THE JOURNAL OF COMPARATIVE NEUROLOGY 300273-286 (1990)

Axonal Regeneration and Sprouting Followinghjuryto the Cerebral-Buccal Connective in the Snailhhutinuficlica ROGER P. CROLL AND MICHAEL W. BAKER Department of Physiology and Biophysics and Department of Psychology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

ABSTRACT Axonal sprouting and regeneration were studied in the land snail Achatina fulica following a unilateral crush to the cerebral-buccal connective. Both normal projection patterns and changes induced by injury were examined with axonal filling techniques. As expected, most staining was lost shortly after the crush when filling across the lesion site. Much of this decrease is attributable to the direct disruption of fiber pathways, but evidence also indicates that a limited amount of retraction of some neurites occurred during the first week. A subsequent, gradual increase in the numbers of stained elements culminated in supernumerary counts of fibers in many pathways and in some novel labeling of cell bodies. Maximum numbers of supernumerary fibers usually occurred 21-28 days after the lesion. Most of these extra neurites and cell bodies subsequently disappeared, and by day 35 the appearance of projections generally returned to within the ranges observed in normal, unlesioned animals. Together the results demonstrate the extent of neuritic regeneration, sprouting, and retraction that occurs in vivo within the gastropod nervous system following injury. The study also indicates the usefulness of such in vivo approaches to understand the long-term processes that contribute to the restoration of morphological and functional integrity. Key words: supernumerary fibers, nervous system, gastropod neurons

Numerous studies suggest that gastropod neurons respond to injury by regenerating lesioned projections and by forming novel connections (Murphy and Kater, ’80; Bulloch and Kater, ’81, ’82; Moffett and Austin, ’82; Bulloch, ‘84; Benjamin and Allison, ’85;Haydon et al., ’87; Murphy et al., ’85; Fredman and Nutz, ’88;Moffett and Ridgway, ’88a,b). In an attempt to understand the physiological mechanisms that trigger sprouting and control axonal guidance and synaptogenesis, much effort has been concentrated on in vitro cell and organ cultures, focusing on the regenerative responses of individually identified neurons. Relatively little effort, however, has been focused on the in vivo regenerative responses of gastropod central neurons. The few studies that have been conducted were again mostly concerned with a very restricted number of identified neurons. Thus, despite many years of intensive study on neural regeneration in gastropods, few studies have yet defined normal, in vivo regeneration and sprouting of larger populations of neurons following a lesion to a central nervous pathway. Questions, therefore, arise regarding the generality of findings based on the few identified neurons that have been studied so intensively in vitro. Do cells respond to injury in a similar manner when studied in vitro and in vivo? Can major findings be generalized outside of o 1990 WILEY-LISS, INC.

the few, best-studied gastropod species? What are the consequences of injury when examined at longer survival times than can be feasibly examined in vitro? Furthermore, given the diversity of responses noted in individual cells, how can one predict the general responses of larger populations? In the present study, we have examined in vivo axonal regeneration following a crush to the cerebral-buccal connective (CBC) of the land snail Achatina fulica. Axonal dye filling (“backfilling”) permitted the description of the normal origins and projections of processes within the connective. Based on changes in these staining patterns, monitored at varying times following nerve crushes, we conclude that axonal sprouting is rapid and not only restores most projections observed in control animals but also produces supernumerary counts of fibers in most pathways during the first few weeks following injury. Most, if not all, of these supernumerary sprouts, however, are transient and tend to disappear with longer survival times. Thus the eventual restoration of nearly normal morphology involves not only rapid and extensive axonal sprouting but also long-term

Accepted July 20, 1990.

R.P. CROLL AND M.W. BAKER

2 74 elimination of large numbers of fibers. Preliminary aspects of this report have appeared elsewhere (Croll et al., '87).

MA'lERULSANDMETHODS Subjecb Adult specimens of A. fulica, with shell lengths ranging from 40 to 60 mm, were obtained from John Takara of Honolulu, Hawaii, from Dr. Ronald Chase at McGill University, or from our laboratory breeding colony. Throughout the study, animals were maintained at 20-24°C on a 12:12 light/dark cycle and were fed fresh lettuce and carrot augmented with a slurry of crushed Purina rat chow in water.

surp;ery Snails were injected with 0.2 ml of 1% succinyl choline in saline in preparation for surgery. As reported by Croll('78) and by Chase and Croll ('Bl), the animals usually became completely flaccid within 5 minutes following the injection. Occasionally, however, a second, smaller injection was needed to produce complete paralysis. A 5-8 mm, middorsal incision was made between the superior optic tentacles. Following another incision through the capitocerebral membrane (Carriker, '461, the underlying esophagus was deflected to one side to expose one of the CBCs, which was then crushed close to either the cerebral or the buccal ganglia with No. 5 jewlers' forceps. In sham-operated animals, one CBC was exposed but not crushed. In all animals, the esophagus and CBC were subsequently replaced and the wound was closed with two to four sutures. Snails were then wrapped loosely in a damp paper towel and returned to their home cage. By the next day, animals had generally recovered sufficiently to crawl, and by the following day no overt abnormalities were observed in their behavior. These procedures resulted in approximately a 90% survival rate.

Axonal dye fills At varying intervals after surgery, animals were sacrificed, and their cerebral and buccal ganglia were removed. In each preparation both CBCs were cut midway between the cerebral and the buccal ganglia. Cut nerve stumps were then drawn into the ends of very fine-tipped pipettes so that one CBC could be dye-filled toward the cerebral ganglia and the other toward the buccal ganglia. Saline within each pipette was replaced with a solution of Ni2+-lysine(1.7 g NiCl2-6H,O and 3.5 g L-lysine free base in 20 ml H,O; Fredman, '87; S.M. Fredman, personal communication). This preparation was maintained at room temperature for 24 hours before the pipettes were removed, and the ganglia were washed in fresh saline. Nickel was precipitated by adding five to ten drops of a saturated alcoholic solution of rubeanic acid to the 10 ml saline bath. After 20-30 minutes the ganglia were transferred to 10%formalin or 4:1 ethanol: glacial acetic acid for fixation overnight and then washed in saline and silver intensified according to Croll('86). Ganglia were mounted whole in Permount (Fisher).

