target discriminatio in regeneratinn g insect sensory nerve · embryo!, exp. morph. vol. 36, 1, pp....

/ . Embryo!, exp. Morph. Vol. 36, 1, pp. 19-39, 1976 19

Printed in Great Britain

Target discrimination in regeneratinginsect 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 append-ages which regenerate readily when amputated during larval life. Their peripherally-locatedsense cells form axons which project centrally as a purely sensory nerve to the terminalabdominal ganglion.

In an attempt to analyze some of the factors which guide a regenerating sensory nerve tocorrect central terminations, implants of homologous, supernumerary terminal ganglia weremade in cricket larvae and the host cerci amputated. The possibility that implants withmultiple nerve stumps might release an attracting substance was considered. Surgical pro-cedures used were (1) implant in posterior abdomen; (2) implant in posterior abdomen,ipsilateral to chronic cereal deprivation; (3) implant in mesothoracic leg socket, adjacent toheterotopically-transplanted regenerated cercus; (4) implant in posterior abdomen, ipsi-lateral host cereal motor nerve sectioned; (5) implant in posterior abdomen, ipsilateral marginof host terminal ganglion wounded. Results were determined after the adult molt, by con-ventional histology or by cobalt chloride filling of regenerated cereal nerves.

In all procedures except (3) and (4), the regenerated afferent nerve bypassed the implant andterminated in the host terminal ganglion. In (3), the regenerated fibers from cereal graftsbypassed the implant; terminations were not found. In (4), some regenerated cereal axonsconnected 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 thecereal motor nerve as a pathway guide but there is as yet no clear evidence for a trophicinfluence from the central nervous system.

INTRODUCTION

The development in arthropods of functional neural connexions between aregenerating sense organ and the central nervous system requires that theregenerating neurons first extend axons from the integument to the centralganglion, and then establish appropriate synaptic connexions within the neuro-pile. This study is concerned with the first of these processes, the establishmentof a pathway between periphery and center.

The abdominal cerci of the cricket, Acheta domesticus, provide appropriatematerial for such studies since they regenerate well, and have been used instudies of central connexion formation (Edwards & Sahota, 1967; Edwards &Palka, 1974; Palka & Edwards, 1974).

1 Author's address: Department of Zoology, University of Washington, Seattle, Washing-ton 98195, U.S.A.

20 MIRIAM McLEAN AND JOHN S. EDWARDS

Cerci are paired elongate mechanosensory appendages arising from thedorso-posterior angles of the abdomen. They lack intrinsic muscles but arecapable of limited movement by means of extrinsic dorsal muscles. Theirlimited mobility is sufficient to facilitate grooming and the support of the femaleby the male during copulation; they execute no evident movement in relation totheir sensory function. The cerci are densely clothed with sensilla of three basictypes, which have been described in detail by Edwards & Palka (1974). Thecereal sensory nerve (N lOd) in the adult contains about 10500 axons (Edwards,1971). This nerve terminates almost entirely ipsilaterally in the terminal abdomi-nal ganglion and provides a major input to the two giant interneurons, 8-1 and9-1, whose axons traverse the length of the ventral cord (Murphey, Mendenhall,Palka & Edwards, 1975). According to present knowledge, the cereal sensorynerve (N lOd) is entirely afferent; although the possibility that it may contain asmall efferent component cannot be dismissed (d'Ajello, Bettini & Casaglia,1972). This nerve is entirely separate from the smaller adjacent cereal motornerve, which branches from N lOv. Essential features of the cereal sensorynerve-giant interneuron system are summarized in Fig. 1.

After repeated cereal amputation or nerve section, degeneration of the proxi-mal stump sheath follows degeneration of the sensory nerve. Thus, there is noobvious physical substrate that might supply contact guidance for regeneratingafferent fibers. In experiments where crickets were deprived of cereal inputduring development by repeated amputation of postmolt regenerate cerci(Palka & Edwards, 1974), the surface of the adult terminal ganglion at the N lOdlocus was smoothly healed and covered with neural lamella. Nevertheless, if suchcrickets were permitted to regenerate cerci toward the end of postembryonicdevelopment, they did so rapidly and with apparently correct afferent termina-tions.

