/. Embryo!, exp. Morph. Vol. 36, 1, pp. 19-39, 1976
Printed in Great Britain
19
Target discrimination in regenerating
insect sensory nerve
By MIRIAM McLEAN AND JOHN S. EDWARDS1
From the Department of Zoology, University of Washington
SUMMARY
The paired abdominal cerci of the cricket Acheta domesticus are mechanosensory appendages which regenerate readily when amputated during larval life. Their peripherally-located
sense cells form axons which project centrally as a purely sensory nerve to the terminal
abdominal ganglion.
In an attempt to analyze some of the factors which guide a regenerating sensory nerve to
correct central terminations, implants of homologous, supernumerary terminal ganglia were
made in cricket larvae and the host cerci amputated. The possibility that implants with
multiple nerve stumps might release an attracting substance was considered. Surgical procedures used were (1) implant in posterior abdomen; (2) implant in posterior abdomen,
ipsilateral to chronic cereal deprivation; (3) implant in mesothoracic leg socket, adjacent to
heterotopically-transplanted regenerated cercus; (4) implant in posterior abdomen, ipsilateral host cereal motor nerve sectioned; (5) implant in posterior abdomen, ipsilateral margin
of host terminal ganglion wounded. Results were determined after the adult molt, by conventional histology or by cobalt chloride filling of regenerated cereal nerves.
In all procedures except (3) and (4), the regenerated afferent nerve bypassed the implant and
terminated in the host terminal ganglion. In (3), the regenerated fibers from cereal grafts
bypassed the implant; terminations were not found. In (4), some regenerated cereal axons
connected with the implant and the majority terminated in the host ganglion.
It is suggested that regenerating cereal afferents may depend in a facultative way on the
cereal motor nerve as a pathway guide but there is as yet no clear evidence for a trophic
influence from the central nervous system.
INTRODUCTION
The development in arthropods of functional neural connexions between a
regenerating sense organ and the central nervous system requires that the
regenerating neurons first extend axons from the integument to the central
ganglion, and then establish appropriate synaptic connexions within the neuropile. This study is concerned with the first of these processes, the establishment
of a pathway between periphery and center.
The abdominal cerci of the cricket, Acheta domesticus, provide appropriate
material for such studies since they regenerate well, and have been used in
studies of central connexion formation (Edwards & Sahota, 1967; Edwards &
Palka, 1974; Palka & Edwards, 1974).
1
Author's address: Department of Zoology, University of Washington, Seattle, Washington 98195, U.S.A.
20
MIRIAM McLEAN AND JOHN S. EDWARDS
Cerci are paired elongate mechanosensory appendages arising from the
dorso-posterior angles of the abdomen. They lack intrinsic muscles but are
capable of limited movement by means of extrinsic dorsal muscles. Their
limited mobility is sufficient to facilitate grooming and the support of the female
by the male during copulation; they execute no evident movement in relation to
their sensory function. The cerci are densely clothed with sensilla of three basic
types, which have been described in detail by Edwards & Palka (1974). The
cereal sensory nerve (N lOd) in the adult contains about 10500 axons (Edwards,
1971). This nerve terminates almost entirely ipsilaterally in the terminal abdominal ganglion and provides a major input to the two giant interneurons, 8-1 and
9-1, whose axons traverse the length of the ventral cord (Murphey, Mendenhall,
Palka & Edwards, 1975). According to present knowledge, the cereal sensory
nerve (N lOd) is entirely afferent; although the possibility that it may contain a
small efferent component cannot be dismissed (d'Ajello, Bettini & Casaglia,
1972). This nerve is entirely separate from the smaller adjacent cereal motor
nerve, which branches from N lOv. Essential features of the cereal sensory
nerve-giant interneuron system are summarized in Fig. 1.
After repeated cereal amputation or nerve section, degeneration of the proximal stump sheath follows degeneration of the sensory nerve. Thus, there is no
obvious physical substrate that might supply contact guidance for regenerating
afferent fibers. In experiments where crickets were deprived of cereal input
during development by repeated amputation of postmolt regenerate cerci
(Palka & Edwards, 1974), the surface of the adult terminal ganglion at the N lOd
locus was smoothly healed and covered with neural lamella. Nevertheless, if such
crickets were permitted to regenerate cerci toward the end of postembryonic
development, they did so rapidly and with apparently correct afferent terminations.
Working with heterotopically-grafted regenerate cerci in cricket larvae,
Edwards & Sahota (1967) found that afferent fibers, arising from successful
grafts at the mesothoracic leg socket, made connexions with the host's central
nervous system. These contacts were found in the vicinity of the giant interneuron axons, the same cells with which the cereal nerve normally synapses in
the terminal ganglion. Moreover, extracellular recordings from abdominal
connectives indicated that the giant axons were excited by stimuli applied to the
heterotopic regenerate cercus.