Analysis Approximately equal numbers of left and right CBCs were crushed in different animals. However, our preliminary analyses revealed no differences in regeneration or

sprouting following crushes to the two sides, so subsequent analyses were performed after collapsing the data across this factor. These procedures thus resulted in two different lesion locations (near the cerebral ganglia or near the buccal ganglia), two relative sides for the fill (ipsilateral or contralateral to the crush), and two directions of filling (into the cerebral or into the buccal ganglia). It was subsequently also found that little difference existed between the fills contralateral to either a distal or a proximal crush. Therefore data were also pooled across this factor. The six types of preparations that resulted from these procedures are depicted in Figures 3A to 8A. The numbers of experimental and control specimens used are shown in Table 1. Two measures were assayed in this study. As described fully in the Results, several distinct populations of cell bodies were labeled following axonal fills of the CBC toward either the cerebral or the buccal ganglia. Changes in the complement of each population were determined at different survival times following surgery. In addition, the numbers of labeled fibers in distinct neural pathways were also counted. Such counts, however, were only easily accomplished in our whole mount preparations when relatively few fibers were involved. Since larger numbers of fibers resulted in ambiguities, a maximum count of 20 was scored for any single pathway. All counts of cell bodies and processes were made directly from a compound microscope at x 400 magnification. Photographs were subsequently made with a Leitz Aristoplan microscope and Wild camera system (MPS46/52). The number of cells in each identified group and the number of fibers in each defined pathway were counted in normal animals, in sham-operated animals, and in experimental animals at varying times after surgery. These analyses permitted the evaluation of very specific neural changes induced by the nerve crush. It soon became apparent, however, that general trends were reflected in all these analyses. To illustrate these trends, we collapsed much of the data across populations for graphic representation. For example, changes in numbers of somata ipsilateral to the crush were illustrated together as were changes in the numbers of somata contralateral to the crush. In cases where pooled data did not accurately reflect the responses of all contributing cell or neurite populations, exceptions are noted in the text. One-way analyses of variance (ANOVAs) on the data for each of the fill types were performed across time. Where significance was found ( P < 0.05) in these overall analyses, individual t tests were performed between the normal counts and the counts at varying times after surgery. TABLE 1. Numbers of Subjects Used in Studies of Normal, Sham-Operated, and Lesioned Animals' Cerebral fill types' Days

I

1 3

4 3 4 4 3 4

7 14 21 28 35

4

Buccal fill types3

I1

111

Iv

V

VI

-

-

4

-

.-

3 3 3 3 3 3

6 8 6 7 6 6

3

3 3 3 3 3 3

4

3 3 4

3

6 8 6 I 7 6

'Experimental fill types are listed for each ganglion acmss the number of animalstestedat ench specific survivaltime. For Day 1 analysis was restricted to fill types I and IV preparations. 'Normal subjects, N = 12; sham-operatedsuhjects,N = 5. 3Normal subjects,N = 11;sham-operatedsubjects, N = 5.

AXONAL REGENERATION AND SPROUTING IN ACHATZNA

2 75

extracellularly across the lesion site. Furthermore, only RESULTS ipsilateral cells with little intervening neuritic volume N o m n a l o r i g i n s a n d p r o j e c t i o n s o f p ~ ~would accumulate appreciable quanties in their somata. A the cerebd-buccal connective similar explanation was proposed by Benfey and Aguayo Axonal filling of each CBC results in the labeling of approximately 65-95 cell bodies within the paired cerebral ganglia (Fig. 1A). Of these cells, only one, the metacerebral giant (MCG) cell (Croll, '85, '87, '88) has been individually identified. The MCG is located near the center of the ventral surface of the ganglion and is by far the largest of the cerebrobuccal neurons. The remainder of the cerebrobuccal neurons are organized into four ipsilateral populations (groups A-D) and two contralateral populations (groups E and F). The positions of the various populations are represented in Figures 1A and 2A, and their normal complementsare tabulated in Table 2. Axons filled from the CBC were also found in all the major trunks emanating from the cerebral ganglia. In fact, all trunks ipsilateral to the fill contained far too many fibers to quantify accurately (i.e., they each contained more than 20 fibers). The numbers of labeled fibers located in contralateral cerebral trunks are shown in Table 2. Labeled fibers crossing the midline in the cerebral commissure formed three distinct fascicles. Their positions are represented in Figure lA, and their complements are shown in Table 2. Axonal fills from the CBC into the buccal ganglia labeled considerably more somata than were observed in the cerebral ganglia (Fig. lB,C). The largest concentration of labeled somata was located along the anterior margin of the ipsilateral ganglion. A particularly large, identifiable buccocerebral cell was reliably located along the lateral margin of the ipsilateral ganglion, adjacent to the medial lateral buccal nerve. This cell will be referred to as the lateral giant buccocerebral (LGBC) neuron. A few moderately sized cells were also labeled in the contralateral ganglion, but the majority of contralateral cells were small and located either within an anterolateral cluster on the dorsal surface or within a cluster near the center of the ventral surface. While the number of ipsilateral, filled cells was too high to quantify accurately (estimated to include roughly 140 neurons), the number of contralateral cell bodies were more easily counted (Table 3). Counts could also be accurately obtained for labeled fibers within most of the buccal nerve trunks. The numbers of labeled fibers in the ipsilateral and contralateral trunks of the ganglia along with the numbers of labeled commissural fibers are shown in Table 3.

Controls As will be reported below, nerve crushes induced several changes in the pattern of staining revealed by axonal filling of the CBC. Much of the decreased numbers of stained elements in animals sacrificed shortly after surgery can be explained in terms of direct damage to fibers in the CBC. To test the completeness of such crushes, CBCs from control specimens were filled across a lesion site proximal to either the cerebral or buccal ganglia within 0-6 hours after a crush. When analyzing these data it became obvious that most counts were reduced to 0 (day 1 in Tables 2 and 3). The few elements that did fill were mostly cell bodies ipsilateral to the lesion. Virtually no contralateral staining was seen after the crush. We assume that fills initiated 0-6 hours following the lesion permitted insufficient time for regeneration of fibers across the lesion to the filling site. It is possible, however, that the cells labeled on that day became so because of a limited amount of dye that diffused

('82) to account for very limited retrograde staining across a crush site shortly after administration of the lesion. Thus, given the limited staining noted on day 1(of less than 15% of the normal number of somata and less than 5% of the normal number of fibers), together with the likelihood of some small amount of extracellular diffusion of dye, we feel that very few CBC fibers survive the nerve crushes. This conclusion is confirmed by direct observations of degenerating axonal segments in the days subsequent to the crush (Croll et al., '87). The finding of supernumerary fibers in many pathways following nerve crushes (see below) likewise needs explanation. Sham operations were, therefore, performed to test for any possible general effects of handling, paralysis, and surgery. Five animals received the identical surgical treatment as the experimental subjects, differing only in the absence of an administered nerve crush. Axonal fills were performed into each CBC at 21 days after surgery. This survival time was chosen since it was found (and reported below) that experimental preparations exhibited the greatest number of supernumerary fibers at approximately this time. As can be seen in Tables 2 and 3, there were no significant differences between the sham-operated animals and the unoperated, normal subjects in the numbers of either labeled cells within the identified groups or in labeled fibers in the pathways. Thus, although generalized effects, which might be associated with surgery, can induce neuritic sprouting in certain gastropod neurons (Bulloch, '841, they appear to be of minimal importance in our analyses. Together, the control procedures strongly suggest that the experimental results, described below, accurately reflect changes in the nervous system resulting from the axotomy of cerebrobuccal and buccocerebral neurons.

