Cell Tiss. Res. 180, 529-536 (1977) Cell and Tissue Research �9 by Springer-Verlag 1977

Regeneration in the Central Nervous System of a Pulmonate Mollusc, Melampus

Christopher H. Price*

Department of Biology, Syracuse University, Syracuse, New York, and the Marine Biological Laboratory, Woods Hole, Massachusetts, USA

Summary. The left cerebral ganglion was ablated f rom 72 anesthetized, adult Melampus bidentatus (Mollusca: Pulmonata). Skin incisions were well healed and normal feeding and locomotion observed four days after surgery. Dissec- tions of animals sacrificed weekly showed that most nerves and connectives regrew within 30 days, attaching to the swollen end of the major labial nerve. The enlarged end of this nerve later developed into a distinctive bud; some of these buds contained cell bodies as soon as 42 days after surgery. As the first known report of central nervous tissue regeneration in molluscs, this study points to the need for controls in experiments involving section or ablation of nervous tissue in molluscs.

Key words: R e g e n e r a t i o n - CNS - Ganglia - Mollusca.

Introduction

The rapid regeneration of tentacles and eyes in molluscs has been known and studied for decades (Nonne, 1925; Ch6tail, 1963; Eakin and Ferlatte, 1973; Gillary, 1974; Hughes, 1976). However, few details are available on the extent of regrowth of associated nerves and peripheral nerve centers. Furthermore, there are no reports of central nervous system tissue regeneration in molluscs (Bullock and Horridge, 1965; Hyman, 1967), despite considerable literature on central and peripheral nervous tissue regeneration in the annelid and ar thropod phyla (Herlant-Meewis, 1961; Guthrie, 1973; Edwards and Palka, 1976).

Send offprint requests to: Dr. Christopher H. Price, Ph.D., University of Texas Medical Branch, Marine Biomedical Institute, 200 University Blvd., Galveston, Tx 77550, USA

* I am grateful to Dr. W.D. Russell-Hunter for his guidance in the course of this work. Support was principally provided by a grant from the National Science Foundation to Dr. Russell-Hunter (Research Grant No. GB-36757 continued as BMS-72-02511-A01)and by two successive grants to the author from the Theodore Roosevelt Memorial Fund of the American Museum of Natural History, New York

530 C.H. Price

In the course of studies on a pu lmona t e mollusc, Melampus bidentatus (Price, 1976), rapid regenerat ion of tentacles and associated nerves from the cerebral ganglia was observed. This suggested that more extensive central nervous tissue regrowth might also occur in Melampus. Because of the impor tance o f p u l m o n a t e s and op is thobranchs in neurophysiological and behavioral research (Kandel , 1976), a s tudy of central gangl ion and nerve regenerat ion was under taken in Melampus that demonst ra tes a remarkable degree of regeneration.

Materials and Methods

Experiments were performed on freshly collected adults (shell lengths 9.5-11.0 ram) maintained in finger bowls in an environmental chamber (22 - 1 ~ C; 15 h light : 9 h dark). Snails were provided with a piece of marsh turf and fed dried lettuce to excess. Anesthesia was induced by sequentially treating animals in 75~ seawater solutions (20-25 min in each solution) of 0.05~ Nembutal (sodium pento- barbital), 0.1~ Nembutal, and 0.1% Nembutal plus 0.2~ MS222 (tricaine methanesulfonate). Fully relaxed snails were secured in a trough of soft wax (in 75% seawater) under a dissecting micro- scope.

In the study of tentacle regeneration, this structure was ablated at the base, leaving underlying tissue intact. In a few cases, once the tentacle was removed, the eye was excised by pulling it away from the tentacle base and cutting the optic nerve.

For the experiments on CNS regeneration, access to the central ganglia and nerves was through a small transverse slit ( < 2 mm) made in the skin on the dorsal side, just posterior to the tentacles. The central nervous system of Melampus (Price, 1977; Fig. 1 A) consists of 11 distinct ganglia joined by connectives that can be stretched about 30~ without breaking. Thus, by inserting an insect pin through the slit and under the cerebral commissure, the paired cerebral ganglia could be pulled out of the body and selected portions of the anterior brain ablated. Removal of a single cerebral ganglion, for example, involved the severance (200-400 ~tm from the ganglion) of all 10 connectives and nerves (Fig. 1 A). Ablated tissue was removed and the remainder of the CNS pushed back into the hemocoel. Stitching was unnecessary and no antibiotics were used. Snails recovered in a large volume of 75% seawater. After showing spontaneous locomotion (in about 1 h), they were placed in standard main- tenance bowls.