Working with heterotopically-grafted regenerate cerci in cricket larvae,Edwards & Sahota (1967) found that afferent fibers, arising from successfulgrafts at the mesothoracic leg socket, made connexions with the host's centralnervous system. These contacts were found in the vicinity of the giant inter-neuron axons, the same cells with which the cereal nerve normally synapses inthe terminal ganglion. Moreover, extracellular recordings from abdominalconnectives indicated that the giant axons were excited by stimuli applied to theheterotopic regenerate cercus.

Thus, regenerating cereal axons are capable of (1) routing a correct coursewithout the guidance of a pre-established path, (2) penetrating a healed ganglionin the correct region, and (3) recognizing appropriate post-synaptic cells in anabnormal region.

These results suggest questions that might be asked to determine the natureand precedence of the cues that allow regenerating sensory axons to connectwith their target organ. For example, can regenerating fibers be diverted fromtheir 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 connexionsof the cercus (CE). Cereal sensory axons (SC) with cell bodies located beneath thecereal integument (CI) send fibers through the cereal sensory nerve (CSN) to theterminal ganglion where they synapse principally with giant interneurons, of whichtwo (MGI, LGI) with their cell bodies (8-1, 9-1) are shown. The cereal motornerve (CMN) arises from nerve 10 v and innervates the extrinsic cereal musculatureat the base of the cercus. GN - genital nerve.

acceptable target? Their response when presented with a homologous, super-numerary ganglion might help to sort out the various possible cues operatingduring afferent regeneration. If the diffusion of 'wound factors' (Bodenstein,1957) from damaged nerve occurs, this tissue could supply such a directionalsignal to the regenerating axons. The release of material from the multiplewound sites of an implanted ganglion might then be sufficient to override othercues that normally influence regenerating fibers. Another question can be posed:In the event that the nerve recognized the implant, would it make contact at theappropriate (N lOd) region, or simply enter the nerve stump first encountered?Finally, how would regenerating axons respond to the presence of a potentiallyincreased central field size?

Working with Acheta domesticus, Rummel (1970) came to the conclusion thatsuccessful regeneration of a cercus depends upon an uninterrupted nerve supply,inferring the dependence of sensory regeneration on a trophic factor distributedby the motor nerve. This hypothesis brings up another question regarding therequirement for an intact cereal motor nerve in sensory regeneration.


Implants of healthy, conspecific tissues generally grow and become tracheatedby the insect host. In the case of ganglia, such grafts often participate in patternsof reciprocal motor and interneuronal innervation with the host system (Boden-stein, 1955, 1957; Guthrie, 1966; Jacklet & Cohen, 1967; Guthrie & Banks,1969). The relationships of host to graft in the case of afferent nerves are lesswell documented.

The observations to be described here are endpoint results; the outcome ofexperiments was not ascertained until after the adult molt. The supernumeraryganglion was never finally accepted as the appropriate target of entire regener-ated cereal nerves, except that we found small populations of regenerated cerealfibers within the implanted ganglion in cases where the host's ipsilateral cerealmotor nerve trunk was cut at the time of implantation.

METHODS

Rearing and development of Acheta domesticus. By providing gravid femalesfrom mass cultures (Fluker's Cricket Farm, Baton Rouge, La.) with receptaclesof damp sand for oviposition, continuous supplies of young crickets were madeavailable.

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 freshlettuce. Stock animals were reared in small groups, experimental animals inisolation.

The number of instars required for development of Acheta domesticus variesaccording to culture conditions. Our animals invariably passed through nineinstars before the final molt to the adult. The duration of stadia is summarizedin Table 1.

Surgery. All operations described below were performed on 6th to 8th stagelarvae at a time before the midpoint of the stadium, that is, before apolysis.Post-apolysis larvae were less able to withstand surgical trauma. The diagramsof Fig. 2 illustrate the five categories of surgery that comprised these experiments.