Type I fi21. into the cerebral ganglia across a crush (Fig.3A) As can be seen in Figure 3, the numbers of both cell bodies and fibers that are labeled when filling across the lesion site into the cerebral ganglia fall markedly in the days immediately following the crush. As previously mentioned (and shown in Table 2), most counts for specimens filled within 6 hours of the lesion were close to 0. Similar counts were recorded on day 3. Despite some variability seen in regenerative responses, it is evident that the nervous system ofAchatina undergoes rapid repair following the crush. For ipsilateral somata, normal levels of staining gradually returned by around day 28 (Fig. 3B). The MCG also generally returned to normal staining over this period. When it first reappeared around day 7 it was very faintly stained, but the intensity of labeling subsequently increased (Fig. 2B,C). The MCG appeared, however, to be variable in its ability to regenerate its CBC axons when crushed near the cerebral ganglion. In several instances, the cell was not labeled until days 21-28. For the contralateral somata, labeling was nearly completely absent for the first 2 weeks following the injury (Fig. 3C). The contralateral cell counts subsequently increased to near the level seen in normal preparations. It should be noted, however, that in several preparations group F failed to reappear even after long survival times. In contrast, group E generally returned to normal numbers in most

R.P. CROLL AND M.W. BAKER

2 76

13 14

17

C

12

\

'%

"

16 Figure 1

'

17

AXONAL REGENERATION AND SPROUTING IN ACHATZNA preparations by day 21. Whether the depressed cell count in group F was due to the death of neurons or to the loss of their neurites within the damaged CBC is presently unknown. As might be expected, the initial return of contralateral cell bodies followed the return of the commissural fibers (Fig. 3D). Following day 1, labeling of the commissural fibers gradually increased and reached a peak at day 21. Interestingly, however, the commissural counts became significantly larger than that normally observed in control animals. These supernumerary commissural fibers were not distributed equally in the different fascicles; they belonged exclusivelyto the posterior fasciculus. The recovery of fibers in contralateral trunks (Fig. 3E) closely paralleled the recovery of commissural fibers. The numbers of labeled fibers gradually returned to near normal levels by around day 14 and then attained supernumerary proportions, with peak values noted at day 28. The occurrence of supernumerary fibers was particularly noticeable in the contralateral CBC, where over ten labeled fibers were often found on day 28, as opposed to the normal complement of about two fibers seen in controls (Table 2). Two possible explanations for the supernumerary fibers exist: First, when buccocerebral axons regenerate across the crush site into the cerebral ganglion, they may produce multiple, parallel branches to replace the original single fibers. Alternatively, the supernumerary fibers may represent newly sprouted, novel projections. These possibilities are not mutually exclusive and likely both contribute to the complement of supernumerary fibers observed here (see below).

TYpeIIfilkintothecerebralgangliaipsilaterd to a c m s h near the buccalganglia (Fig.4A) In this preparation, the total number of labeled, ipsilatera1 cell bodies remained relatively stable for the duration

~

~~

Fig. I. Camera lucida tracings of typical cerebral and buccal ganglia following axonal filling of the cerebral-buccal connective (CBC). A. Ventral view of the cerebral ganglia. The singularly identifiable MCG is located in the center of the cerebral ganglion ipsilateral to the filled CBC. Other cell bodies ipsilateral to the fill are organized into four discrete populations. Group A is located between the CBC and the lateral edge of the ganglion. Group B is comprised of smaller cells located anterior to the CBC. Group C is located between the metacerebra1 giant cell (MCG) and the CBC. Group D, which is located along the posterior margin of the ganglion, extends from the ventral to the dorsal surface; hence only a portion of its cellular complement is depicted here. Groups E and F are located in the contralateral cerebral ganglion in positions corresponding to those of groups D and C, respectively. Fiber numbers in the ganglionic trunks (labeled 1-7) and the commissural tracts (8-10) should be taken only as indicating relative densities (see Table 2 for the names of the different pathways and for actual cell and fiber counts). B: Ventral view of the buccal ganglia following axonal filling of the CBC. The majority of ipsilaterally labeled somata are located along the anterior margin of the ganglion. The lateral giant buccacerebral (LGBC) cell (asterisk) is located laterally and approximately mid-distance along the anteroposterior axis. A few small and moderately sized contralateral cells are located near the center of the ventral surface. Fibers shown exiting ganglionic nerve trunks and in the commissure (labeled 11-18) indicate relative densities only (see Table 3 for names of pathways and actual fiber and cell counts). C : Dorsal view of the buccal ganglia following axonal filling of the CBC. Ipsilateral, labeled cells occupy most of the anterior portion of the ganglion. Considerably fewer contralateral neurons are found dorsally, and most of these reside within a cluster near the center of the ganglion. Bar = 300 pm.

277

of the study (Fig. 4B), although occasionally decreased numbers of somata in groups A and C were noted in the first week. The MCG also sometimes failed to fill or was only faintly labeled at this point. These shifts from control values were short-lived and the cell counts for both groups A and C and the appearance of the MCG returned to normal by day 14 and remained stable thereafter. In contrast to the relative stability in ipsilateral soma staining, three to four times the number of normally labeled somata were observed in the ganglion contralateral to the fill by day 7 (Fig. 4C), and nearly all of these were located in group E. By day 14, the contralateral cell counts returned to nearly normal levels, although a second supernumerary count was also noted on day 28. Again the extra somata were restricted to within group E, and the counts returned to normal by day 35. The numbers of labeled fibers in both the commissural tracts (Fig. 4D) and in the contralateral nerve trunks (Fig. 4E) increased gradually over the first 14 days following the lesion. The number of labeled fibers in commissural tracts remained elevated for the duration of the study, but the number of labeled fibers in contralateral roots returned toward nearly normal levels by day 35. It should be noted that the increased fiber counts occurred during a time when one might, in fact, have expected decreased counts because of the degeneration of severed axonal segments of buccocerebral neurons. Together, these results indicate that probably only a few cerebral cells (occasionallythe MCG and neurons in groups A and C) retract their s o n s from the CBC followinga crush near the buccal ganglion. Such retraction is variable and short lived. The results also indicate that a major effect of the nerve crush is to induce sprouting of other cells into the damaged CBC. Specifically, the cells of group E appear to respond to damage of a CBC by sprouting new processes directed toward the injury. By the second week the distribution of labeled somata returns to relatively normal, but the numbers of labeled fibers in different pathways continue to mount, and only later is there a trend back to control values. Again, it is unknown to what degree the increased numbers of labeled fibers represent supernumerary collaterals of original projections or newly sprouted, novel projections.

