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AMER. ZOOL., 28:1109-1122 (1988) Structural Repair and Functional Recovery Following Cerebral Ganglion Removal in the Pulmonate Snail Melampus1 STACIA B. MOFFETT AND RICHARD L. RIDGWAY2 Department of Zoology, Washington State University, Pullman, Washington 99164-4220 SYNOPSIS. Regeneration of the nervous system of Melampus following cerebral ganglion removal proceeds through tract, bud, and ganglion stages. Each stage can represent a terminal condition in some animals. Early events of regeneration appear to include roles for chemotactic and growth-promoting agents and axonal guidance by preferential adhesion to a connective tissue sheath. This latter proposed mechanism accounts for the observed sequence in which the neural elements unite in the tract stage and for the pattern of failures that result when the sheath is disrupted. In the tract stage of regeneration, communication through the site of the missing ganglion is restored within the central nervous system, and between neurons of non-excised ganglia and the denervated periphery. Some behavioral recovery results. The bud stage of regeneration is characterized by neuropil development and associated swelling at the site of confluence of the tracts. Serotonin immunohistochemistry of bud stage preparations and retrograde dye transport via bud nerves show tracts and numerous synaptic varicosities, but the neuron somata that are labeled are located in other ganglia. Ultrastructural examination of late bud/early ganglion stage tissue reveals the presence of small undifferentiated cells. By six to seven months postoperative, some snails have clearly reached a ganglion stage of regeneration characterized by the appearance of differentiated neurons within the bud. The origin of these new neurons is currently under investigation. Koritzansky and Hartwig, 1974; Hulsebosch and Bittner, 1981) and in a single mollusc, the snail Melampus bidentatus (Moffett and Austin, 1982). Why these few species should be able to replace nerve cells while other species cannot, is outside the scope of this brief review (for a summary of the main hypotheses see Hulsebosch and Bittner, 1980). However, of the many factors that could contribute to the ease with which a species would be able to regenerate neurons, Melampus can claim several: (1) a primitive evolutionary position among pulmonate gastropods, (2) a long lifespan, (3) a potential to add neurons into adulthood, and (4) a propensity for its tissues to respond to neural induction. Melampus is a high intertidal salt marsh snail belonging to the lower order of pulmonate gastropods, the Basommatophora. It is unusual among pulmonates in retaining the primitive feature of laying large numbers of small eggs that hatch to a planktonic veliger stage and eventually 1 From the Symposium on Nervous System Regener- undergo a metamorphosis into the adult ation in the Invertebrates presented at the Annual Meet- form (Apley, 1970; Russell-Hunter el al., ing of the American Society of Zoologists, 27-30 1972). This reproductive strategy is augDecember 1986, at Nashville, Tennessee. mented by an iteroparous life cycle, i.e., * Present address: Dept. Med. Physiol., HSC, Univ. one in which the parents live on to reproof Calgary, Alberta T2N 4N1 Canada. INTRODUCTION In terms of their capacity to regenerate, molluscs are probably best known for being able to replace various body parts. Tentacles, eyes, siphon, penis, and regions of the foot, mantle, and shell, can all be regenerated to a degree by most molluscs (Hyman, 1967). There is evidence of, if not a requirement for, the influence of neural factors on regeneration in each of these cases; damage to the nervous system itself is typically repaired along with the associated body part. Many animal species are unable to regenerate central nervous system (CNS) tissues in the absence of body part replacement and it is especially rare to encounter the ability to replace severely damaged or lost neurons in adults (Hulsebosch and Bittner, 1980). In invertebrates this ability has been documented only in a few annelid species (Herlant-Meewis, 1962; 1109 1110 S. B. MOFFETT AND R. L. RlDGWAY duce again and again (Calow, 1978). Melampus bidentatus is a slow growing species with a lifespan of greater than 3 yr in field populations (Apley, 1970); under controlled laboratory conditions we have maintained individuals for more than 6 yr. Primitive features are also evident in the anatomy of the digestive and reproductive tracts of Melampus (Morton, 1955; Hubendick, 1978) and, more importantly for this review, in the nervous system. The CNS of pulmonate gastropods consists of eleven ganglia (five paired and one unpaired) plus the commissures and connectives uniting them. A primitive characteristic of the Melampus nervous system is the unfused ganglia and relatively long connectives (Price, 1977a, 1979). Each ganglion consists of a medullary neuropil surrounded by a cortical rind of neurons which in turn is encapsulated by a connective tissue sheath. The largest neurons of gastropod ganglia are usually the most peripheral and these appear to remain constant in number throughout life. Their large size is primarily attributable to polyploidy (Lasek and Dower, 1971; Boer et al, 1977). Neuron addition into adulthood has been noted in the CNS of molluscs, such as in the opisthobranch gastropod Aplysia californica, but this addition is apparently confined to clusters of smaller cells bordering on the neuropil (Coggeshall, 1967). Recent evidence suggests that this type of neuron addition also occurs in Melampus (May etal., 1987). Melampus is well-adapted to withstand the extremes of temperature, salinity, and oxygen tension associated with its harsh environment (McMahon and Russell-Hunter, 1981). Moreover, studies in our laboratory continue to demonstrate the remarkable regenerative capacity of Melampus tissues, which includes the ability to produce supernumerary sensory structures under the influence of implanted ganglia (Moffett and Austin, 1981). This demonstrates that adult animals have the ability to completely replace body parts, so regeneration of ganglia may simply represent one aspect of a generalized capacity to access developmental information in response to injury. In this article we review what is currently known about cerebral tract and ganglion regeneration in Melampus and present various possibilities for the mechanisms involved. EARLY EVENTS OF GANGLION REGENERATION Effects of cerebral ganglion ablations The paired cerebral ganglia are among the most important integrative centers in pulmonate gastropods. Much of the information the animal receives about its environment passes through sensory tracts associated with these ganglia (Janse, 1974). Cerebral neurons direct feeding behavior (Pentreath et al., 1982), locomotion (Snyder and Moffett, 1987), tentacle and head movements (Lever et al., 1978), and the whole body withdrawal response (Benjamin et al., 1985). Growth and reproduction are also under control of cerebral ganglion neurons (Joosse and Geraerts, 1983). The survival time is brief when both cerebral ganglia of Melampus are excised (Price, 19776). However, if both cerebral ganglia are removed and subsequently reimplanted, or removed and replaced by a pair of cerebral ganglia from a donor, the snails not only survive but recover behaviors indicative of regeneration of cerebral circuitry (Moffett and Snyder, 1985; Moffett, unpublished data). Support of survival and regenerative growth by a single intact cerebral ganglion or by a pair of implanted cerebral ganglia suggests that factors produced by these ganglia are critical for nervous system regeneration. This interpretation is supported by Wong and co-workers (Wong et al., 1984) who have shown that cultured neurons of the pond snail Helisoma trivolvis require media conditioned with soluble and surface-active factors from central ganglia for neurite outgrowth. The initial tissue response to ganglion ablation in molluscs is a period of reorganization, lasting about a week, that is characterized by reactive (chromatolytic) cell bodies in the non-excised ganglia and evidence of degeneration in the neuropil regions, cut nerves, and connectives (Borovyagin et al, 1972; Moffett, 1980). The degenerate tissue gradually disappears as regenerative growth begins. The challenge then becomes that of directing GANGLION REGENERATION IN MELAMPUS 1111 FIG. 1. Diagrammatical representation of the degree and sequence of nervous system regeneration in the early period following removal of a left cerebral ganglion in Melampus bidentatus. A. Dorsal view of the CNS with focus on the relative position of neural tracts (numbered structures) severed as a result of left cerebral ganglionectomy (area delineated by the box). B-D. Examples of tissue regrowth exhibited by animals sacrificed 14 days (B), 28 days (C), and 70 days (D) post-lesion. The sequence of tissue regrowth leading to ganglion reformation is suggested by the addition of regenerating neural tracts (shaded structures) to