For ganglion transfer experiments, two animals were immobilized by chillingto about 4 °C, then supported venter up by means of filter paper strips pinnedon each side to a thin wax plate attached to a covered plastic container of icewater. In all experiments except those illustrated in Fig. 2C, the host's penulti-mate sternite was freed on three sides with fine sharp scissors and braced openwith a pin. To prevent desiccation of tissues and coagulation of hemolymph, asaline based on cricket hemolymph (Levine, 1966) was applied. The terminalganglion 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 autogenousterminal ganglion, and toward the origin of a forthcoming cereal nerve re-generate. The flap of cuticle was then repositioned, excess fluid blotted off, andthe wound allowed to seal by hemolymph coagulation.

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3-54-54-54-75-86-96-117-119-14

Approx. 6 months

44456679

10—


Table 1. Development o/Acheta domesticus under controlled conditions*

Range of Cumulativeduration Mean no. duration

Instar (days) of days/stadium (days)

4+18±1

12±117±223 + 229±236±345 ±355 ±5

* 27-28 CC; 16-h L/8-h D photoperiod (incandescent lamp); approx. 70% relativehumidity.

Host animals for the experiments diagrammed in Fig. 2B were reared withoutone cercus by means of repeated extirpation up to the 7th or 8th stage. This wasdone by removing the regenerated cereal 'button' just after each molt.

Control experiments to determine the response to cereal motor nerve sectionwere done by cutting the entire motor root, N lOv, at its exit from the terminalganglion (see Fig. 1). This nerve divides about halfway down its length to supplygenitalia with one branch and cereal base musculature with the other. Isolatingand sectioning only the cereal motor branch in a larval animal is technicallydifficult and requires more extensive surgery than sectioning the entire root nearthe source.

In the implant experiments diagrammed in Fig. 2D, E, nerve section orwounding of the host ganglion was done just before inserting the implant. Hostganglia were wounded either by sectioning the roots of N 8 (see Fig. 1) orpuncturing at the midlateral margin, ipsilateral to the implant.

For the heterotopic implant-cereal graft experiments (Fig. 2C), donors hadbeen cercectomized two molts earlier, so that they bore small regenerate cerci.After amputating the host's mesothoracic leg at the coxofemoral joint, the donorterminal ganglion was inserted through the wound. The donor cereal regeneratewas amputated and applied to the host's leg socket and held until it adhered bycoagulated hemolymph.

No antiseptic precautions were found necessary, other than cleaning instru-ments in 70 % ethanol.

Host animals were cercectomized by grasping the cercus at the base with fineforceps and pulling it cleanly away. The cereal motor nerve was left intact bythis operation.

Nerve tracing and histology. Animals were chilled to immobility and injectedwith paraformaldehyde-glutaraldehyde fixative (Edwards & Palka, 1974)


Fig. 2. Surgical procedures used in this study. (A) Simple terminal ganglion (TG)implant with concurrent ipsilateral cercectomy. (B) TG implant, host rearedwithout ipsilateral cercus, bilateral cercectomy concurrent with implantation.(C) Heterotopic TG implant and cereal graft on mesothoracic leg base. (D) TGimplant, 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 toone hour, the animal was dissected and the nerve tissue immersed in fixative for1 to 2 h at room temperature.

Simultaneous fixation and staining of tissues by immersion in a solution of0-5% 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 blueand fibers pink. The distortion produced by this treatment was reduced byprefixing tissues as described above for 1-5 days, then transferring them to thethionin-formalin solution for 2 days. Tissues were embedded in paraffin andsectioned at 8-15 /im.

Sections pre-treated with thionin were dewaxed and cleared in toluene andmounted in Permount. Sections not previously stained were hydrated andstained in 0-1 % aqueous toluidine blue.


Fig. 3. Dissection of adult with simple ganglion implant (procedure A). Implant(Im), in host 36 days, is well tracheated, with multiple connexions to nearbytracheal trunks (Tr). Scale: 0-5 mm.