Thus, regenerating cereal axons are capable of (1) routing a correct course
without the guidance of a pre-established path, (2) penetrating a healed ganglion
in the correct region, and (3) recognizing appropriate post-synaptic cells in an
abnormal region.
These results suggest questions that might be asked to determine the nature
and precedence of the cues that allow regenerating sensory axons to connect
with their target organ. For example, can regenerating fibers be diverted from
their normal course and termination by the presence of another potentially
Regenerating insect sensory nerve
21
Fig. 1. Schematic diagram of Acheta terminal ganglion (TG) and neural connexions
of the cercus (CE). Cereal sensory axons (SC) with cell bodies located beneath the
cereal integument (CI) send fibers through the cereal sensory nerve (CSN) to the
terminal ganglion where they synapse principally with giant interneurons, of which
two (MGI, LGI) with their cell bodies (8-1, 9-1) are shown. The cereal motor
nerve (CMN) arises from nerve 10 v and innervates the extrinsic cereal musculature
at the base of the cercus. GN - genital nerve.
acceptable target? Their response when presented with a homologous, supernumerary ganglion might help to sort out the various possible cues operating
during afferent regeneration. If the diffusion of 'wound factors' (Bodenstein,
1957) from damaged nerve occurs, this tissue could supply such a directional
signal to the regenerating axons. The release of material from the multiple
wound sites of an implanted ganglion might then be sufficient to override other
cues that normally influence regenerating fibers. Another question can be posed:
In the event that the nerve recognized the implant, would it make contact at the
appropriate (N lOd) region, or simply enter the nerve stump first encountered?
Finally, how would regenerating axons respond to the presence of a potentially
increased central field size?
Working with Acheta domesticus, Rummel (1970) came to the conclusion that
successful regeneration of a cercus depends upon an uninterrupted nerve supply,
inferring the dependence of sensory regeneration on a trophic factor distributed
by the motor nerve. This hypothesis brings up another question regarding the
requirement for an intact cereal motor nerve in sensory regeneration.
22
MIRIAM McLEAN AND JOHN S. EDWARDS
Implants of healthy, conspecific tissues generally grow and become tracheated
by the insect host. In the case of ganglia, such grafts often participate in patterns
of reciprocal motor and interneuronal innervation with the host system (Bodenstein, 1955, 1957; Guthrie, 1966; Jacklet & Cohen, 1967; Guthrie & Banks,
1969). The relationships of host to graft in the case of afferent nerves are less
well documented.
The observations to be described here are endpoint results; the outcome of
experiments was not ascertained until after the adult molt. The supernumerary
ganglion was never finally accepted as the appropriate target of entire regenerated cereal nerves, except that we found small populations of regenerated cereal
fibers within the implanted ganglion in cases where the host's ipsilateral cereal
motor nerve trunk was cut at the time of implantation.
METHODS
Rearing and development of Acheta domesticus. By providing gravid females
from mass cultures (Fluker's Cricket Farm, Baton Rouge, La.) with receptacles
of damp sand for oviposition, continuous supplies of young crickets were made
available.
All animals were housed in a controlled-environment chamber at 27-28 °C,
about 70% relative humidity, with a photoperiod of 16-h light and 8-h dark;
they were fed on 'Little Friskies' dried cat food (Carnation Co.) and fresh
lettuce. Stock animals were reared in small groups, experimental animals in
isolation.
The number of instars required for development of Acheta domesticus varies
according to culture conditions. Our animals invariably passed through nine
instars before the final molt to the adult. The duration of stadia is summarized
in Table 1.
Surgery. All operations described below were performed on 6th to 8th stage
larvae at a time before the midpoint of the stadium, that is, before apolysis.
Post-apolysis larvae were less able to withstand surgical trauma. The diagrams
of Fig. 2 illustrate the five categories of surgery that comprised these experiments.
For ganglion transfer experiments, two animals were immobilized by chilling
to about 4 °C, then supported venter up by means of filter paper strips pinned
on each side to a thin wax plate attached to a covered plastic container of ice
water. In all experiments except those illustrated in Fig. 2C, the host's penultimate sternite was freed on three sides with fine sharp scissors and braced open
with a pin. To prevent desiccation of tissues and coagulation of hemolymph, a
saline based on cricket hemolymph (Levine, 1966) was applied. The terminal
ganglion of the donor animal was rapidly excised and rinsed in Levine's solution.