the presumptive ganglion bud (asterisks) in each of the examples. Abbreviations and numbering: left and right cerebral ganglia (LCe, RCe), cerebral commissure (1), tentacle nerve (2), optic nerve (3), cerebral tube/external peritentacular nerve (4), anterior (internal) labial nerve (5), median labial nerve (6), labial artery (7), cerebrobuccal connective (8), cerebropedal connective (9), cerebropleural connective (10). Not shown is the very thin statocyst (or static) nerve which runs parallel to the cerebropedal connective. Redrawn from Price (19776) with modifications based on observations of the authors. the regrowing axons to the area where the new ganglion will be established. Axonal guidance by soluble factor gradients Price (19776) discovered CNS regeneration in Melampus in the course of investigating the effects of ganglion ablation on reproductive behavior. He observed that after unilateral cerebral ganglionectomy, the cut distal end of the medial labial nerve (MLN; L, of Price, 19776) seemed to serve as a focal point toward which the regenerating nerves and connectives grew. We have confirmed these findings as well as Price's suggestion that the regrowing tracts arrive at the presumptive ganglion bud site in a fairly predictable temporal sequence as shown in Figure 1. The initial bud is a swelling formed by the fused distal stumps of the three labial nerves. A thin cerebral commissure is usually reestablished by 14 days following ganglionectomy (Fig. IB). Within 30 days the transected cerebropleural and cerebropedal connectives (and statocyst nerve) fuse to form a single large tract that joins the developing bud (Fig. 1C). By 70 days post-ablation the last of the severed tracts, the cerebrobuccal connective and the tentacle and optic nerves have reconnected (Fig. ID). The points of attachment of these last structures are highly variable so that only rarely does the new ganglion have the same spatial arrangement of tracts and nerves that is characteristic of control ganglia. Price (19776) hypothesized that a diffus- 1112 S. B. MOFFETT AND R. L. RlDGWAY ible substance might be released from the cut end of MLN to attract the regenerating neural tracts. This hypothesis is bolstered by reports that neurites can indeed be guided by gradients of soluble molecules {e.g., nerve growth factor: Letourneau, 1978) and by evidence that in several pulmonate gastropods the sheath of MLN serves as a neurohaemal area for growth hormone-producing neurosecretory cells. In the pond snail Lymnaea stagnalis these cells are known as "Light-Green Cells" (LGC) from their appearance when stained with alcian blue/alcian yellow dyes (Joosse and Geraerts, 1983). We have shown that cells probably homologous to the LGC are present in Melampus and that their axons likewise project to the sheath of MLN (Ridgway, 1987). The growth hormone has yet to be fully characterized, but immunohistochemical and chromatographical evidence suggest that it may be related to the vertebrate neuropeptide somatostatin (Schot et al., 1981; Grimm-Jorgenson, 1983a, b; Ridgway and Moffett, 1987). Moreover, recent experiments have shown that exogenous somatostatin can enhance outgrowth and electrical coupling of regenerating molluscan neurons, possibly by lowering intracellular free calcium concentrations (Grimm-Jorgenson, 1987; Bulloch, 1987). the form and connectivity of the nervous system in addition to their role in chemical synaptic transmission (for a recent review see Kater and Haydon, 1987). An even broader morphogenetic role for neurotransmitters during regeneration is suggested by studies on transected flatworms. The trauma of sectioning is thought to abruptly lower intracellular free calcium levels within the animal, causing a rise in serotonin and noradrenaline concentrations (Martelly and Franquinet, 1984). This sets in motion a cascade of intracellular molecular events leading to the initiation of DNA synthesis around 12 hr after transection. Likewise, the elevation of dopamine leads to the initiation of RNA synthesis 18-24 hr after transection (Martelly and Franquinet, 1984). A similar sequence of events may underlie the cellular activation observed early in the regeneration of axotomized neurons of Melampus and other organisms. Axonal guidance by substrate adhesion The tract regeneration following ablation of one cerebral ganglion in Melampus does not follow the shortest path to the presumptive bud site, as would be expected if guidance by a soluble factor gradient were the primary mechanism. Recent observations