Postoperative animals were fixed weekly in Bouin's solution (Pantin, 1964) and partially dissected to expose the CNS. Drawings and photomicrographs were made of normal and regenerated tissue which was then prepared for histological examination. Properly oriented, wax-embedded material was sectioned at 7 ~tm and stained with standard Mayer's hematoxylin and eosin (National Cancer Institute, 1972).

Results

After ext i rpat ion of tentacles, tissue regenerat ion was rapid. Wi th in one week after surgery, cone-shaped, unp igmented buds 0 . 3 - 0 . 4 m m long had formed; at three weeks, tentacles were nea r -normal length (1 .5 -2 .50mm) and pigmented. F u n c t i o n a l tentacle retract ion was observed by the end of the second week in mos t snails, suggesting that some nerve and muscle regrowth had occurred. In a few cases a b n o r m a l regrowth resulted in double (in 2 of 60 tentacle ablat ions) and bifurcated (3/60) single tentacles. In 4 of 5 cases in which an eye was removed with the tentacle, the eye regenerated within 6 weeks and was topologically in- dis t inguishable f rom the no rma l eye.

Three experiments on CNS regenerat ion were carried out. In the first, the cerebral commissure (CC) was removed from 14 snails by cuts at each ganglion,

CNS Regeneration in Melampus (Mollusca)

CA) (B) 14 DAYS

(C) 28 DAYS

cc

531

(D) 70 DAYS

Fig. 1A-D. Diagram of CNS of Melampus showing regrowth of ablated tissue. Dashed lines: cuts made to remove cerebral commissure (CC). Wavy lines: severance of major nerves to remove left cerebral ganglion (LCG; shaded area). B-D Examples of regrowth following ablation of LCG. Drawings made from relaxed, fixed, and partially dissected animals sampled at 14, 28, and 70 days after surgery. End of major labial nerve (L1) is focal area for regrowth until recognizable "bud" is formed. Note that CC shortens and thickens with time. CPC and CPLC connectives fuse near their origins at pedal and pleural ganglia and then send a single connective anteriorly to bud area. BG buccal ganglion; CBC cerebral-buccal connective; CPC cerebral-pedal connective; CPLC cerebral-pleural connective; L1, L 2 and L 3 labial nerves; LPAG left parietal ganglion; O optic n.; PT peritentacular n.; RCG right cerebral ganglion; RPG right pedal ganglion; RPLG right pleural ganglion; Ttentacular n.; VG visceral ganglion

as shown by dashed lines on F igure 1 A. The commissu re regrew and r econnec ted b o t h gangl ia , usual ly wi th in 16 days. Ini t ia l ly , the C C was 2 to 3 t imes longer t han normal , bu t it t h i ckened a n d became shor te r du r ing the next few weeks. There was no m o r t a l i t y in this group.

The second exper iment involved the r emova l o f b o t h cerebra l ganglia . O f the an imals used in this s tudy, 6 0 ~ (6/10) died wi thin 3 days; the o thers l ived for up to 30 days. N o regenera t ion o f nervous t issue was obse rved in these animals .

532 C.H. Price

Table 1. Regeneration of nervous tissue after ablation of the left cerebral ganglion from 69 snails (10-11 mm shell length). The postoperative time (indicated by a + ) at which each regenerative event occurred is the median for 4-6 snails sampled at random per week. The large labial nerve (L1; see Fig. 1) was the focal area for regrowth; most nerves and connectives reattached near the swollen end of L I . See Figure 1 for abbreviations and text for details of the regenerative process

Event Days post-surgery

2 4 7 14 21 28 42 70

Activity Locomotion Feeding Feces produced Egg laying (first noted)

Wound healing Incision plugged (with amebocytes) Incision healed Skin repigmented

Regeneration L2

L3 CC CPC-CPLC fusion

Fusion reconnects to bud

T O CBC "Bud" size

(as ~ normal ganglion size)