In some experiments, regenerated cereal nerves were filled with cobaltchloride in order to visualize their terminations. The cereal nerve was cut at thebase of the cercus, then the terminal ganglion including about 1 mm of connec-tives and other associated nerve stumps was excised from the host. The distalcereal 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 thepart of the tissue not exposed to CoCl2 during treatment was immersed inSchneider's Drosophila medium. Tissues were fixed for 1 to 3 h in aldehydefixative, 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 inTable 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 timesoccasionally exceeded the normal range. Regenerated cerci were indistinguish-able from those of control animals without implants.

Implanted ganglia invariably became attached, usually by means of scar orconnective tissue, to the body wall near the point where they were originally

Tab

le 2

Que

stio

nF

ig. n

o.O

pera

tion

No.

of

anim

als

oper

ated

on

No.

of

succ

essf

ulop

era-

tions

Impl

ant

surv

ival

(sur

gery

tofix

atio

n)(d

ays)

Res

ults

Will

reg

ener

ate

cere

al

2Aaf

fere

nts

conn

ect

with

asu

pern

umer

ary

impl

ante

dte

rmin

al g

angl

ion?

Wou

ld n

erve

stu

mps

of

im-

2Bpl

ant

be m

ore

attr

activ

e to

rege

nera

te C

SN t

han

heal

edce

real

ner

ve e

ntry

reg

ion

ofh

ost

TG

?

Sinc

e re

gene

rate

aff

eren

t 2C

fibe

rs f

rom

a h

eter

otop

icce

rcus

are

cap

able

of

' rec

og-

nizi

ng' t

he p

osts

ynap

tic c

ell

at a

n at

ypic

al s

ite (

Edw

ards

& S

ahot

a, 1

967)

wou

ld t

his

still

be

the

pref

erre

d co

n-ne

xion

if

an i

mpl

ante

d T

Gw

ere

avai

labl

e in

the

sam

ere

gion

?

(1)

Impl

ant

term

inal

gang

lion

(TG

);re

mov

e ip

sila

tera

lce

rcus

(2)

Impl

ant

TG

in

host

rea

red

with

out

ipsi

late

ral

cerc

us;

rem

ove

cont

rala

tera

lce

rcus

and

ips

ilate

ral

cere

al '

bu

d'

(3)

Impl

ant

TG

and

graf

t re

gene

rate

cerc

us h

eter

oto-

pica

lly t

o m

eso-

thor

acic

leg

sock

et

1418

-44

Reg

ener

ate

cere

al s

enso

ryne

rve

(CSN

) es

tabl

ishe

d co

n-ne

xion

s on

ly w

ith h

ost

(aut

ogen

ous)

TG

. N

eura

lco

nnex

ions

bet

wee

n ho

stan

d im

plan

ted

TG

s in

thr

eeca

ses

8-23

R

egen

erat

e C

SN a

s ab

ove.

No

neur

al c

onne

xion

s be

twee

nho

st a

nd i

mpl

ante

d T

Gs

13-2

2 H

eter

otop

ic r

egen

erat

e C

SNby

pass

ed i

mpl

ante

d T

G.

Neu

ral

conn

exio

ns b

etw

een

impl

ante

d T

G a

nd h

ost

Mes

o-G

; be

twee

n im

-pl

ante

d T

G a

nd h

ost

mus

cles

o w o

Tab

le 2

(co

nt.)

Que

stio

nFi

g. n

o.O

pera

tion

No.

of

anim

als

oper

ated

on

No.

of

succ

essf

ulop

era-

tions

Impl

ant

surv

ival

(sur

gery

to

fixat

ion)

(day

s)R

esul

ts1 I'

Doe

s th

e ho

st c

erea

l m

otor

nerv

e (C

MN

) pr

ovid

e a

guid

ance

cue

for

the

ipsi

late

ral

rege

nera

te C

SN ?

Is d

isru

ptio

n of

the

reg

ener

ate

CSN

pat

hway

a c

onse

quen

ceof

hos

t ip

sila

tera

l T

G d

amag

ean

d no

t ne

cess

arily

a s

peci

ficre

spon

se t

o N

10

v se

ctio

n?