performed with the help of Dr. Keith We are currently investigating the ability Snyder indicate that the initial guidance of somatostatin and related substances to for many of the regrowing tracts is proact as chemotactic or growth-promoting vided by a connective tissue sheath (capiagents in Melampus CNS regeneration, but tocerebral membrane). It covers the CNS there are many other substances, such as and fans out like a web between many of serotonin, dopamine and glutamate, that the nerves. It surrounds much of the could potentially serve a neurotrophic esophagus and extends into the periphery, function or provide guidance for regen- providing protection and mechanical superating axons. For example, while gluta- port for the nervous system. We first noted mate has been shown to enhance neurite the role of the sheath in cerebral commisoutgrowth and electrical synaptogenesis by surotomy regenerates (Figs. 2-6). We found specific axotomized buccal ganglion neu- that when the sheath is interrupted as little rons in Helisoma, serotonin and dopamine as possible, regeneration is rapid: a long can inhibit regenerative activities (Hay- thin connection is usually present within don et al, 1987; Bulloch and Jones, 1988). 7-10 days (Fig. 2B) and a distinct commisFurthermore, experimental depletion of sure formed by 1 mo postoperative (Fig. serotonin during embryogenesis alters the 4). The regenerate commissure gradually morphology and connectivity of identified thickens (and shortens) so that by 1 yr it Helisoma neurons (Goldberg and Kater, closely resembles control tissue (compare 1985). Taken together, these findings Figs. 3 and 5). When the sheath was cut strongly suggest that common neurotrans- far anterior, the axons growing out of the mitters play a significant role in regulating ganglia followed the cut edge forward (Fig. GANGLION REGENERATION IN MELAMPUS 1113 B FIG. 2. Position of the cerebral commissure (arrows) and anterior portion of the connective tissue sheath (shaded areas) as they appear in control preparations (A), in regenerate preparations after transection of only the cerebral commissure (B), and in regenerate preparations after transection of the cerebral commissure plus tearing of the connective tissue sheath (C). In both B and C, regenerating axons from the left and right cerebral ganglia (LCe, RCe) follow the sheath margin to reestablish the commissure. 2C). When the sheath was torn extensively, the forward-growing tracts often failed to meet, and the commissure never formed (Fig. 6). The sheath is usually the substrate chosen by the earliest regenerating axons in preference to the underlying esophageal surface or other tissues adjacent to the ganglia. Subsequent regenerating axons often appear to select the surfaces of the "pioneer" axons as a substrate but may also make use of the sheath. The importance of the sheath can readily be extended to the situation following cerebral ganglion ablation. When a ganglion is removed there are 10 major tracts that are severed as shown in Figure 7. Six of these tracts (plus the statocyst nerve) are held in place and are united by the sheath. All of these tracts tend to use the sheath as a substrate for growth toward the ganglion bud site. The other four tracts have their connection with the sheath, as well as their CNS connections, severed by ganglionectomy. These tracts are left as free entities following ganglionectomy and cannot grow along a sheath edge to unite with the rest of the nervous system. We feel this is the main reason for the longer times required for their reunification with the bud (recall Fig. 1) and the high variability in their final junction site. The preference of regenerating tracts in Melampus for the connective tissue sheath may relate to differences in the adhesive properties of surfaces available to axonal growth cones. Studies in other animals have demonstrated that the filopodial extensions of growth cones randomly explore surfaces in the vicinity of the axon tip with the result being growth in the direction of greater adhesivity (Letourneau, 1985). It is impossible to say at present what molecule^) provide the greater adhesivity in the case of Melampus regeneration because the constituents of the sheath are still incompletely described. Ultrastructural and histochemical studies have revealed that the sheath consists primarily of an extracellular matrix (ECM) of collagen and other products derived from fibroblast-like cells and possibly glia (Ridgway, 1988). The "other products" probably include