r o w n e c k a n d d o u b l e s b a c k u p o n i tse l f ; t h e o t h e r (in Fig . 9) has a b r o a d base w h i c h b r a n c h e s in to t h r e e f ibers

(DF) . T h e s e n e u r i t e s c o n t a i n m i t o c h o n - d r i a , p o l y s o m e s , ER, m i c r o t u b u l e s , g lyco- gen g ranu le s , a n d cored a n d c lea r ves ic les . A dense s h e a t h c o m p o s e d of l a m e l l a e of


FIG. 6. Basal region of a ganglion cell (GC) with dendritic fiber (DF, partially outlined in ink). ER, endoplasmic reticulum; G, Golgi cisternae; M, mitochondria; MT, microtubules; P, polysome; PC, pigmented cells; PS, presynaptic fiber; V, synaptic vesicles, x 14 000.

FIG. 7. Enlargement of rectangle in Fig. 6. DF, dendritic fiber; PS, presynaptic fiber with synaptic junction (arrow). × 32 000.

FIG. 8. Ganglionic dendrite (DF) with two branches (B1, B2). ER, endoplasmic reticulum; PS, presynaptic fiber; V, vesicles. Arrows, thickened plasmalemma. × 27 000.

supportive cells (S, Fig. 9) encases a ganglionic cell body, but the encapsulation is broken by the dendrites. Most of the latter are naked and lie adjacent to neurites of sensory cells. Each dendrite terminates within the neural region of the retina. For example, Fig. 8 illustrates a dendritic stalk which divides into two branches

(B1 and B2). Along one process (B1) are short stretches of thickened plasmalemma (arrows, Fig. 8) with dense material on their cytoplasmic side and neural vesicles nearby. The regions of the neurite are believed to be synapses because they re- semble synaptic junctions elsewhere in H. aspersa, as in the swelling (neuropile)

FIO. 9. Basal half of ganglion cell (GC) with three dendrites (DF). L, lysosome; M, mitochondria; N, nucleus; S, dense pigmented cell sheath around ganglion cell; V, vesicles. × 21 000.

FIG. 10. Axon of ganglion cell (GC) before penetrating the capsule. A, axons of sensory cells; GR, glycogen granules; M, mitochondria; MT, microtubules; S, sheath of pigmented cell; SP, spines on axon. × 23 000.

FIG. 11. Enlargement of spine (SP) in Fig. 10 showing synaptic region (arrow). × 27 000.

221


of the optic nerve (10). Another synaptic region is shown in Fig. 6 (see rectangle) and in an adjacent section (Fig. 7). Neural Vesicles in a neighboring sensory cell fiber (PS, presynaptic) lie near the cell membrane opposing the dendritic plasmalemma (arrow).

Spines differ from the above neurites. (1) They do not branch, and (2) they extend only a few micrometers. They are found attached to the cell soma, but they can also occur on the axon (SP, Figs. 10-11). Most spines measure less than 2 #m in length and about 0.5 tim in diameter, and they are often hooked or irregularly shaped (SP, Fig. 10). Their cytoplasm contains the same organelles and inclusions found in the longer dendrites. Like the latter, the spines are in close proximity to nearby sensory fibers. A possible synaptic junction (arrow, Fig. 11) finds cored and clear vesicles near the cell membrane of the fiber adjacent to the spine (SP).

Axon. As the single broad axon leaves from the basal end of the cell's axis, it is ensheathed by electron-dense extensions of pigmented cells (S, Fig. 10) until it reaches the capsule and enters the optic nerve (Fig. 1). The axon within the retina measures over 3 #m in diameter, and it has cytoplasmic structures similar to those in the cell soma (Fig. 10). There is a decrease in the number and kind of organelles as the axon tapers and departs from the eye. It appears nearly empty in the optic nerve except for neurotubules and a few vesicles (Fig. 12). Within the swelling of the optic nerve, the axon is easily identified by its large size 1.5-2.0 #m in diameter and relatively few organelles (A, Fig. 13). In contrast, neighboring neural fibers from the sensory cells are less than 0.5 #m in diameter, and they have many vesicles and mitochondria. Other components of the nerve are glial cells which are characterized by bundles of tonofilaments, glycogen granules, desmo- somes, and lobate nuclei (Fig. 13). They are often associated with the large axons of ganglion cells.