It was then placed in the host's hemocoel, posterolateral to the autogenous
terminal ganglion, and toward the origin of a forthcoming cereal nerve regenerate. The flap of cuticle was then repositioned, excess fluid blotted off, and
the wound allowed to seal by hemolymph coagulation.
Regenerating insect sensory nerve
23
Table 1. Development o/Acheta domesticus under controlled conditions*
Instar
1
2
3
4
5
6
7
8
9
A
Range of
duration
(days)
3-5
4-5
4-5
4-7
5-8
6-9
Mean no.
of days/stadium
4
4
4
5
6
6
7
9
10
—
Cumulative
duration
(days)
4+1
8±1
12±1
17±2
23 + 2
29±2
36±3
45 ±3
55 ±5
6-11
7-11
9-14
Approx. 6 months
* 27-28 CC; 16-h L/8-h D photoperiod (incandescent lamp); approx. 70% relative
humidity.
Host animals for the experiments diagrammed in Fig. 2B were reared without
one cercus by means of repeated extirpation up to the 7th or 8th stage. This was
done by removing the regenerated cereal 'button' just after each molt.
Control experiments to determine the response to cereal motor nerve section
were done by cutting the entire motor root, N lOv, at its exit from the terminal
ganglion (see Fig. 1). This nerve divides about halfway down its length to supply
genitalia with one branch and cereal base musculature with the other. Isolating
and sectioning only the cereal motor branch in a larval animal is technically
difficult and requires more extensive surgery than sectioning the entire root near
the source.
In the implant experiments diagrammed in Fig. 2D, E, nerve section or
wounding of the host ganglion was done just before inserting the implant. Host
ganglia were wounded either by sectioning the roots of N 8 (see Fig. 1) or
puncturing at the midlateral margin, ipsilateral to the implant.
For the heterotopic implant-cereal graft experiments (Fig. 2C), donors had
been cercectomized two molts earlier, so that they bore small regenerate cerci.
After amputating the host's mesothoracic leg at the coxofemoral joint, the donor
terminal ganglion was inserted through the wound. The donor cereal regenerate
was amputated and applied to the host's leg socket and held until it adhered by
coagulated hemolymph.
No antiseptic precautions were found necessary, other than cleaning instruments in 70 % ethanol.
Host animals were cercectomized by grasping the cercus at the base with fine
forceps and pulling it cleanly away. The cereal motor nerve was left intact by
this operation.
Nerve tracing and histology. Animals were chilled to immobility and injected
with paraformaldehyde-glutaraldehyde fixative (Edwards & Palka, 1974)
24
MIRIAM McLEAN AND J O H N S. EDWARDS
Fig. 2. Surgical procedures used in this study. (A) Simple terminal ganglion (TG)
implant with concurrent ipsilateral cercectomy. (B) TG implant, host reared
without ipsilateral cercus, bilateral cercectomy concurrent with implantation.
(C) Heterotopic TG implant and cereal graft on mesothoracic leg base. (D) TG
implant, ipsilateral cereal motor nerve sectioned, concurrent bilateral cercectomy.
(E) TG implant, ipsilateral host TG wounded, concurrent bilateral cercectomy.
through the lateral mesothoracic intersegmental membrane. After one-half to
one hour, the animal was dissected and the nerve tissue immersed in fixative for
1 to 2 h at room temperature.
Simultaneous fixation and staining of tissues by immersion in a solution of
0-5% thionin in 10% formalin for a period of 1 to 4 days (Ehrlich, in Gurr,
1953) provided a method for nerve tracing in which cell bodies were stained blue
and fibers pink. The distortion produced by this treatment was reduced by
prefixing tissues as described above for 1-5 days, then transferring them to the
thionin-formalin solution for 2 days. Tissues were embedded in paraffin and
sectioned at 8-15 /im.
Sections pre-treated with thionin were dewaxed and cleared in toluene and
mounted in Permount. Sections not previously stained were hydrated and
stained in 0-1 % aqueous toluidine blue.
Regenerating insect sensory nerve
25
Fig. 3. Dissection of adult with simple ganglion implant (procedure A). Implant
(Im), in host 36 days, is well tracheated, with multiple connexions to nearby
tracheal trunks (Tr). Scale: 0-5 mm.
In some experiments, regenerated cereal nerves were filled with cobalt
chloride in order to visualize their terminations. The cereal nerve was cut at the
base of the cercus, then the terminal ganglion including about 1 mm of connectives and other associated nerve stumps was excised from the host. The distal
cereal nerve stump was immersed in 200 mM CoCl2 (aq.) at 5 °C for 18 to 24 h.