glycoproteins, proteoglycans, and glycosaminoglycans organized into a network having a specific structure and composition conducive to filopodial adhesion. A similar hypothesis has been formulated to explain the rapid and directed outgrowth of neurites from leech neurons cultured on CNS capsule ECM extract as compared to the slower, less directed, or unsuccessful growth exhibited by neurons plated on concanavalin A, poly-(L-)lysine, fibronectin, or laminin (Chiquet and Acklin, 1986). The composition of the sheath ECM may therefore determine the rapidity of early neurite outgrowth and provide the necessary adhesion while soluble factor gradients and target influences may determine the specific direction of the outgrowth. Summary of the early events of ganglion regeneration The first response to cerebral ganglion removal in Melampus is a brief period of degeneration and reorganization followed by the outgrowth of axons belonging to 1114 S. B. MOFFETT AND R. L. RlDGWAY B / **.<* 6A FIGS. 3-6. Examples of neural regeneration in animals where the anterior portion of the connective tissue sheath was torn during cerebral commissure transection. In each example, A is an osmium tetroxide infiltration whole mount (plastic embedment) showing the position of the cerebral commissure (CC) whereas B is a crosssection through the same commissure (asterisks) showing the position of the connective tissue sheath (S). Figure 3 is a control preparation. Figures 4 and 5 are successful cerebral commissure regenerates sacrificed at 1 mo post-lesion and 1 yr, respectively. Figure 6 is a failed cerebral commissure regenerate sacrificed at 4 mo post-lesion. The large arrowhead in 6A points to a thin, anteriorly directed growth that upon ultrastructural examination was found to consist of only a very few neurites as shown in 6B. Magnification bars: 3A, 4A, 5A, 6A = 100 /im; 3B, 4B, 5B = 20 Mm; 6B = 1 Mm. Moffett and Ridgway (unpublished data). neurons having their somata in non-excised ganglia. Nerves and central tracts united by incorporation within the connective tissue sheath grow together first, with the sheath providing the preferred substrate for initial neuritic growth and the distal stump of the median labial nerve serving as a focus for ganglion bud formation. The result of these early events is reestablishment of central connections through the site of the missing ganglion and between remaining central neurons and the denervated periphery. This is the tract stage of ganglion regeneration. 1115 GANGLION REGENERATION IN MELAMPUS FIG. 7. Position of neural tracts (numbered structures) relative to the connective tissue sheath (shaded areas) in situ (A) before and (B) after removal of the left cerebral ganglion (LCe). The sheath normally overlies the CNS and fans out like a web, incorporating the cerebral commissure (1), labial nerves (5,6, and 7), cerebropedal connective (9), statocyst nerve (not shown), and cerebropleural connective (10). When the left cerebral ganglion is removed the sheath holds these tracts in place and provides a substrate for their regrowth (see text). Tracts not normally incorporated by the sheath, such as the tentacle nerve (2), optic nerve (3), cerebral tube/external peritentacular nerve (4), and cerebrobuccal connective (8) are left isolated in the blood space as a result of ganglion removal. 100-r UJ O 51 80- 60-• ID O 40-- S 20- N 0 -112-24 (n = 10) 6-9 <n=16) > 24 MONTHS FIG. 8. Regeneration in snails with one cerebral ganglion removed was evaluated upon sacrifice two to more than 24 mo after the lesion. The results were arbitrarily grouped into four time periods. The state of repair was categorized as: no communication through the site of the missing ganglion (N), tract stage (T), bud stage (B), or ganglion stage (G). The distribution within these four categories was plotted as percent of the total number (n) of animals examined in each postoperative time period. 