Sensory Cell II

The slender, elongate type II sensory cell is found intermingled with type I receptoral cells and supportive cells. In any given cross section through an eye, there may be only one or two cells of type II compared to 20-30 of type I. Both kinds of sensory cells have the same general size and shape, but SC II have several unique characteristics. First, short, irregularly dis- posed microvilli extend from its apical surface (Fig. 14). They are 3-4 #m in length, more than 0.1 #m in diameter, and often twisted (Fig. 15). By comparison, SC I have long (10 ~m), slender (less than 0.1 #m in diameter), and regularly arranged villi, which touch the undersurface of the lens. Intervillous spaces are numerous around the type II villi and filled with a granular humor (H, Fig, 14). Cilia are scarce in retinal cells of H. aspersa. Short cilia are rarely observed in sensory cells and only occasionally in pigmented cells. The 4 #m long cilium in F ig . 14 is the longest one that I have seen in any sensory cell of H. aspersa. The ciliary membrane is clearly independent of neighboring villi which arise directly from the plasma mem- brahe of the cell.

The shaft of a SC II cell is concave at its apex, and its electron-lucent cytoplasm has a scattering of mitochondria, vesicles and microtubules. On the other hand, the dense, dome-shaped tip of type I is rich in mitochondria, ER, vesicles, and other organelles (SC I, Fig. 14). In the pigmented zone of the retina, a SC II is columnar, and it varies from 1 to 2 mm in diameter. By contrast, a SC I consist of many narrow columns formed by intrusions or folds of supportive cells (arrows, Fig. 14).

The chief feature of the basal half of a type II cell (Fig. 16) is the complete absence of a massive aggregation of photic vesicles, so typical of a sensory cell type I. The broad column of cytoplasm of the former has numerous Golgi bodies, ER, ribosomes, glycogen granules, mitochon-


FIG. 12. Part of large ganglionic axon (A) in optic nerve flanked by smaller sensory cell axons (SC). MT, microtubules; V, vesicles. × 29 000.

Fla. 13. Segment of neural swelling or neuropile of optic nerve with large axon (A) of ganglion cell and smaller fibers filled with neural vesicles (V) and mitochondria (M). D, desmosome; GLN, glial cell nucleus; LP, liposomes; TF, tonofilaments. × 12 000.

d r i a , a n d l y sosomes . S o m e Golgi c i s t e r n a e a re f i l l ed w i t h an e l e c t r o n - d e n s e m a t e r i a l (F ig . 17). In t he v i c i n i t y of Golgi b o d i e s a re d e n s e - c o r e d , l i g h t l y g r a n u l a r , a n d c lea r ves ic les . T h e l a t t e r d i f fer f rom p h o t i c ves i -

cles of SC I in b e i n g i r r e g u l a r in s h a p e a n d v a r y i n g in s ize f rom 500 to 1200 • in d i a m e t e r . A l t h o u g h cored ves ic les occur in SC I, t h e y a re m o r e c o m m o n in SC II. T h e two k i n d s of sensory cel ls can no t be


Fro. 14. Distal ends of sensory cell type II (SC II), sensory cell type I (SC I), and parts of several pigmented cells (PC). C, cilium; H, humor; M, mitochondria; MV~, microvilli of SC I; MVH, microvilli of SC II; PG, pigment granules. Arrows, columns of SC I. × 7000.

Fro. 15. Enlargement of microvilli (MV) of sensory cell II (SC II). H, humor, x 25 000.

d i s t i n g u i s h e d by the i r nuc le i which are of s im i l a r size, shape, a n d c h r o m a t i n dis t r i -

b u t i o n . Neu r i t e s of SC II show cons ide rab le

v a r i a t i o n in size a nd c o n t e n t a long the i r pa thway . In a favorab le sect ion a s ingle axon (ou t l ined in ink, Fig. 18) m a y be seen l eav ing ba sa l l y from the cell soma. I t


16i

Fi(x 16. Nuclear region of sensory cell II (SC II). ER, endoplasmic reticulum; G, Golgi cisternae; L, lysosome; M, mitochondria; N, nucleus; PC, pigmented cells; V, vesicles, clear and cored, x 18 000.

FIG. 17. Golgi cisternae (G) in sensory cell type II (SC II). CV, clear vesicles; DV, dense-cored vesicles; GV, lightly granular vesicles; M, mitochondrion; R, ribosomes, x 30 000.

m e a s u r e s f rom 0.5 to over 1 t im in d i a m e t e r a long i ts 20-30 # m l eng th . P a r t of th i s axon a t h ighe r m a g n i f i c a t i o n (Fig. 19) c o n t a i n s

ER, m i t o c h o n d r i a , m i c r o t u b u l e s , g ranu les , a n d c l ea r a n d cored ves ic les . W i t h i n t he s p i n e (SP, Fig. 19) a re n e u r a l vesic les , one


Fro. 18. Axonal outflow (A) of sensory cell type II (SC II, outlined in ink). N, nucleus; PV, photic vesicles in sensory cell type I. × 7500.