Levine's saline (1966) was used for rinsing and sulfide development, and the
part of the tissue not exposed to CoCl2 during treatment was immersed in
Schneider's Drosophila medium. Tissues were fixed for 1 to 3 h in aldehyde
fixative, then prepared for paraffin histology. Sections were cut at 10 to 20 fim.
and processed with Timm's intensification method as modified by Tyrer & Bell
(1974).
RESULTS
The data from each of five categories of experiments are summarized in
Table 2. All regenerated cereal sensory nerves grew to the autogenous (host)
ganglion, except where the ipsilateral cereal motor nerve trunk was sectioned
(category (4)).
Experimental animals grew and molted normally, although maturation times
occasionally exceeded the normal range. Regenerated cerci were indistinguishable from those of control animals without implants.
Implanted ganglia invariably became attached, usually by means of scar or
connective tissue, to the body wall near the point where they were originally
2A
2B
2C
Would nerve stumps of implant be more attractive to
regenerate CSN than healed
cereal nerve entry region of
hostTG?
Since regenerate afferent
fibers from a heterotopic
cercus are capable of ' recognizing' the postsynaptic cell
at an atypical site (Edwards
& Sahota, 1967) would this
still be the preferred connexion if an implanted TG
were available in the same
region?
Fig. no.
Will regenerate cereal
afferents connect with a
supernumerary implanted
terminal ganglion?
Question
(2) Implant TG in
host reared without
ipsilateral cercus;
remove contralateral
cercus and ipsilateral
cereal 'bud'
(3) Implant TG and
graft regenerate
cercus heterotopically to mesothoracic leg socket
(1) Implant terminal
ganglion (TG);
remove ipsilateral
cercus
Operation
14
Heterotopic regenerate CSN
bypassed implanted TG.
Neural connexions between
implanted TG and host
Meso-G; between implanted TG and host
muscles
13-22
8-23
Regenerate cereal sensory
nerve (CSN) established connexions only with host
(autogenous) TG. Neural
connexions between host
and implanted TGs in three
cases
Regenerate CSN as above. No
neural connexions between
host and implanted TGs
Results
18-44
Implant
No. of
No. of
survival
animals successful (surgery to
operated operafixation)
on
tions
(days)
Table 2
o
o
w
2D
2B
Does the host cereal motor
nerve (CMN) provide a
guidance cue for the
ipsilateral regenerate CSN ?
Is disruption of the regenerate
CSN pathway a consequence
of host ipsilateral TG damage
and not necessarily a specific
response to N 10 v section?
(5) Implant TG,
wound ipsilateral
margin of host TG
or cut N 8; remove
both cerci
(4) Implant TG, cut
ipsilateral host
N 10 v; remove
both cerci
Operation
10
16-17
18-33
* One with normal regenerate cercus, one with heteromorphic structure.
Fig. no.
Question
Implant
survival
No. of
No. of
animals successful (surgery to
fixation)
operated operations
on
(days)
Table 2 (cont.)
In each case, some regenerate
CSN fibers went to implanted TG, most to host
TG; entire regenerate CSN
contacted implant at least
superficially on way to host
TG. Neural contacts between
host and implanted TGs;
neuromuscular contacts
between implant and host
muscle
Regenerate CSN established
connexions only with the
host TG. Neural connexions
between host and implanted
TGs
Results
I'
1
28
MIRIAM MCLEAN AND JOHN S. EDWARDS
Regenerating insect sensory nerve
29
inserted. They increased in size in parallel with the autogenous terminal ganglion,
and were amply tracheated with outgrowths from the host system (Fig. 3).
Although there were varying degrees of internal disorganization and local
degeneration, implanted ganglia produced fiber outgrowth volumes consonant
with their length of time in the host. These outgrowths came from motor or
mixed nerve or connective stumps; cereal sensory nerve stumps, where recognizable, had degenerated. Attachments between fibers originating from implants
and host muscle occurred in most cases.
Simple implants (Fig. 2 A). In all eight cases, the regenerated cereal sensory
nerve established connexions in the appropriate region of the host terminal
ganglion. Even in situations where the base of the regenerated cercus was
clearly nearer to the implant than to the host ganglion, we found no regenerated
afferent fibers contacting the implant.
Implant in 'deprived' host (Fig. 2B). In all four adults the ipsilateral cereal
sensory nerve made contact only with the host terminal ganglion. No neural
connexions linking the implanted and host ganglia were found.
Heterotopic implant with cereal graft (Fig. 2C). Of the five animals subjected
to this operation, three lost the grafted cercus before or during the subsequent
molt. Two animals retained grafts, but the cereal form was maintained in only
one (Fig. 4 A). Serial sections of the heteromorphic structure in the other animal
revealed no sensory cells. Sections from the animal with the successful heterotopic cercus revealed that the afferent fiber bundle formed from sensory cells
in the graft had bypassed the implanted ganglion and had grown toward the
host's ventral cord (Fig. 4B).