Type IIIfilk into the cerebral ganglia contralateralto a crush near the cerebralor buccal ganglia(Fig.5A) When the CBC is filled contralateral to a crush near either the cerebral or buccal ganglia, the number of labeled somata ipsilateral to the fill decreases during the first 14 days after the lesion (Fig. 5B). These changes are almost exclusively the results of changes in the number of cells labeled in group D. Groups A-C did not change significantly over the course of this study. The total number of somata contralateral to the fill (ipsilateral to the lesions) increased greatly following the crush (Fig. 5 0 . Once again, this overall ganglionic count did not represent the responses of all the component populations. The supernumerary response was restricted to a distinct population of cells, group E. As before, labeling returned to normal on days 28-35. A trend toward supernumerary elements was also seen in the commissural fibers (Fig. 5D) and in the number of fibers in contralateral trunks, with both elements peaking around days 21-28 (Fig. 5E). A return to normal levels was evident by day 35.

2 78

Fig. 2. Morphology of normal and regenerating ipsilateral cerebral neurons shown in whole mounted ganglia. The MCG is marked by an asterisk, population A by an arrowhead, and group B by the open arrow. The C group, comprising five to six moderately sized cells located between the CBC and the MCG, is unmarked. A: A representative normal cerebral hemiganglion following axonal filling of the CBC. The plane of focus captures groups A, B, and C, together with the ventral commissure. Lying prominently in the center of the ganglion is the MCG. Group D is obscured by tissue thickness. Similarly, the posterior and dorsal commissural tracts are out of the plane of focus. B, C : Variability in regenerative responses. Both show regenerating cerebral ganglia 1week after an ipsilateral crush (type I fill). In B only the MCG is faintly visible. Numerous fine fibers are also seen entering the

R.P. CROLL AND M.W. BAKER

ganglion. In contrast, C shows a more progressed state of regeneration. Clearly visible is the full complement of cells from group C, and partly obscured against the dark labeling of the CBC lies the cells of group A (group B is present but below the plane of focus). A faintly stained MCG can also be discerned. Fiber staining has also resumed in the ventral commissure, neuropilar regions, and ipsilateral ganglionic trunks. D An ipsilateral cerebral ganglion shown 3 weeks after a contralateral crush close to the buccal ganglion (type I11 fill). Dense fiber labeling is seen in all trunks, commissural tracks, and the central neuropilar region, showing an extensive and profuse supernumerary response. Cells of groups A, €5, and C are present but obscurred by the dense labeling. Bar = 300 pm.

AXONAL REGENERATION AND SPROUTING IN ACHATZNA TABLE 2. Numbers of Somata and Fibers Labeled in the Cerebral Ganglia Following Axonal Filling of the CBC' Normal Ipsilateral cerehral soma populations Group A Group B Group C Group D Contralateral cerebral soma populations Group E Group F Contralateral cerehral trunks 1. Tentacular nerve 2. External labial nerve 3. Medial labial nerve 4. Interior labial nerve 5. Cerebral-bud connective (CBC) 6 . Cerebral-pedal conneetive 7. Cerebral-pleural connective Commissural tracts 8. Posterior fasciculus 9. Ventral anterior fasciculus 10. Dorsal anterior fasciculus

TABLE 3. Numbers of Somata and Fibers Labeled in the Buccal Gangha Following Axonal Filling of the CBC'

Day 1

Sham

4.2 (0.4) 23.3 (1.1) 5.3 (0.3) 34.7 (2.4)

3.7 (0.8) 21.3 (1.3) 4.7 (0.4) 29.0 14.5)

2.5 (1.4) 6.5 (3.7) 2.0 (1.2) 2.7 (2.5)

3.1 (0.7) 2.9 (0.6)

3.2 (1.0) 3.0 (0.4)

0.0 (0.0) 0.0 (0.0)

5.4(1.1) 1.8 (0.5) 7.5 (1.8) 9.4(1.7)

5.7 (1.4) 1.5 (0.8) 9.0 (1.8) 9.0 (2.1)

0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)

2.2 (0.5)

3.6 (1.1)

0.0 (0.0)

0.9 (0.4)

0.8 (0.5)

0.0 (0.0)

3.3 (0.6)

6.2 (2.3)

0.0 (0.0)

1.2(0.1)

2.3 (1.4)

0.0 (0.0)

9.7 (0.5)

9.5 (1.0)

1.5 (0.5)

ND

2 79

ND

ND

'Means m d . in pxenthews, standard deviatiun. lor n wm d unqwraud!. &mqcratcd and eqprimenrd suhjccfs thdt &6 hours prpvlouily had reeived a crush UI thr ('BC p n u o d U) thr fill site. The numhera shuwn befurr the names of the rerrbral trunks imd wmmiiiurd tracts romspond 10 the Intx.hg in Figure 1 Recause the crrphrd ganglia w t ~ i niutinvb. , viewed from the ventral surface. vhangs in thc dc1r.d anterior fasnculus of the cerebral coiimiisurp uew not determintd SUJA twu-wdy ANOVA performed on these dnta indicatrd no si~mlimi ddYermw between 1he normal and s h m - o p m u dsubjens P 0 05, However. a mnilar cumparism hi~wirmnnrinal and day I-perawd animals inhwted a statetically .significant diffirrnw P c 0 01, Furthermore, pairwiw cumpnnnins in&mtcd signiRcant &lferenu$ iii t.wh of tlir indiwdud counts of wll populations and t i k r numbers it u'st. P c 001

-.

In summary the data in Figure 5 support the contention that a crush to the CBC induces widespread sprouting in cerebral neurons and fibers. As before, the maximal effect is seen during days 21-28. The more rapid return to normal counts seen in this preparation, when compared with the ones discussed previously, presumably reflects the fact that a lesion to the CBC contralateral to the fill causes less severe and less direct damage to the labeled fibers.

TypeIVfilkintobuccalgaqlia across a crush (Fig.6A) In addition to the previously mentioned axonal fills into the cerebral ganglia, we performed similar analyses of changes in the buccal ganglia followingCBC lesions. On day 1 all counts of labeled fibers in ipsilateral and contralateral trunks, labeled fibers in the buccal commissure, and labeled contralateral somata were reduced to 0 or near 0 (Table 3; Fig. 6B-E). Subsequently, the numbers of labeled processes and cell bodies quickly reappeared so that most counts were nearly normal between days 14 and 21. The identified buccal neuron LGBC also reappeared during this time. When first seen at day 7, labeling was faint, but over the next week full labeling returned. As was seen in analogous cerebral preparations (type I), supernumerary processes were evident with longer survival times. The numbers of labeled fibers in the commissure (Fig. 6C) and in contralateral trunks (Fig. 6E) exceeded normal levels on days 21-35. Supernumerary counts were particularly evident in the CBC, where numbers increased fivefold from a normal complement of around three. Thus the regeneration and supernumerary sprouting evident within the cerebral ganglia were also observed in the buccal ganglia.