Fins. 19 20. Enlargements of two regions of axon (A) in Fig. 18. GR, glycogen granules; MT, microtubules; PC, pigmented cells; TF, tonofilaments; V, vesicles. × 26 000.

Fla. 21. Parts of several neurites in the neural region of the retina, some filled with cored vesicles (DV), others with clear vesicles (CV). CA, intrusions of capsular matrix deep within neural layer of eye; FP, footpieces of pigmented cells. × 19 000.


of which is very close to the axolemma. In another part of the same fiber (Fig. 20), there are clusters of clear vesicles and a few cored ones. Bordering this segment of the axon on the reader's left is a part of a supportive cell (PC) with a bundle of tonofilaments and glycogen granules. Other neurites have accumulations of cored vesicles (Fig. 21), but these fibers could not be associated with either type of sensory cell. I could not detect any differ- ence between the axons of the two kinds of sensory cells. Many projections of capsular matrix extend deeply into the neural region of the retina (CA, Fig. 21); they may serve as vascular channels.

A rough estimate of sensory cell type I was made in a previous study (12), in which counts of nerve fibers from cross sections of two different optic nerves to- taled 2500 and 3800. If type II is approximately 3-5% of type I, there would be 75-200 of the former. These results and other features of the four types of retinal cells are summarized in Table I.

DISCUSSION

Ganglion Cells

The idea of an optic ganglion in gastropod eyes was first introduced by Leydig in

1857 (23) and later Henchman (17) described a ganglion in Limax maximus as "a funnel-shaped enlargement of the optic nerve containing oval nuclei." Smith in 1906 (28) concluded that these findings were misinterpretations of connective tis- sue nuclei which did not stain with methy- lene blue whereas the neurites did. In 1920 Eisenmann (13) illustrated ganglion cells in the eyes of two stylommatophoran pulmonates (Helix and Arion) as scattered cells along the optic nerve, outside the retina.

Eakin, Brandenburger, and Westree (12) studied the forementioned enlarged area of the optic nerve which we designated a neural swelling or neuropile. We believed that the nuclei present in this structure belong to glial cells because the surrounding cytoplasm contains tonofilaments and glycogen granules, characteristic of supportive cells. They lack common neural structures such as microtubules and vesicles. The synapses within the swelling are con- sidered en passant junctions between axons of sensory cells. In an experiment on H. aspersa, optic nerves were severed below the swellings. The proximal segments (closest to the brain) showed signs of de- generation within 48 hr. We concluded

TABLE I

COMPARISON OF CELL TYPES IN RETINA OF Helix aspersa

Cell types Shape Microvilli Vesicles Nucleus Axon Cytoplasmic Estimated features number

Supportive pigmented none

Sensory, type I

Sensory, type II

Ganglion

elongate multiple columns (see text)

elongate, convex apex, multiple columns

elongate, concave apex, single column

ovoid

Short, s tubby

long, slender, extend to lens

short, irregular

none

uniform, 800 ~ photic, some cored

500-1200 ~, irregular, c lear&cored

cored, some clear

ellipical, basal

ovoid, basal

ovoid, basal

spherical, central

slender, clear and cored vesicles

slender, clear and cored vesicles

broad, few vesicles,

dense, tonofilaments glycogen granules

dense

electron- lucent

electron- lucent

2500- 3800

about 75-200

about 12


that the fibers in the optic swelling and nerve originate in the retina (10). Newell (24) claims, however, that there are second-order neurons behind the eye of a pro- sobranch gastropod, Littorina littorea. Each so-called bipolar neuron is believed to synapse with one visual cell. An ultrastruc- tural study is needed to confirm this re- port.

The large ganglion cells described in this study were found within the retina, not outside in the optic nerve as discussed above. No large cells have been observed heretofore in the retina of Helix by light and electron microscopists (9, 25, 26). Ac- cording to Bullock and Horridge (5), ganglion cells in the brain of Helix range from medium to giant size (20 to over 200 tLm in diameter) with a nucleus about two-thirds the size of the cellular diameter. Each possesses a stout axon (over I #m in diameter) which is most often surrounded by several layers of neuroglial processes. The large electron-lucent cells in the retina of H. aspersa here described have these characteristics.

Another general feature of ganglion cells is synapses along their dendrites. These cell junctions are poorly developed as is true of synapses of Helix in general (6, 10, 14, 16). Often it is difficult to distinguish synaptic junctions and their polarity because cored and clear vesicles occur on both sides of the synapse. Moreover, synaptic membranes vary in thickness and density. These synapses could provide the anatomical basis for interactions between many photoreceptoral cells and a ganglion cell allowing summation of excitations. This possible function needs to be sup- ported by electrophysiological studies like those on a nudibranch, Hermissenda crass- icornis, by Dennis (7) and those on a sea hare, Aplysia californica, by Jacklet (19).