1116 S. B . MOFFETT AND R. L . RlDGWAY FIG. 9. Transmission electron micrographs of regenerate left cerebral ganglia of Melampus. A. Neuropil region of a 6 mo regenerate ganglion bud with neuritic processes bearing synaptic vesicles and neurosecretory granules. B. A cluster of small undifferentiated cells in a 6 mo regenerate ganglion bud. C. Small differentiated cells of a 12 mo regenerate ganglion. D. Large differentiated neuron of a 12 mo regenerate ganglion bearing numerous cytoplasmic neurosecretory granules. Samples were prepared for electron microscopy as described by Moffett and Austin (1982). Magnification bars: All bars are equal to 1 nm. LATER EVENTS IN GANGLION REGENERATION Budformation The morphology of ganglion regeneration has suggested three distinct stages: tract formation, bud formation and gan- glion formation (Price, 19776; Moffett and Austin, 1982; Moffett and Snyder, 1985). Each stage can represent a terminal con(Fig. 8). The d i t i o n in s o m e anjmais sequence of events in formation of the tract stage has been described above. Despite the loss of the ipsilateral sensory, motor GANGLION REGENERATION IN MELAMPUS 1117 FIG. 10. Serotonin-like immunoreactivity in whole mounts of normal and regenerate left cerebral ganglia of Melampus. A. A control cerebral ganglion contains 30-35 immunoreactive cells and a large number of efferent and afferent processes. B. A 9 mo regenerate ganglion bud with profuse sprouting and growth of neurites originating from neuron somata located in non-excised ganglia. C. A 15 mo regenerate ganglion with a cluster of small cell bodies. This large cluster contains at least four immunoreactive cells (large arrowheads); one cell having an axon (small arrowheads) that could be followed into the adjacent neuropil. Samples were prepared by the indirect immunofluorescence (A, B) and indirect immunoperoxidase (C) methods as described by Ridgway (1988). Abbreviations: cell bodies (CB), cerebral commissure (CC), cerebropedal connective (CPeC), median labial nerve (MLN), neuropil (NP), tentacle nerve (TN). Magnification bars: A = 50 fim; B = 50 /mi; C = 10 urn. and interneuronal elements, a limited recovery of behaviors mediated via cerebral pathways is seen as early as 3—4 wk after ganglion removal. This roughly coincides with recovery of the normal extended appearance of the tentacle on the operated side, and the ability of this tentacle to respond to stimulation with withdrawal (Moffett and Snyder, 1985). Bud formation results from neuropil development and associated swelling at the site(s) of confluence of the regenerating 1118 S. B. MOFFETT AND R. L. RlDGWAY nerves and central tracts (Moffett and Austin, 1982; Ridgway and MofFett, 1985). This occurs over a relatively long period and is the typical condition encountered in animals sacrificed 2-9 mo after ganglionectomy. Ultrastructural examination of bud stage tissue reveals an organized neuropil region with many areas rich in neurosecretory granules and synaptic vesicles (Fig. 9A). Axonal projections traced by retrograde dye transport into the bud from bud peripheral nerves reveal cells in other central ganglia that project through the bud, but no somata within the bud (Moffett and Snyder, 1985). Immunohistochemical studies employing antisera to serotonin show numerous varicosities ramifying within the bud neuropil, but again no labelled cell bodies (Fig. 10B). Nevertheless, the behavior of snails in this stage of recovery typically includes robust tentacle and labial withdrawal responses normally mediated by cerebral neurons. Electrophysiological recordings combined with acute transections that isolated the bud from the rest of the CNS indicate that neuropil development within the bud is in some instances sufficient to mediate reflexes on the operated side. (Moffett and Snyder, 1985). The bud stage therefore supports good behavioral recovery while relying on the growth of neurites and elaboration of synapses by neurons having their somata in non-excised central ganglia and the periphery. Is the observed behavioral recovery in the bud stage simply the result of regrowth of neurons whose axons normally project into the excised ganglion or are novel projections sent into the bud to compensate for neurons lost due to ganglion ablation? In the CNS of control snails, retrograde dye transport (Kahan and Moffett, 1979) and serotonin immunohistochemistry (Ridgway, 1988) have shown that a percentage of the axons in cerebral nerves normally arise from cells with bilateral or contralateral somata (Fig. 10A). Thus, the substrate for some recovery of function is present after ganglion removal, even if regeneration consists only of replacement of normal projections by these cells. Whether novel projections are made during ganglion regeneration in Melampus is presently under investigation but studies in several other gastropod species support such a possibility. Following removal of the right pleural ganglion in Lymnaea, regrowing neurites of an identified dopaminergic neuron of the right pedal ganglion demonstrate highly specific regeneration to original target areas but also extend into regions of the CNS not normally occupied