Ipsilateral buccal somata Contralateral buccal somata Ipsilateral buccal trunks 5. Cerebral-bud connective (CBC) 11. Medial buccal nerve 12. Supertidal pharangeal nerve 13. Anterior lateral buccal nerve 14. Medial lateral buccal nerve 15. Posterior lateral buccal nerve 16. Salivarynerve 17. Esophageal nerve 18. Commissural fibers Contralateral b u d tru& 5. Cer ebr al- bud mnnective (CBC) 11. Medial buccal nerve 12. Superficial pharangeal nerve 13. Anterior lateral buccal nerve 14. Medial lateral buccal nerve 15. Posterior lateral buccal nerve 16. Salivarynerve 17. Esophageal nerve

Normal

Sham

Day 1

138 67.8 (8.5)

ND 65.6 (8.51

ND 4.5 (3.3)

ND 7.7 (1.3)

> 20.0 10.1 (1.6) 6.6(1.6) 3.4 (1.3) 2.4 (1.1) 3.7 (0.3) 13.9(1.2) 3.4 (0.11) 4.1 (0.6) 1.5 (0.7) 2.2 (0.8) 1.1 (0.5) 0.7 (0.5) 0.0 (0.0)

1.0 (0.7)

ND

ND

9.3 (1.1) > 20.0 8.0 (2.0) 8.3 (0.6) 5.3(1.5) 2.6 (0.6) 4.3 (0.5) 13.6 (1.5)

0.2 (0.21 0.6 (0.1) 0.7 (0.7) 0.5 (0.5)

3.0(1.0) 4.5 (0.7) 2.3 (0.6) 3.0 (1.0) 0.6 (0.5) 0.6 (0.5) 0.0 (0.0) 0.3 (0.5)

0.0 (0.0) 0.0 (0.01 0.0 (0.0) 0.0 (0.01 0.0 (0.0) 0.0 (0 0) 0.0 (0.0) 0.0 (0.0)

0.0 (0.0) 0.2 (0.2)

0.0 (0.0) 2.7 11.7)

'Means and, in parentheses, standard deivations for normal, sham-opeated, and experimental subjeds that 0-6 hours previously had received a crush to the CBC proximal to the till site. The numbers shown before the names of the buccal trunks and the commissural fibers correspond to the labeling in Figure 1. I n most preparations the buccal ganglion and the CBC ipsilateral to the fd were labeled so darkly that accurate counts were not possible and therefore not determined (ND).The value given for the ipsilateral b u d somata therefore is only an estimate based on a single sample in which the ganglia was stained with Ni2+-lysinebut not silver intensified. A two-way ANOVA performed on these data inmcated no significant difference between the normal and sham-operated subjeds (P > 0.05). However, a similar comparison between normal and day 1-prated animals indicated a statistidy significant difference (P< 0.01). Furthermore, paimisecomparisons indicated significant differences in each of the individual counts of cell populations and fiber numbers (t test; P < 0.01) (excluding the contralateral salivary nerve, in which no fiber labeling is present normally and at day 1).

Type Vfilk intothebuccalgangliaipsjlateralto

acrushnearthecerebdganglia No significant changes occurred in the labeling of ipsilateral trunk fibers following the crush when the CBC was filled proximal to a crush near the cerebral ganglia (Fig. 7B). In contrast, a marked increase in commissural fiber numbers (Fig. 7C) and contralateral trunk fiber numbers (Fig. 7E) were seen beginning on days 3 and 14 respectively. This supernumerary staining subsequently diminished, and by day 35 normal numbers were once again evident. The LGBC, like the MCG in the analogous cerebral preparation, was often faint or absent during the first 2 weeks following the crush. Thereafter normal staining resumed. Similarly,a decline in labeling occurred among the contralateral buccal neurons during the first 2 weeks following the injury. Thereafter numbers began to recover, although a depressed count was still evident by day 35 (Fig. 7D). Taken together, these results suggest that the crush caused both retraction of several axons from the damaged CBC and simultaneous sprouting of new projectionsthrough the commissure and out the buccal trunks contralateral to the lesion and fill. The sprouting appeared to be of sufficient magnitude to predominate over the retraction and any possible degeneration of severed cerebrobuccal axons. As seen in other preparations, fiber counts returned toward normal levels by day 35.

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contralateralto a crush near the buccal or cerebdganglia Following a crush to the contralateral CBC, the number of labeled fibers increased in the nerve trunks ipsilateral to

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sural fasciculi. E: Mean number of labeled fibers in trunks contralateral to the fill. Days following the lesion are noted on the abscissae, and SEM are indicated by brackets. Values shown on day 1are derived from Table 1. Values found in normal, unoperated subjects are indicated at the beginning of the abscissae by N. Asterisks denote significant differences between normal and experimental preparations at days indicated (t test; P < 0.05)

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Fig. 4. Type I1 fill into the cerebral ganglia isilateral to a crush near the buccal ganglia. A Diagrammatic representation of the ganglia and lesion site. The dye-filled location is represented by the darkened portion of the CBC and the arrow, and the crush site is indicated by the horizontal outlined break in the CBC. B: Mean number of somata labeled ipsilateral to the fill (ANOVA, P > 0.05). C: Mean number of somata labeled contralateral to the fill. D: Mean number of labeled

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fibers in the ventral anterior and the posterior cerebral commissural fasciculi. E: Mean number of labeled fibers in trunks contralateral to the fill. Days following the lesion are noted on the abscissae, and SEM are indicated by brackets. Values found in normal, unoperated subjects are indicated at the beginning of the abscissae by N. Asterisks denote significant differences between normal and experimental preparations at days indicated (t test; P < 0.05).