Motor nerve section. Preliminary operations were done on 16 animals to
determine survival rates. In these animals both motor nerves (N lOv) were
sectioned, both cerci removed, and a ganglion was implanted. Two reached the
last molt; most dying within 4 days after surgery. A control group of 12 animals
had only one N lOv sectioned, and one or both cerci removed. In animals that
reached adulthood, we found both motor and sensory cereal nerves had
regenerated and made anatomically correct connexions. The only abnormality
found upon histological examination was a mixing of fibers from sensory and
motor bundles near the cereal base (Fig. 4D). More proximally, the two major
bundles separated and were attached at normal positions on the terminal
FIGURE 4
(A) Adult female with heterotopic TG implant (procedure C) and cereal graft (Ce)
to mesothoracic leg base. Scale: 1 mm. (B) Horizontal section to mesothorax showing implanted ganglion (Ig) and base of cercus below, with sensory nerve (N) entering leg base. Toluidine blue. Scale: 100 /*m. (C) Base of normal regenerate cercus
showing separate cereal motor (CM) and cereal sensory (CS) nerves. Arrow indicates
direction of cercus tip. Thionin stain. Scale: 100/*m. (D) Base of regenerate cercus
in animal with cereal motor nerve cut three instars earlier (procedure D), showing
mixed motor and sensory nerve (SMN). Thionin stain. Scale: 100 /im.
30
MIRIAM McLEAN AND JOHN S. EDWARDS
A
Tr
Fig. 5. Sketches of host and implanted ganglion and their relationships. (A) Ipsilateral nerve lOv cut at time of ganglion implantation. Implant (Im) has massive
neuroma to left. Regenerate cereal sensory nerve (CSN) reaches host ganglion (HG)
via implant. (B) Ganglia from animal with lateral wound (W) on host ganglion (HG)
ipsilateral to implanted ganglion (Im). Neural connexion formed between neuroma
at wound site and implant, which is heavily tracheated (Tr). Cereal sensory nerves
(CSN) from normal (left) and regenerate (right) cerci connect with host ganglion
(HG).
ganglion. Cerci regenerated by these animals were indistinguishable from normal
regenerates.
Implant with cut motor nerve (Fig. 2D). Five out of ten operated crickets
became apparently healthy adults. Tissues from two of these were processed
with the thionin method, and cobalt preparations of the ipsilateral cereal
sensory nerve were made with three.
Both cereal motor and sensory nerves regenerated, but the sensory nerves had
attached to both the implanted and the host terminal ganglia. A sketch of the
ganglia from one of these adults in Fig. 5 A represents a typical pattern of
connexions seen in this category.
Regenerated fiber bundles were associated with small glial cells and were not
Regenerating insect sensory nerve
C
B
Fig. 6 (A and B): Horizontal 10 /tm paraffin sections, CoCl2 treatment, of host and
implanted ganglion in host with previously cut N lOv (procedure C). Implant in
host 33 days. Sections A and B separated vertically by about 20/*m. Pathway of
regenerated cereal sensory nerve (CSN and arrow) connects with implanted ganglion
(IG) by way of neuroma the remainder of the CSN diverts to the host ganglion (HG).
Scale: 100/.im. (C) Projection of cobalt-filled regenerate cereal sensory fibers in
28-day implant ganglion; 20/tm paraffin section. Scale: 100/tm. (D) Periphery of
healthy 33-day implant ganglion. PN, intact perineurial sheath. CB, neuron cell body.
10/tm paraffin section, glutaraldehyde fixation, osmium staining. (E) Degenerating
25-day implant ganglion, perineurial-neuropile zone, invaded by hemocytes (H),
which may be distinguished from glial cells (G) by the density of their nuclei. 10 /*m
paraffin section, toluidine blue stain. Scale for D and E: 10/*m.
31
32
MIRIAM McLEAN AND JOHN S. EDWARDS
D
Regenerating insect sensory nerve
33
as clearly delineated as bundles in normal material. In every case it was possible
to trace the regenerated sensory nerve from the base of the ipsilateral cercus to
contact first with the implant, and further to contact with the host terminal
ganglion (Fig. 6A and B). The great majority of afferent fibers appeared to
terminate in the host ganglion but examination of CoCl2-filled tissues revealed
a few fiber terminations in the implant (Fig. 6C).
CoCl2 projections in host ganglia were not as complete as those typically seen
in the case of normally developed sensory neurons. This would be expected,
since afferent projections from regenerated cerci in control animals are less
dense than those from normal cerci, the reduced quantity of fibers evidently
reflecting the time available for regeneration (Fig. 6C).