2D 2B

(4)

Impl

ant

TG

, cu

tip

sila

tera

l ho

stN

10

v; re

mov

ebo

th c

erci

10

(5)

Impl

ant

TG

,w

ound

ips

ilate

ral

mar

gin

of h

ost

TG

or c

ut N

8;

rem

ove

both

cer

ci

18-3

3 In

eac

h ca

se,

som

e re

gene

rate

CSN

fib

ers

wen

t to

im

-pl

ante

d T

G, m

ost

to h

ost

TG

; en

tire

rege

nera

te C

SNco

ntac

ted

impl

ant

at l

east

supe

rfic

ially

on

way

to h

ost

TG

. Neu

ral c

onta

cts

betw

een

host

and

im

plan

ted

TG

s;ne

urom

uscu

lar

cont

acts

betw

een

impl

ant

and

host

mus

cle

16-1

7 R

egen

erat

e C

SN e

stab

lishe

dco

nnex

ions

onl

y w

ith t

heho

st T

G. N

eura

l co

nnex

ions

betw

een

host

and

im

plan

ted

TG

s

* O

ne w

ith n

orm

al r

egen

erat

e ce

rcus

, on

e w

ith h

eter

omor

phic

str

uctu

re.

28 MIRIAM MCLEAN AND JOHN S. EDWARDS


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 localdegeneration, implanted ganglia produced fiber outgrowth volumes consonantwith their length of time in the host. These outgrowths came from motor ormixed nerve or connective stumps; cereal sensory nerve stumps, where recogniz-able, had degenerated. Attachments between fibers originating from implantsand host muscle occurred in most cases.

Simple implants (Fig. 2 A). In all eight cases, the regenerated cereal sensorynerve established connexions in the appropriate region of the host terminalganglion. Even in situations where the base of the regenerated cercus wasclearly nearer to the implant than to the host ganglion, we found no regeneratedafferent fibers contacting the implant.

Implant in 'deprived' host (Fig. 2B). In all four adults the ipsilateral cerealsensory nerve made contact only with the host terminal ganglion. No neuralconnexions linking the implanted and host ganglia were found.

Heterotopic implant with cereal graft (Fig. 2C). Of the five animals subjectedto this operation, three lost the grafted cercus before or during the subsequentmolt. Two animals retained grafts, but the cereal form was maintained in onlyone (Fig. 4 A). Serial sections of the heteromorphic structure in the other animalrevealed no sensory cells. Sections from the animal with the successful hetero-topic cercus revealed that the afferent fiber bundle formed from sensory cellsin the graft had bypassed the implanted ganglion and had grown toward thehost's ventral cord (Fig. 4B).

Motor nerve section. Preliminary operations were done on 16 animals todetermine survival rates. In these animals both motor nerves (N lOv) weresectioned, both cerci removed, and a ganglion was implanted. Two reached thelast molt; most dying within 4 days after surgery. A control group of 12 animalshad only one N lOv sectioned, and one or both cerci removed. In animals thatreached adulthood, we found both motor and sensory cereal nerves hadregenerated and made anatomically correct connexions. The only abnormalityfound upon histological examination was a mixing of fibers from sensory andmotor bundles near the cereal base (Fig. 4D). More proximally, the two majorbundles 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 show-ing implanted ganglion (Ig) and base of cercus below, with sensory nerve (N) enter-ing leg base. Toluidine blue. Scale: 100 /*m. (C) Base of normal regenerate cercusshowing separate cereal motor (CM) and cereal sensory (CS) nerves. Arrow indicatesdirection of cercus tip. Thionin stain. Scale: 100/*m. (D) Base of regenerate cercusin animal with cereal motor nerve cut three instars earlier (procedure D), showingmixed motor and sensory nerve (SMN). Thionin stain. Scale: 100 /im.


A

Tr

Fig. 5. Sketches of host and implanted ganglion and their relationships. (A) Ipsi-lateral nerve lOv cut at time of ganglion implantation. Implant (Im) has massiveneuroma 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 neuromaat 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 normalregenerates.

Implant with cut motor nerve (Fig. 2D). Five out of ten operated cricketsbecame apparently healthy adults. Tissues from two of these were processedwith the thionin method, and cobalt preparations of the ipsilateral cerealsensory nerve were made with three.