by branches of this cell (Allison and Benjamin, 1985). The longevity, specific morphology (e.g., varicosities), and electrophysiology of these projections suggest that novel, permanent synaptic connections are formed (Benjamin and Allison, 1985). Interestingly, direct damage is not necessary to elicit outgrowth by molluscan neurons (Bulloch, 1984; Allison and Benjamin, 1986; Bulloch and Jones, 1988), though in such cases the projections are most often transient. The extent to which these novel projections can compensate for neurons lost during ganglionectomy remains unclear. The ga,7iglion stage By definition, the regeneration of a ganglion would require the addition of neurons to the bud. In control ganglia, a rind of neurons just beneath the sheath is visible as distinct cells and cell clusters that give the ganglion an overall yellow coloration. We know from ultrastructural examination of late bud/early ganglion stage regenerates that the first non-glial cells that are detected are small, morphologically undifferentiated cells usually present in clusters (Fig. 9B). These presumptive neurons (or neuron precursors) are not encountered earlier than 5 mo after ganglion removal (Moffett and Austin, 1982). By 7 mo, a population of differentiated neurons can be present, characterized by neurosecretory granules or synaptic vesicles, extensive Golgi apparatus and rough endoplasmic reticulum (Fig. 9C, D). In some instances of longer-term regeneration following cerebral ganglionectomy, we have found small, serotonin immunoreactive somata (Fig. IOC). Thus, new cells expressing the same phenotype as some of the lost neurons can arise, indicating that replacement of specific cells is possible in Melampus. The highly successful regenerated gan- GANGLION REGENERATION IN MELAMPUS glion appears to have all the features of a normal, albeit slightly smaller ganglion, complete with lobes and identifiable cell clusters (Moffett and Austin, 1982). The question arises as to how a nervous system that had adjusted to the absence of a ganglion over a period of 6 mo can accommodate to the addition of newly generated neurons. The problem can be posed by considering the situation encountered by new motor neurons. In order to be effective, these neurons must grow out and assume control of peripheral structures that were innervated by other neurons in their absence, and attract the sensory and interneuronal inputs that had presumably been concentrated upon the remaining neurons after the original target cells were removed. Experiments employing implanted ganglia have shed light upon the ability of the Melampus nervous system to accommodate additional neurons (Moffett, 1980). One or more cerebral ganglia implanted into the hemocoel of a snail with an intact nervous system can remain viable and form connections to the periphery and exchange information with the host nervous system, as revealed by recordings from the nerves and ganglion surface. Implanted ganglia are able to innervate regions of host tissues that are already innervated, and the doubly innervated region tends to enlarge. This is particularly apparent when a host snail's tentacle is innervated by an implanted ganglion. Induction of supernumerary sensory structures appears to represent further evidence of the stimulatory and organizing effect that the nervous system exerts on other tissues (Moffett and Austin, 1981). 1119 following implantation of ganglia in Melampus has shown that increasing the mass of neural tissue can elicit an increase in size of the target organ. Cerebral ganglion removal produces a mismatch between the mass of cerebral nervous tissue and the mass of the tissues that are normally innervated by cerebral neurons. This mismatch may set in motion the cellular growth required to create a new balance, e.g., the generation of new ganglion neurons. Another possible source of new neurons could be the neurons or other cell types present in the non-excised central ganglia. In annelids, migration of undifferentiated stem cells from existing ganglia is the mechanism whereby regenerating ganglia are populated (Herlant-Meewis, 1962). We know that there is an increase in the number of cells in certain cell clusters well on into adulthood in Melampus (May et al., 1987) and this phenomenon may be common among gastropods and other invertebrates (Hauser and Koopowitz, 1987). Since glia, pigment cells and some classes of neurons increase in number during development, migration via commissure or connectives would either supply the numbers of post-mitotic