Implanted ganglia appeared to be healthy, and in general their symmetry was
well preserved (Fig. 6D). In one case, the motor nerve (N lOv) of the implant
made contact with muscles of the host's cereal base, ipsilateral and parallel to
the afferent nerve from the regenerated cercus to the implant.
The striking results of this group of experiments indicated that the cereal
motor nerve - or at least N lOv - influenced the growth direction of sensory
fibers, but whether the influence was direct or secondary was still unclear.
Ganglion implant with simultaneous wounding of host terminal ganglion
(Fig. 2E). This operation gave rise to moderate distortions of overall morphology and neuropile patterns of the host ganglion. Fig. 5B is a diagrammatic
sketch made from a dissection of one of these animals: the relationships were
similar in the other two cases. In every instance, host and donor ganglia were
complexly interconnected by way of an outgrowth between the initial wound
site of the host ganglion and one or more motor or connective trunks of the
implanted ganglion (Fig. 7C). However, tracing of serial sections revealed that
all the regenerate cereal sensory fibers bypassed the implant and terminated ih
the ipsilateral portion of the host ganglion (Fig. 7D). Since there was a varying
degree of disorganization of the host neuropile brought about by wounding,
pattern abnormalities appeared in the CoCl2-filled afferent terminations.
Non-sensory host-implant connexions. As indicated in Table 2, neural contacts
between host and implanted ganglia occurred in four out of five categories.
These connexions were formed by way of cut nerve stumps or damaged areas
of ganglion. Outgrowths from implants occasionally resulted in neuromas
FIGURE 7
(A and B) Cobalt preparations of cereal sensory nerves in terminal ganglion, whole
mounts. Dashed line is midline. (A) Normal adult. (B) Adult with regenerated
cercus. Scale: 100 jum. (C and D) Animals with wounded host ganglia (treatment E).
(C) Nerve connexions (arrow) between host (HG) and implanted ganglion (IG).
Wound region of host ganglion at left (W). Implant in host 32 days. Toluidine blue
stain. Scale: 100 /*m. (D) Projection of regenerated cereal sensory nerve into host
TG. Implant in host 28 days. 10/*m paraffin sections. Scale: 100/tm.
3
EMB 36
34
MIRIAM McLEAN AND JOHN S. EDWARDS
(Fig. 6 A) which often served as channels for interconnexion between host and
implant.
Neural connexions between the implant and host muscle were observed in
three categories. The quantity of these contacts appeared to be related posit'vely
to the health of the implant and to the amount of the time in the host. The only
known specificity exhibited in this class of connexions was the single case
mentioned above in which the regenerated N lOv of the implanted ganglion
made contact with muscles of the host's cereal base.
Cellular migrations, proliferations; degenerative reactions. Nerve cells within
implants usually appeared normal histologically (Fig. 6D). In two cases from
the first category of experiments (Fig. 2 A), and in one from the second category
(Fig. 2B), degenerative changes in the implants were pronounced. In most
animals of the first category variable numbers of small cells were dispersed
throughout the implants and their outgrowths (Fig. 6E). These appeared to be
mainly small glial cells, but on the basis of comparison with hemolymph
smears, also included hemocytes.
Summary of results
(1) In all surviving animals, normal regenerate cerci were produced.
(2) With the single exception of cereal afferents in experiments in which
N lOv was sectioned, regenerated cereal sensory fibers bypassed the implant to
terminate in the host terminal ganglion.
(3) When implantation was combined with section of host N lOv, regenerating
cereal afferents were diverted to the implanted ganglion, and a few fibers
terminated within it.
(4) In no instance did regenerate afferent (host) cereal nerves terminate
entirely in the implanted ganglion.
(5) In instances where contacts were made with the implanted ganglion,
afferent fibers from the regenerate cercus did so in the appropriate region, that
is, through part of the old sensory nerve stump.
(6) Implants were always attached to host tissues. They were supplied with
tracheae, grew in size during maturation of the host, and put forth nerve outgrowths.
(7) Nerve-muscle contact between donor and host, respectively, was found
frequently. Nerve contacts between donor and host ganglia occurred in over
half the cases.
(8) Nerve outgrowths from donor ganglia arose from motor or connective
stumps or from other wounds; sensory cereal nerve stumps of implants, where
recognizable, were degenerate.
(9) Glial cells responded to the changed circumstances of these experiments
by accumulating in greater than normal numbers at sites of trauma, regeneration, and abnormal growth, and by atypical migrations within implants.