Both cereal motor and sensory nerves regenerated, but the sensory nerves hadattached to both the implanted and the host terminal ganglia. A sketch of theganglia from one of these adults in Fig. 5 A represents a typical pattern ofconnexions seen in this category.

Regenerated fiber bundles were associated with small glial cells and were not

Regenerating insect sensory nerve

C

31

BFig. 6 (A and B): Horizontal 10 /tm paraffin sections, CoCl2 treatment, of host andimplanted ganglion in host with previously cut N lOv (procedure C). Implant inhost 33 days. Sections A and B separated vertically by about 20/*m. Pathway ofregenerated 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 in28-day implant ganglion; 20/tm paraffin section. Scale: 100/tm. (D) Periphery ofhealthy 33-day implant ganglion. PN, intact perineurial sheath. CB, neuron cell body.10/tm paraffin section, glutaraldehyde fixation, osmium staining. (E) Degenerating25-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 /*mparaffin section, toluidine blue stain. Scale for D and E: 10/*m.


D


as clearly delineated as bundles in normal material. In every case it was possibleto trace the regenerated sensory nerve from the base of the ipsilateral cercus tocontact first with the implant, and further to contact with the host terminalganglion (Fig. 6A and B). The great majority of afferent fibers appeared toterminate in the host ganglion but examination of CoCl2-filled tissues revealeda few fiber terminations in the implant (Fig. 6C).

CoCl2 projections in host ganglia were not as complete as those typically seenin the case of normally developed sensory neurons. This would be expected,since afferent projections from regenerated cerci in control animals are lessdense than those from normal cerci, the reduced quantity of fibers evidentlyreflecting the time available for regeneration (Fig. 6C).

Implanted ganglia appeared to be healthy, and in general their symmetry waswell preserved (Fig. 6D). In one case, the motor nerve (N lOv) of the implantmade contact with muscles of the host's cereal base, ipsilateral and parallel tothe afferent nerve from the regenerated cercus to the implant.

The striking results of this group of experiments indicated that the cerealmotor nerve - or at least N lOv - influenced the growth direction of sensoryfibers, 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 morpho-logy and neuropile patterns of the host ganglion. Fig. 5B is a diagrammaticsketch made from a dissection of one of these animals: the relationships weresimilar in the other two cases. In every instance, host and donor ganglia werecomplexly interconnected by way of an outgrowth between the initial woundsite of the host ganglion and one or more motor or connective trunks of theimplanted ganglion (Fig. 7C). However, tracing of serial sections revealed thatall the regenerate cereal sensory fibers bypassed the implant and terminated ihthe ipsilateral portion of the host ganglion (Fig. 7D). Since there was a varyingdegree 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 contactsbetween host and implanted ganglia occurred in four out of five categories.These connexions were formed by way of cut nerve stumps or damaged areasof ganglion. Outgrowths from implants occasionally resulted in neuromas

FIGURE 7

(A and B) Cobalt preparations of cereal sensory nerves in terminal ganglion, wholemounts. Dashed line is midline. (A) Normal adult. (B) Adult with regeneratedcercus. 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 bluestain. Scale: 100 /*m. (D) Projection of regenerated cereal sensory nerve into hostTG. Implant in host 28 days. 10/*m paraffin sections. Scale: 100/tm.3 EMB 36


(Fig. 6 A) which often served as channels for interconnexion between host andimplant.

Neural connexions between the implant and host muscle were observed inthree categories. The quantity of these contacts appeared to be related posit'velyto the health of the implant and to the amount of the time in the host. The onlyknown specificity exhibited in this class of connexions was the single casementioned above in which the regenerated N lOv of the implanted ganglionmade contact with muscles of the host's cereal base.

Cellular migrations, proliferations; degenerative reactions. Nerve cells withinimplants usually appeared normal histologically (Fig. 6D). In two cases fromthe first category of experiments (Fig. 2 A), and in one from the second category(Fig. 2B), degenerative changes in the implants were pronounced. In mostanimals of the first category variable numbers of small cells were dispersedthroughout the implants and their outgrowths (Fig. 6E). These appeared to bemainly small glial cells, but on the basis of comparison with hemolymphsmears, 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 toterminate in the host terminal ganglion.