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Fig. 5. Type I11 fill into the cerebral ganglia contralateral to a crush near either the cerebral or buccal ganglia. A: Diagrammatic representation of the ganglia and lesion site. The dye-filled location is represented by the darkened portion of CBC and the arrow, and the crush sites are indicated by the horizontal outlined breaks in the CBC. B: Mean number of somata labeled ipsilateral to the fill. C: Mean number of somata labeled contralateral to the fill. D: Mean number of labeled

fibers in the ventral anterior and the posterior cerebral commissural fasciculi. E: Mean number of labeled fibers in trunks contralateral to the fill. Days following the lesion are noted on the abscissae, and SEM are indicated by brackets. Values found in normal, unoperated subjects are indicated at the beginning of the abscissae by N. Asterisks denote significant differences between normal and experimental preparations at days indicated (t test; P < 0.05).

the fill (Fig. 8B), in the commissure (Fig. 8C), and in the trunks contralateral to the fill (Fig. 8E). These counts all returned toward normal levels by the end of the study. Some of this increase in the number of neurites may be easily explained by the newly labeled cerebrobuccal cells that project out the CBC following such crushes, as revealed in type 111 fills (see above; Fig. 5 ) . The number of somata labeled in the ganglion contralateral to the fill (ipsilateral to the lesion) fell during the first few days following the crush (Fig. 8D), but after the first week normal staining resumed. The results summarized in Figure 8 support the conclusion that the sprouting of new neurites is a widespread response to neural injury in the gastropod nervous system. This sprouting first becomes apparent shortly after the lesion. Supernumerary counts are evident for several weeks following the lesion, but a return toward normal levels is generally apparent by day 35. In this preparation evidence for a small degree of axonal retraction was manifested in the form of a slight drop in the number of contralateral buccal neurons labeled shortly after the crush.

in the projection patterns of relatively large numbers of cells following a nerve crush in the land snail Achatzna. However, we also report several observations that could not be predicted from earlier studies. To compare our observations with those previously reported in the literature, it is convenient to examine the specific events that mediate the observed changes in labeling patterns. One of the first events noted following the nerve crush was a limited retraction of neurites. Such retraction could most readily be observed as a drop in the labeling of LGBC and of contralateral somata in the buccal ganglion during the first week after injury in type V preparations. The decreased labeling of neurons belonging to groups A and C and of the MCG in the cerebral ganglia in type I1 preparations is also consistent with initial neurite retraction from the lesion site. It should be noted, however, that not all cells demonstrated neurite retraction. Previous studies have shown that retraction can be dependent on the location of the crush relative to the somata of the lesioned cells. For example, it has been well documented in Helisoma that crushing a nerve close to a ganglion results in the retraction of the proximal axon stumps of identified neurons (Murphy and Kater, '80; Bulloch and Kater, '81, '82; Hadley et al., '82). Distal crushes cause little or no retraction. Clearly, however, our results cannot be easily explained only by such location dependency. Both type I1 and V preparations demonstrated retraction followingaxotomy distal to the cell body. Furthermore, fills contralateral to the lesion into both the cerebral and the buccal ganglia (Types I11 and VI) suggest that initial retraction is not restricted to the damaged side. The causes for neurite retraction by cells

DISCUSSION Most investigations of regeneration within the gastropod central nervous system have focused on selected identified neurons, under either cell or organ culture paradigms (Bulloch and Kater, '81, '82; Murphy et al., '851, or occasionally in uivo (Benjamin and Allison, '85; Cohan et al., '87). Many of the findings from these previous studies can be successfully extended toward understanding changes

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Fig. 6. Type IV fill into the buccal ganglia across the crush site. A: Diagrammatic representation of the ganglia and lesion site. The dye-filled location is represented by the darkened portion of the CBC and the arrow, and the crush site is indicated by the horizontal outlined break in the CBC. B. Mean number of labeled fibers in trunks ipsilateral to the fill. C : Mean number of laheled fibers in the buccal commissure. D: Mean number of somata labeled contralateral to the

fill. E:Mean number of labeled fibers in trunks contralateral to the fill. Days following the lesion are noted on the abscissae, and SEM are indicated by brackets. Values shown on day 1are derived from Table 2. Values found in normal, unoperated subjects are indicated at the beginning of the abscissae by N. Asterisks denote significant differences between normal and experimental preparations at days indicated (t test; P < 0.05).

following distal axotomy or even when no direct injury was incurred are not apparent. The observations, however, are consistent with the possibility of more global influences such as shifts in connectivity caused by rapid sprouting of novel projections (see below) that in turn destabilize and perhaps displace existing projections. The results are also consistent with the possibility of the types of presynaptic retraction from injured neurons reported in vertebrates (Acheson et al., '42; Mathews and Nelson, '75; Mendell et al., '76; Purves, '76; Sumner, '76). The sprouting of new processes, like the retraction of certain pre-existing ones, began soon after injury. A gradual recovery in staining was already noticeable by days 3-7, and by days 14-21 most fiber pathways and cell numbers were restored to near the ranges seen in normal and control animals. The subsequent emergence of supernumerary neurites in different pathways, however, serves as ample evidence of continued sprouting for at least another week. Two likely possibilities may explain the extensive supernumerary counts: Several parallel fibers might replace the originals lost through injury, or novel projections may be sprouted. Evidence supports the possibility of contributions from both mechanisms. Numerous studies have shown that gastropods can regenerate many small parallel fibers to replace the larger original ones lost through injury (Murphy and Kater, '80; Benjamin and Allison, '85; Murphy et al., '85). In fact, it has been previously reported that the MCG in Achatina responds to axotomy in this manner (Croll et al., '87). Thus one might expect greater than normal numbers of fibers within defined pathways several weeks after the injury. Interestingly, however, previous

literature indicates that even though numerous small sprouts may originate from the same proximal stump, they do not necessarily all show equal fidelity to the original projection pathway. New collateral projections extending from the axonal stump are often seen extending both anterogradely and retrogradely (Murphy et al., '85). Similar sprouting has also been reported in lamprey central neurons (Hall and Cohen, '88).In the present study, a crush to the CBC might, therefore, be expected to induce widespread sprouting from proximal stumps in both anterograde and retrograde directions and presumably among both cerebrobuccal and buccocerebral neurites. Thus labeling of supernumerary neurites growing toward the normal targets, as well as the labeling of aberrant neurites, may at least partly account for our results. The staining of new cell bodies following the lesion indicates that sprouting is also induced in cells that do not normally have axons in the CBC. For example, increased numbers of contralateral cerebral somata were observed in type I1 preparations by 7 days after the lesion. Increased numbers of cerebral somata also send processes into the CBC contralateral to the lesion in the weeks following the crush as seen in type I11 preparations. These findings are consistent with numerous other reports of novel projections induced by injury to the gastropod nervous system (Murphy and Kater, '80; Bulloch and Kater, '81; Hadley et al., '82; Benjamin and Allison, '85). Interestingly, however, such novel sprouting was not as widespread in this study as might have been expected. In fact, most of the novel soma labeling in the cerebral ganglia was restricted to groups D and E. Apparently, signals indicating damage to one of the CBCs induce general