The large size of the ganglionic axon would facilitate rapid conduction of impulses to the brain. The axons of photosen- sory cells in the optic nerve being smaller, and generally having no individual wrap-

ping of glial elements, might be slower conductants.

Receptor Cells

The presence of two types of sensory cells as in H. aspersa is not unique among molluscan eyes. The eye spots of a scallop, Pecten maximus, possess two-layered retinas: a distal one whose cells have flattened cilia (9 + 0 axoneme) which stand on edge in a row, and proximal layer whose cells have short irregular microvilli (1). Moreover, in the single-layered retinas of the elliptical eyes of a sea hare, Aplysia punctata, Hughes (18) reports two types of receptor cells: one with an equal number of unmodified cilia (9 + 2 axoneme) and irregular microvilli (usually 15), and another which has a large tuft of long microvilli with an occasional cilium among them. Both kinds of cells in A. punctata have a similar shape and the same organelles, including large accumulations of clear spherical vesicles.

Land (22) discusses the above mentioned two kinds of receptors (ciliary and microvillar) of gastropod and bivalve mol- lusks in relation to neural responses and behavioral movements. Electrical recordings from a single fiber of the proximal layer of the eye of Pecten indicated excita- tory, depolarizing responses to light (1). These microvillar receptors are said to give "on" responses which are associated with orientation movements. An animal either moves toward or more commonly away from regions of high light intensity. Bud- denbrock (4) concluded that a change in orientation in Helix pomatia was depend- ent on a light-sensitivity of the eyes. If he was correct, sensory cell type I in H. aspersa with its distal array of long villi might belong to the "on" response type of sensory cell. The reader is referred to Eakin (8) for further discussion of light sensitivity of microvilli.

Electrophysiological studies have shown that the distal ciliary cells in the eyes of Pecten (1), the ciliary cells in siphonal eyes


of a cockle, Cardium edule (2), and the ciliary cells in the dorsal eyes of a gastropod, Onchidium verruculatum, (29) give a distinct "off" response to a decrease in illumination or to a shadow. The unmodified ciliated cells in A. punctata may also produce a similar reaction as suggested by Hughes (18). FSh (15) found Helix pomatia to withdraw its body into the shell with a sudden drop in light intensity, but he thought that the eyes were completely insensitive to shadow. A region of the mantle just in front of the shell was believed to be the most sensitive area to shadow. It would be useful to examine this region for receptor cells.

Is sensory cell type II in H. aspersa photosensitive? Cilia are rare in this type. Although an extensive search was not made of each SC II cell to determine the presence or absence of cilia, enough sections were examined to state that SC II does not possess numerous cilia, one per cell at the most. Therefore, it should not be called a ciliated cell as are those in Pecten, Cardium, or Aplysia. The few cilia present in the eye of H. aspersa probably play little or no role in photoreception. Both types of sensory cells in this pulmonate are microvillar. Sensory cell I, with long regularly arranged villi and large masses of photic vesicles, probably plays the dominant role in photoreception, responding to an in- crease in light. The function of the other sensory cell (II), with short irregular villi and clusters of cored vesicles, may be in- volved in a shadow response or other func- tions mentioned below.

Jacklet (19) has found the optic nerve of isolated eyes of Aplysia californica to produce spontaneous neural impulses in the dark which have a circadian rhythm. This nervous activity has been associated with a third type of retinal cell in Aplysia called the secondary cell (20, 21). The latter numbers 950 and differs from the 3700 receptor cells by its location in the retina, size of nucleus, and staining properties in the light microscope (21). No study of the

fine structure of secondary cells has been made. SC II in H. aspersa may respond in a way similar to that proposed for the secondary cells of A. californica. Another possible function for the SC II may be one of circulating within the optic lumen vari- ous nutrients or breakdown products from photoexcitation.

Note added in proof. Recently, C. J. Stoll investi- gated the fine structure and neural responses of the eyes of a fresh water snail, Lymnaea stagnalis (Proc. Kon. Ned. Akad. Wetensch. Ser. C, 76,406 and 414, 1973). Although I knew of Stoll's articles, they were not available when this paper was written. He described so-called secondary neurons in the retina of this snail, in addition to sensory and supportive cells. The nuclei of the secondary neurones were said to lie in a ganglionlike region of the retina together with axons of sensory cells. No synapses were found. In the second study Stoll reported that recordings from the optic nerve revealed "on" responses, as in Pecten (see my discussion), when an eye of Lymnaea was illuminated.

I am grateful to Dr. Richard M. Eakin for his stimulation and counsel during this investigation, for his critical reading of the manuscript, and for support from his grant-in-aid from the U.S. Public Health Service (GM 10292).

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