neuron precursors needed or provide a stem cell population that could increase by mitosis within the regenerating ganglion. One observation about ganglion regeneration that is apparent from the data in Figure 8 is that some snails do not progress beyond the tract or bud stage even if allowed more than 2 yr of regeneration time. The proportion of snails exhibiting We do not yet know where the cells that regeneration of a ganglion does not change are destined to become new neurons arise. between the 12-24-mo period and the over One possibility is that there is a recapitu- 24-mo period, whereas in the 6-9-mo lation of developmental events, such as period the bud stage rather than the ganthose recently described for Aplysia glion stage predominates. We therefore (McAllister et al., 1983; Jacob, 1984). Neu- plan to study snails in the 6-9-mo period ron precursors divide in the body wall and of regeneration to determine the origin(s) migrate to establish the central ganglia, of the new neurons. with the cerebral ganglia being the first formed. These migratory cells are thought Summary of the later events of to have withdrawn from the cell cycle. ganglion regeneration However, there is evidence that at least The later stages of regeneration seem to some post-migration mitosis occurs within be dependent upon the success of earlier the developing ganglia (see Fig. 9 ofJacob, developments, including first the unifica1984). The growth of sensory structures tion of most or all the central tracts, the 1120 S. B. MOFFETT AND R. L. RlDGWAY addition of a neuropil to form a bud, and the population of the bud by neuron somata to produce a ganglion. In instances in which the cerebral commissure failed to unite the regenerating tracts of the left side with the intact right cerebral ganglion, for instance, regeneration never has been found to progress beyond the tract or possibly the bud stage. This implies that physical guidance is an important mechanism in promotion of regeneration in Melampus. Neuron somata have rarely been found in situations in which unification of nerves and central tracts had produced an elongated structure or one with more than one site of enlargement. We therefore think that a favorable architecture may promote the development of interactions within the regenerating bud that in some way may be required for more advanced stages of regeneration. Clearly, replacement of neurons is a longterm process which cannot depend upon the relatively short-term stimulation of regenerative growth that is associated with injury. CONCLUSION The phenomenon of CNS regeneration in Melampus provides us with an opportunity to determine what is required for successful repair of injury involving neuron loss. Regeneration following ganglion removal proceeds through a sequence of stages, and behavioral analysis indicates that attainment of each successive stage affords a greater measure of recovery. This report and others in this symposium have dealt with factors that stimulate neuron differentiation and growth and are involved in guidance and the formation of connections. New techniques in the areas of biochemical analysis, cell culture, electrophysiology, and imaging, should allow the identification of the factors responsible for repair at each stage of recovery. Besides being intrinsically interesting, we can hope that investigations on nervous system regeneration in invertebrates such as Melampus will shed light on basic phenomena that may aid in promotion of the process in mammals. ACKNOWLEDGMENTS We thank Dr. Keith Snyder for his contributions to the work presented and Dr. David F. Moffett, Ronald May, and Tamara Howard for critical reading of the manuscript. We also thank Daniel Austin for contributing Figure 9B. This research was supported by grants from the National Institutes of Health (5 R01 NS 14333 and 1 R01 NS 22896) to S. B. Moffett and by Grants-in-Aid of Graduate Research from Sigma Xi and Washington State University to R. L. Ridgway. REFERENCES Allison, P. and P. R. Benjamin. 1985. Anatomical studies of central regeneration of an identified molluscan interneuron. Proc. R. Soc. London B 226:135-157. Allison, P. and P. R. Benjamin. 1986. Stimulation of neuritic outgrowth in an undamaged molluscan interneuron. J. Exp. Biol. 122:447-451. Apley, M. L. 1970. Field studies on life history, gonadal cycle and reproductive periodicity in Melampus bidentatus(Pu]monala: Ellobiidae). Malacologia 10:381-397. Benjamin, P. R. and P. Allison. 1985. 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