Regenerating insect sensory nerve
35
DISCUSSION
Target discrimination in regenerating nerves
Experimental studies of neural regeneration in orthopteroid insects, exploiting
their capacity for sensory and motor regeneration and for sustaining implanted
ganglia, have demonstrated the vigor of nerve growth and specificity in the
restoration of functional connexions. It is clear that severed motor nerves
regenerate in immature and adult stages (Bodenstein, 1957) and can recognize
their correct target (e.g. Jacklet & Cohen, 1967; Pearson & Bradley, 1972) or
their homologue from another segment (Young, 1972), and that supernumerary
grafted limbs will acquire motor innervation even though the normal system is
intact (Sahota & Edwards, 1969).
Implanted ganglia have been shown to provide channels for motor innervation
and can innervate muscle (Guthrie, 1966; Guthrie & Banks, 1969). None of
these studies addressed the question of the factors that determine the pathway
of axons between their origin and target organs.
With sensory systems, similarly, the capacity for regeneration has been amply
demonstrated, but with the exception of Wiggles worth's (1953) demonstration
of the tendency of ingrowing sensory axons to follow pre-existing pathways, the
mechanisms of pathway determination have received little attention.
In our experiments sensory and motor connexions were made in the appropriate locations, as evaluated by endpoint histological examination, although
some pattern abnormalities developed. Regenerated afferent and efferent fibers
intermingled in the distal pathway; however they apparently became sorted out
and formed topographically normal terminations. Hamburger (1929) observed
that in the development of amphibian limbs the tips of pioneering nerve fibers
growing out of motor and sensory centers take different routes through the
tissue matrix, diverging at a considerable distance before reaching muscles or
skin, respectively.
If the situations in amphibians and insects are comparable with respect to
separation of types of growing nerves, it would be difficult to imagine how a
regenerating cereal afferent fiber might depend for directional cues upon a
motor nerve which, although running parallel to the sensory pathway, is
normally entirely separate from it throughout its course. Further, the regenerating motor and sensory axons linking two regions grow in opposite directions.
Yet the findings of this report show that, in the absence of the normal motor
nerve at the onset of sensory regeneration, afferent fibers innervate a supernumerary ganglion which they would bypass were the motor nerve intact.
The presence of an intact motor nerve is not required as a 'local guidepost'
for the regenerating sensory nerve to penetrate the ganglion in the appropriate
locus. Indeed, afferent fibers always entered the ganglion at the proper place,
whether the ganglion was an implant with no normal CNS connexions, or an
autogenous ganglion with the motor nerve severed at its origin. It may be that
3-2
36
MIRIAM McLEAN AND JOHN S. EDWARDS
pioneering regenerate afferents could be guided by adjacent motor fibers without making permanent attachments to them. Once the first group of sensory
fibers had terminated and separated from the motor nerve, then afferent fibers
developing in the following molts would be guided by those already in place.
Exploratory behavior of pioneer fibers in growth and regeneration has long
been known to occur in vertebrates. Since time-course observations were not
made in our studies, we cannot state that there were no temporary contacts
formed by regenerating axons which were subsequently aborted. Regenerating
afferent fibers might have first contacted the implant and then dissociated to
grow further until they reached the host ganglion. Why then were persistence of
sensory contacts in the implant associated only with experiments involving cut
and subsequently regenerated motor nerves ? Since motor fibers tend to make
attachments between host and implanted nerve tissue, it seems possible that the
regenerating host N lOv could have made temporary contact with the implant,
thus providing a guidance cue for pioneering afferents.
In their work with developing optic systems in Daphnia, Lopresti, Macagno &
Levinthal (1973) pointed out that the growth cone is not necessarily a feature of
all growing axons. They found that the lead axons from retinal ganglion cells
growing in to meet lamina neuroblasts were the only ones of the group to
produce growth cones. Furthermore, they suggested that this transient structure
may be functionally associated with the recognition of cell surfaces or spatial
relations by pioneer fibers. This idea is supported by a more recent paper
(Lopresti, Macagno & Levinthal, 1974), where transiently formed gap junctions
were found at these contact points.
The question remains, if cereal afferents depend upon intact motor nerve for
pathway guidance, how are they able to regenerate to their normal locations
when motor nerves are cut? The postulated dependence on contact with motor
nerves could be facultative and not absolute. We can suggest two possible
mechanisms: regenerating motor axons may have reached the periphery before
cereal afferents terminated in the ganglion, thus providing pathway guidance.
Alternatively, it may be that there exists a hierarchy of cues available to a
regenerating nerve, so that in the absence of a primary cue (in this case, the
intact motor nerve) a secondary one provides the requisite factor. Such a
secondary cue might be factors diffusing from the stumps of cut nerves.