+ +

+" +d

lO%

+

@d

+

..[.. b _[_c

+ +

15-20%

a cerebral commissure (CC) 3 times normal length b CC 2X normal length c CC 1.5X normal length d CPC and CPLC fuse first and then send one connective to bud

The third experiment dealt with the ablation of the left cerebral ganglion (left cerebral extirpation, LCE) in 72 snails. (Preliminary work showed that extirpation of the right cerebral ganglion resulted in a similar sequence of events.) A summary of the temporal progression of events during recovery, healing, and regrowth is presented in Table 1 and Figure 1 B-D. Mortality was less than 5 ~ (3/72) during 10 weeks, a rate approximating that found in untreated laboratory stocks. Normal locomotion and feeding were observed in all LCE snails within 5 days after surgery. Aside from a tendency to make more turns to the right than to the left (only during the first 2-3 weeks after surgery), these animals showed no obvious behavioral distinctions between LCE and normal snails. The LCE snails produced egg masses as soon as 28 days after surgery.

The severed end of the largest labial nerve (L 1, Fig. 1) seems to serve as the focal area of regrowth and reconnection with other nerves and connectives. Snails examined five days after the LCE operation had L l nerves with swollen ends. The nerves that regenerated most quickly (usually labial nerve 2 and the cerebral

CNS Regeneration in Melampus (Mollusca) 533

Fig. 2. Photomicrograph of 7 pm section of regenerated tissue (stained with hematoxylin and eosin) from snail sacrificed 28 days after ablation of left cerebral ganglion. Note bundles of axons covered with darkly stained connective tissue; "bud" area (b) beginning to form. Single connective P/PL resulting from fusion of regrowing pedal and pleural connectives. For abbreviations, see Figure 1. Scale bar 50 gm

commissure) typically grew into this "bud" area. Labial nerve 1 in Melampus is probably homologous to the "medial labial nerve" in the freshwater pulmonate Lymnaea; the sheath surrounding this nerve in Lymnaea has been identified as a probable neurohemal area (Joosse, 1964). The presence of such an area in L 1 of Melampus may relate to its role as the focal area for regeneration.

The cerebral commissure grows laterally f rom the right cerebral ganglion and after two weeks has rejoined the L 1 region on the left side of the animal. At this t ime the commissure is about three times its normal length. As noted earlier, however, it shortens and thickens (Fig. 1 B-D) so that by the sixth week the CC is usually near-normal in length (0.3mm). The cerebral-pleural (CPLC) and cerebral-pedal (CPC) connectives fuse into a single connective, which then grows forward to join the L 1 bud area. In three snails, a thin nerve was found to originate near the area of the CPLC-CPC fusion (Fig. 1 C); the distal termination of this nerve was not found. Optic nerves and cerebral-buccal connectives were slow to regrow and were identified with certainty in only four animals (at 70 days).

While some regeneration of nervous tissue was found in all LCE snails, the rate and extent of regrowth were highly variable among animals, especially with respect to the bud area at the end of nerve L I . In some snails no true bud appeared, and the area remained a simple swelling long after other regrown nerves and

534 C.H. Price

connectives had attached. In other LCE snails, the bud increased in size, up to 20~ (at 70 days) of normal cerebral ganglion dimensions, and became an increas- ingly distinct structure. Several of these bud areas were examined histologically. In the earlier stage (swollen ending), the tissue was largely composed of bundles of nerve fibers surrounded by connective tissue (Fig. 2). No nuclei or cell bodies were visible and most of the internal area resembled the neuropile of a normal ganglion. In two well-developed buds observed at later stages (one at 42 and one at 70 days post-surgery), the neuropile-like area was enlarged and bordered in places by groups of small (< 7 Ixm diameter) cell bodies. Some nuclei were scattered about the neuropile. Neither the origin nor the function of the grouped or scattered cells is known.