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labeled contralateral to the fill. E: Mean number of labeled fibers in trunks contralateral to the fill. Days following the lesion are noted on the abscissae, and SEM are indicated by brackets. Values found in normal, unoperated subjects are indicated at the beginning of the abscissae by N. Asterisks denote significant differences between normal and experimental preparations at days indicated (t test; P < 0.05).

sprouting by members of both groups D and E, with some of the novel projections re-routed toward the undamaged side while other processes project toward the injury itself to restore innervation to the damaged side. While it is clear that aberrant and supernumerary projections occur following a crush to the CBC, questions remain as to the eventual fate of such sprouts. Once established, are these features stable? Recent investigations have demonstrated that the multiple collateral fibers that replace originals lost through injury can persist for weeks or months (Murphy et al., '85; Allison and Benjamin, '85). Similarly, while novel projections are reported to occur quickly following neural damage, retraction of such projections can be either very prolonged or absent (Cohen et al., '87). In contrast to these studies, our results argue against such stability for novel sprouts. We observed a return of most normal morphological projections between 4 and 5 weeks after injury. Of those features not fully restored to normal levels by the end of the study, most showed a definite trend toward a normal morphology. Those features not fully restored by this time therefore presumably required only a slightly longer period of recovery. Our results of eventual retraction of aberrant projections are not without support from the literature. Maetzold and Bulloch ('861,for instance, reported such retraction of novel projections caused by osmotic disturbances in Helisoma. Our observation of long-term elimination of aberrant projections is also consistent with studies on tentacular regeneration in Achatina (Chase and Kamil, '83). Regeneration of neurons within the sensory epithelial pad and underlying digitate ganglion of the tentacle was complete by 20 weeks

after amputation, but the organization of these elements appeared chaotic during earlier stages. The more orderly and essentially normal later appearance of the tentacles was hypothesized to arise from the selective loss of misdirected fibers. These reports are reminiscent of shifting patterns of projections and connectivity that have been so well describedin vertebrates during development and following injury (e.g., Brown et al., '76; Hubel et al., '77; Jackson and Diamond, '81; for a review see Purves and Lichtman, '85). Thus our findings are consistent with the hypothesis that many of the same regulatory processes thought to be involved in the formation, reformation, and maintenance of connectivity in vertebrates may have analogous counterparts operating within the invertebrate nervous systems (Murphey, '86). While our observations clearly demonstrate initial retraction of existing neurites, sprouting of new projections, and eventual elimination of supernumerary sprouts, the results shown here concerning the degeneration of severed distal neurites are much less conclusive. Previous literature suggests that such degeneration can be rapid and widespread in molluscs (Borovyagin et al., '72). In fact, Croll et al. ('87 and unpublished data), have found that severed neurites of the MCG in Achatina degenerate completely by 7 days after a crush to the CBC. However, if such degeneration is rapid and widespread, one might expect to have seen a rapid decrease in the number of fibers in trunks and commissural tracts after buccocerebral fibers were severed in type I1 preparations or after cerebrobuccal fibers were severed in type V preparations. No such results were observed in either case. These anomolies, however, might

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Fig. 8. Type VI fill into the buccal ganglia contralateral to a crush near either the buccal or the cerebral ganglia. A Diagrammatic representation of the ganglia and lesion site. The dye-filled location is represented by the darkened portion of the CBC and the arrow, and the crush sites are indicated by the horizontal outlined breaks in the CBC. B: Mean number of labeled fibers in trunks ipsilateral to the fill. C: Mean number of labeled fibers in the buccal commissure. D: Mean

number of somata labeled contralateral to the fill. E: Mean number of labeled fibers in trunks contralateral to the fill. Days following the lesion are noted on the abscissae, and SEM are indicated by brackets. Values found in normal, unoperated subjects are indicated at the beginning of the abscissae by N. Asterisks denote significant differences between normal and experimental preparations at days indicated (t test;P < 0.05).

be explained in one of several ways. First, the number of cerebrobuccal cells with processes in buccal roots may be small. While the MCG is known to make such projections, it is unknown if other cells have similar morphologies. Likewise nothing is known about the anatomy of buccocerebral cells. Thus, while degeneration might have occurred, it could have been easily missed in our analyses. The possibility also arises that the degeneration of severed fibers was masked by simultaneous sprouting of new fibers. Indeed, evidence discussed above suggests that sprouting does occur very rapidly. Finally, it also remains possible that some of the degeneration might be delayed. Murphy et al. ('85) reported that degeneration of severed gastropod neurites did not occur for at least 28 days in vitro. Muller and Carbonetto ('79) reported delayed in vivo degeneration in leeches. Together these results suggest that some of the decreased fiber counts noted on days 28-35 in our study may be due to such delayed degeneration rather than to retraction of supernumerary projections. Obviously future efforts must focus more attention on this question of degeneration, especially in light of the reported roles of persistent severed fibers in regeneration in other organisms (Muller and Carbonetto, '79; Hoy et al., '67; Macagno et al.,

discussion but not explicitly mentioned is the general completeness of the restoration to a normal morphology. Such complete restoration is, in fact, inadequately conveyed by the quantitative analyses presented in this study. Generally, without resorting to detailed cell and fiber counts, it was difficult to differentiate between normal, unoperated animals and those following 5 weeks of recovery. Furthermore, the general completeness of the regeneration seen in this report was unexpected, since previous reports had indicated that not all gastropod neurons regenerate their lost axons (Fredman and Gage, '86; Fredman and Nutz, '881, and in fact axotomy may even cause the death of certain neurons (Moffett and Ridgway, '88b). Our observations of normal counts of labeled somata in the weeks following the lesion indicate that only relatively few neurons may occasionally died or otherwise fail to regenerate their original projections. Thus our general conclusion closely parallels that of Chase and Kamil ('831,who similarly noted nearly complete restoration of cellular morphology following regeneration of the tentacle tip in Achatina. Since comprehensive studies have so rarely been conducted, it is presently unclear if Achatina is especially proficient in regeneration or if such findings of essentially complete restoration would be generalized to other species. Thus, while many of our findings are consistent with past reports, some other findings discussed above appear to contradict our expectations based on past literature. The reasons for the unexpected findings are presently unclear. Presumably, at least part of the explanation involves species differences and/or well known differences in the regenerative responses of individual neurons. Unfortu-

'85).