Terminal contact formation. The evidence presented above suggests that once a
regenerating axon has established a contact, then the fibers following are
guided into the same pathway. However, at the level of synaptic terminals, other
processes must be operating, for only a small proportion of cereal afferents in
cut motor nerve experiments remained in the implant, as judged by CoCl2
techniques.
Reduction in dendritic fields after axotomy has been reported in work with
vertebrates (e.g. Cerf & Chacko, 1958; Sumner & Watson, 1971). Another postaxotomy phenomenon is that of' somatic stripping', the loss of synapses on cell
Regenerating insect sensory nerve
37
bodies and proximal dendrites. This has been measured electrophysiologically
(Kuno & Llinas, 1970a, b; Mendell, Munson & Scott, 1974), and observed
ultrastructurally in the form of glial displacement of synaptic terminals on
axotomized motoneurons (Blinzinger & Kreutzberg, 1968; Kerns & Hinsman,
1973). In our experiments, the damage in the form of loss of axon volume of
giant interneurons of the implant could have resulted in a deficit or deactivation
of synaptic sites for incoming fibers.
Trophic effects in cereal regeneration. A trophic role of efferent axons has been
inferred by Drescher (1960) who reported that antennae do not regenerate in
Periplaneta americana if deprived of all neural connexions with the brain, and by
Schoeller (1964) who concluded that differentiation of transplanted antennal
disks of antennae, compound eyes and palps of Calliphora erythrocephala
required innervation from the host.
An extensive exploration of the trophic role of the central nervous system in
the regeneration of cerci in Acheta domesticus is closely relevant to the present
study and must be critically assessed in relation to our own findings.
Rummel (1970) reported the effects of surgical intervention on cereal regeneration in Acheta domesticus larvae. The object of the study was to determine
the influence of the nervous system in the regeneration of sensory appendages.
Separation of the cercus from its nerve supply was accomplished by a deep
incision through the body wall in the middle of the angle formed by the cercus
and the adjacent abdominal margin. Presumably both motor and sensory nerves
were severed by this operation, but Rummel did not distinguish between the
two. Cerci degenerated disto-proximally until regeneration set in. The regenerated cercus was formed partly from the existing cereal base; in some cases
a small regenerate was formed in the wound region, just medial to the normallysituated cercus. Rummel suggested that maintenance of a normal cercus
depended on the presence of an uninterrupted nerve supply, and that production
of a regenerate must await the arrival of regenerated nerves in the wound area.
A comparable operation, but with the insertion of barriers of mica, plastic
film, or aluminum foil yielded more varied results. The aim was to prevent
normal regeneration or induce supernumerary regenerates by diverting nerves
from their normal course. Mortality of operated animals was higher, and cereal
regeneration reduced. Various abnormalities included distal degenerative
changes in the contralateral cercus. Rummel inferred the need for a centrifugally
moving substance from the central nervous system, requiring neural connexion
between the central ganglion and the site of regeneration following the well
established vertebrate model. Several questions arise from this interpretation.
First, the innervation of the cereal musculature, the only possible pathway for
a neurogenic stimulus to cereal regeneration since sensory fibers arise from the
cercus itself, is entirely extrinsic to the cercus and has no known terminations
on the epidermis in the tissue from which the sensory regenerate arises.
Secondly, the diminution of cereal regeneration obtained by interposition of
38
MIRIAM McLEAN AND JOHN S. EDWARDS
barriers could be due to the diversion of hemolymph and tracheation by the
barrier and scar tissue. The cerci lack pulsatile organs at their base which ensure
hemolymph circulation in other elongate appendages such as antennae, and
may thus be sensitive to alterations of patterns of hemolymph flow which could
lead to local stagnation and thus to tissue necrosis.
A simple method for testing the importance of the motor nerve in cereal
regeneration would be to remove the host terminal ganglion, thereby preventing
efferent regeneration. This experiment was attempted with 7th, 8th and 9th
stage larvae but none survived beyond ten days and none molted. Presumably
the loss of regulating functions such as control of water balance accounted for
the mortality. Cutting all nerves to the host terminal ganglion and leaving it in
situ gave the same negative results in four animals. A decisive test of the need
for centripetal innervation by means of such simple surgical intervention thus
seems to be precluded. The less drastic expedient of severing the cereal motor
nerve alone at its exit from the terminal ganglion had no significant effect on the
time span or quality of cereal regeneration. We conclude that firm evidence for
a trophic role of central innervation in the development of cereal regenerates
has not yet been demonstrated.
This work was supported by Developmental Biology Training Grant HD-00266 from
NICHHD and by research grant NB 07778. We thank Drs John Palka, Eldon Ball and
Robert Seecoff for critical reading of the manuscript and for helpful discussion.
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(Received 3 November 1975; revised 19 February 1976)