Discussion

This study demonstrates, for the first time, the regeneration of parts of the central nervous system (CNS) in a mollusc. Regeneration of peripheral nervous tissue in invertebrates has been investigated as a part of the regrowth of injured, autotomiz- ed, or experimentally ablated appendages (e.g., insects, Guthrie, 1973; Edwards and Palka, 1976; crustaceans, Holland and Skinner, 1976; Weis, 1976). Nervous centers and neuroendocrine organs of some crustaceans can apparently regenerate, such as the eyestalk ganglion and sinus gland (von Bruddenbrock, 1954; Vernet, 1969). Annelids, on the other hand, can regenerate entire body segments and associated CNS ganglia (Herlant-Meewis, 1961). Intraganglionic structure is faithfully reconstituted, including the differentiation of specialized neurosecretory cells which are essential to later stages of complete segment regeneration (Clark et al., 1962; Herlant-Meewis, 1962). Longer-term studies on molluscs may reveal similar capacities.

There is considerable interest in the regeneration and plasticity of CNS tissue, in both clinical (Stein et al., 1974; Guth, 1975) and comparative neurobiological (Guthrie, 1973) research. Findings from regenerative processes in the CNS of invertebrates are not usually applicable to vertebrates; some of the fundamental mechanisms, however, may well be shared (Edwards and Palka, 1976). There is no longer any doubt about regeneration in the CNS of mammals (i.e., axonal regrowth and collateral sprouting; e.g., Burnstock and Costa, 1975; Svengaard et al., 1976). As pointed out by Goldstein (1976), the primary problem is identifying conditions that promote such regeneration. Studies on the conditions attending extensive invertebrate regeneration may therefore be useful in designing ex- periments for maximizing regeneration in higher animals.

Among the many questions under study is the mechanism by which specific connections are established between neurons in both developing and regenerating neural tissue. A major problem in this area is the need to identify individual cells, a difficult task in vertebrates. The arthropod CNS has a relatively small number of cells and can be at least partially mapped (Cohen and Jacklet, 1967). Studies on the regeneration of nervous tissue in cockroaches and other insects (Guthrie, 1973; Edwards and Palka, 1976) have contributed much to the current under- standing of neurogenesis and the formation of neural connections.

CNS Regeneration in Melampus (Mollusca) 535

The CNS of pulmonate and opisthobranch molluscs is also of relatively simple and consistent organization, contains identifiable and giant neurons (e.g., Frazier et al., 1967), and can be maintained in vitro for considerable periods (Dewhurst and Weinreich, 1974). The rapidity of reconnection of commissures and nerves (and probable reestablishment of functional electrophysiological pathways) de- monstrated in the present work on Melampus is likely to exist in other snails. This clearly indicates that controls should be used in experiments involving the section or ablation of molluscan nervous tissue. At the same time, such CNS regeneration in molluscs seems amenable to studies on neural development and function, as well as on the electrophysiology and neuroanatomy of regeneration.

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behavior (J.C. Fentress, ed.), pp. 167-185. Sunderland-Massachusetts: Sinauer Associates 1976 Frazier, W.T., Kandel, E.R., Kupfermann, I., Waziri, R., Coggeshall, R.E.: Morphological and

functional properties of identified neurons in the abdominal ganglion of Aplysia caliJbrnica. J. Neurophysiol. 30, 1288-1351 (1967)

Gillary, H.L.: The regenerating eye of Strombus: anatomy and electrophysiology. Amer. Zool. 12, 251 (1972)

Goldstein, M.: The current status of research on growth and regeneration in the central nervous system. Surg. Neurol. 5, 157-160 (1976)

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Holland, C., Skinner, D.M. : Interactions between molting and regeneration in the land crab. Biol. Bull. 150, 222-240 (1976)

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536 C.H. Price

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relations of Melampus bidentatus Say (Mollusca: Pulmonata). Ph.D. Thesis, Syracuse University, 171 pp. (1976)

Price, C.H. : Morphology and histology of the central nervous system and neurosecretory cells in Melampus bidentatus Say (Gastropoda: Pulmonata). Trans. Amer. micr. Soc. 96, (1977) (in press)

Stein, D.C., Rosen, J.J., Butters, N., eds.: Plasticity and recovery of function in the central nervous system. New York: Academic Press 1974

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Vernet, G.: R6g6n6ration de cellules neuros6cr&rices et de la glande du sinus apr~s ablation partielle du p6doncle oculaire chez Pachygrapsus marmoratus (Fabricius). Ann. Endocr. (Paris) 30, 261-266 (1969)

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Accepted February 1, 1977