Taken in toto, the comprehensive nature of our study permitted us to evaluate the generality of injury-induced responses across different classes of neurons projecting to ciimerous targets. As discussed above, it allowed for very specific comparisons of such population responses to the types of responses previously noted in identified cells. One comparison, however, which is inherent in the preceeding

AXONAL REGENERATION AND SPROUTING IN ACHATINA nately, as previously mentioned, so much of our knowledge of gastropod neural regeneration is derived from observations of relatively few cells in only a few species that the contributions of these factors are difficult to assess. It should be noted, however, that certain species differences might be significant. For example, each MCG in Achatina is known to possess several parallel axons within the CBC (Croll et al., '871, whereas only a single stouter axon is present in such well-studied species as Helisoma (Murphy et al., '851, Lymnuea (Croll and Chiasson, '89), and Aplysia (Weiss and Kupfermann, '76). Given these species differences, it is perhaps not surprising that differences might also arise in the regulation of fiber numbers following injury. Furthermore, in the present study we were able to examine axonal regeneration by cells that are much smaller than the identified neurons that are normally the subjects of most studies. In fact, in terms of sheer numbers, these populations of smaller cells predominated in our analyses. Since gastropod cells lying within discrete clusters may undergo very different ontogenies than the larger identifiable cells (Croll and Chiasson, '891, it is possible that their regenerative responses may differ as well. Thus, while the eventual restoration of labeling in the present study appeared to be generalized across both large and small cell bodies, more subtle differences influencing the time course of events or the relative numbers of fibers might arise from cell-specificdifferences. While our approach to understanding regeneration in gastrogods has several advantages over previous lines of investigations, it, like all avenues of research, has its own inherent weaknesses. For instance, use of whole mount preparations limits the resolution of the very finest fibers and the numeration of large numbers of closely packed fibers. Furthermore, we are left with questions of the fate of severed axonal segments, of the functionality of regenerated and sprouted fibers, and of the physiological and biochemical cues that might initiate sprouting and retraction. Obviously, a complete understanding of the responses of such a simple nervous system to injury will arise from a fully integrated approach ranging from the molecular and ultrastructural levels through behavioral analysis. The present detailed study of changes in populations of neurons after injury provides one means of broadening our perspective in the direction of such an integrated approach.

ACKNOWLEDGMENTS This work was supported by an operating grant from NSERC (Canada) to R.P.C. We thank Raym0ndY.S. Lo and Judith Skeat for help with some of the surgery and Bernard J. Chiasson for useful criticisms on an earlier version of this report,

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Chase, R., and R.P. Croll (1981) Tentacular function in snail olfactory orientation. J. Comp. Physiol. 143Ar357-362. Chase, R., and R. Kamil(1983)Morphology and odor sensitivity of regenerated snail Achatina fulica tentacles. J. Neurobiol. 14t34-50. Cohan, C.S., P.G. Hadon, A.J. Mercier, and S.B. Kater (1987) Formation, maintenance, and functional uncoupling of connections between identified Helisoma neurons in situ. J. Neurobiol. 18:329-341. Croll, R.P. (1978) A Neuroethological Study of Plasticity of Food-Finding Behavior of the African Land Snail Achatina fulica. PhD thesis, Montreal: McGill University. Croll, R.P. (1985) Search for the metacerebral giant cell in diverse gastropods. SOC.Neurosci. Abstr. 11:627. Croll, R.P. (1986) Modified cobalt staining and silver intensification techniques for use with whole-mount gastropod ganglion preparations. J. Neurobiol. 17:569-576. Croll, R.P. (1987) Identified neurons and cellular homologies. In M.A. Ali (ed): Nervous Systems in Invertebrates. New York: Plenum, pp. 41-59. Croll, R.P. (1988) Distribution of monoamines within the central nervous system of the juvenile pulmonate snail, Achatina fulica. Brain Res. 4602949. Croll, R.P., M.W. Baker, R.C. Swetnam, and R.Y.S. Lo (1987) Regeneration and neural plasticity following axotomy of an identified gastropod neuron. SOC.Neurosci. Abstr. 13:974. Croll, R.P., and B.J. Chiasson (1989) Postembryonic development of serotoninlike immunoreactivity in the central nervous system of the snail, Lymnaea stagnalis. J. Comp. Neurol. 280:122-142. Fredman, S.M. (1987) Intraceliular staining of neurons with nickel-lysine. J. Neurosci. Methods 2Ot181-194. Fredman, S.M., and P.G. Gage (1986)A conditioning lesion promotes axonal regeneration and synapse formation in a giant neuron in Aplysia. Neurosci. Abstr. 12r278. Fredman, S.M., and P.G. Nutz (1988) Regeneration of identified neurons and their synaptic connections in the central nervous system of Aplysia. Am. Zool. 28t1099-1108. Hadley, R.D., R.G. Wong, S.B. Kater, D.L. Barker, and A.G.M. Bulloch (1982) Formation of novel central and peripheral connections between molluscan central neurons in organ cultured ganglia. J. Neurobiol. 13.217-230. Hall, G.F., and M.J. Cohen (1988) The pattern of dendritic sprouting and retraction induced by axotomy of lamprey central neurons. J. Neurosci. 8:3584-3597. Haydon, P.G., D.P. McCobb, and S.B. Kater (1987) The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesisby serotonin. J. Neurobiol. 18:197-215. Hoy, R., G.D. Bittner, and D. Kennedy (1967) Regeneration in crustacean motoneurons: Evidence for axonal fusion. Science 156t251-252. Hubel, D.H., T.N. Wiesel, and S. LeVay (1977) Plasticity of ocular dominance Lond. LBiol.1 columns in the monkey striate cortex. Philos. Trans. R. SOC. 278:377409. Jackson, P.C., and J. Diamond (1981) Regenerating axons reclaim sensory targets from collateral nerve sprouts. Science 214:926-928. Macagno, E.R., K.J. Muller, and S.A. DeRiemer (1985) Regeneration of axons and synaptic connections by touch sensory neurons in the leech central nervous system. J. Neurosci. 5.2510-2521. Maetzold, D.J., and A.G.M. Bulloch (1986) Sprouting by undamaged adult molluscan neurons: Putative role for changes in haemolymph osmoregulation. J. Exp. Biol. 122t427-432. Mathews, M.R., and V.H. Nelson (1975) Detachment of structurally intact nerve endings from chromatolytic neurons of rat superior cervical ganglion during the depression of synaptic transmission induced by post-ganglionic axotomy. J. Physiol. (Lond.) 245:91-135.

286 Mendell, L.M., J.B. Munson, and J.G. Scott (1976) Alterations of synapses on axotomixed motoneurons. J. Physiol. (Lond.) 255:67-79. Moffett, S., and D.R. Austin (1982) Generation of new cerebral ganglion neurons in the snail Melampus: An ultrastructural study. J. Comp. Neurol. 207: 177-182. Moffett, S.B., and R.L. Ridgway (1988a) Structural repair and functional recovery following cerebral ganglion removal in the pulmonate snail Melampus. Am. Zool. 28:1109-1122. Moffett, S.B., and R.L. Ridgway (1988b) Central ner