(3) When implantation was combined with section of host N lOv, regeneratingcereal afferents were diverted to the implanted ganglion, and a few fibersterminated within it.

(4) In no instance did regenerate afferent (host) cereal nerves terminateentirely 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, thatis, through part of the old sensory nerve stump.

(6) Implants were always attached to host tissues. They were supplied withtracheae, grew in size during maturation of the host, and put forth nerve out-growths.

(7) Nerve-muscle contact between donor and host, respectively, was foundfrequently. Nerve contacts between donor and host ganglia occurred in overhalf the cases.

(8) Nerve outgrowths from donor ganglia arose from motor or connectivestumps or from other wounds; sensory cereal nerve stumps of implants, whererecognizable, were degenerate.

(9) Glial cells responded to the changed circumstances of these experimentsby accumulating in greater than normal numbers at sites of trauma, regenera-tion, and abnormal growth, and by atypical migrations within implants.


DISCUSSION

Target discrimination in regenerating nerves

Experimental studies of neural regeneration in orthopteroid insects, exploitingtheir capacity for sensory and motor regeneration and for sustaining implantedganglia, have demonstrated the vigor of nerve growth and specificity in therestoration of functional connexions. It is clear that severed motor nervesregenerate in immature and adult stages (Bodenstein, 1957) and can recognizetheir correct target (e.g. Jacklet & Cohen, 1967; Pearson & Bradley, 1972) ortheir homologue from another segment (Young, 1972), and that supernumerarygrafted limbs will acquire motor innervation even though the normal system isintact (Sahota & Edwards, 1969).

Implanted ganglia have been shown to provide channels for motor innervationand can innervate muscle (Guthrie, 1966; Guthrie & Banks, 1969). None ofthese studies addressed the question of the factors that determine the pathwayof axons between their origin and target organs.

With sensory systems, similarly, the capacity for regeneration has been amplydemonstrated, but with the exception of Wiggles worth's (1953) demonstrationof the tendency of ingrowing sensory axons to follow pre-existing pathways, themechanisms of pathway determination have received little attention.

In our experiments sensory and motor connexions were made in the appro-priate locations, as evaluated by endpoint histological examination, althoughsome pattern abnormalities developed. Regenerated afferent and efferent fibersintermingled in the distal pathway; however they apparently became sorted outand formed topographically normal terminations. Hamburger (1929) observedthat in the development of amphibian limbs the tips of pioneering nerve fibersgrowing out of motor and sensory centers take different routes through thetissue matrix, diverging at a considerable distance before reaching muscles orskin, respectively.

If the situations in amphibians and insects are comparable with respect toseparation of types of growing nerves, it would be difficult to imagine how aregenerating cereal afferent fiber might depend for directional cues upon amotor nerve which, although running parallel to the sensory pathway, isnormally entirely separate from it throughout its course. Further, the regenerat-ing 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 motornerve at the onset of sensory regeneration, afferent fibers innervate a super-numerary 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 appropriatelocus. Indeed, afferent fibers always entered the ganglion at the proper place,whether the ganglion was an implant with no normal CNS connexions, or anautogenous ganglion with the motor nerve severed at its origin. It may be that

3-2


pioneering regenerate afferents could be guided by adjacent motor fibers with-out making permanent attachments to them. Once the first group of sensoryfibers had terminated and separated from the motor nerve, then afferent fibersdeveloping in the following molts would be guided by those already in place.

Exploratory behavior of pioneer fibers in growth and regeneration has longbeen known to occur in vertebrates. Since time-course observations were notmade in our studies, we cannot state that there were no temporary contactsformed by regenerating axons which were subsequently aborted. Regeneratingafferent fibers might have first contacted the implant and then dissociated togrow further until they reached the host ganglion. Why then were persistence ofsensory contacts in the implant associated only with experiments involving cutand subsequently regenerated motor nerves ? Since motor fibers tend to makeattachments between host and implanted nerve tissue, it seems possible that theregenerating 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 ofall growing axons. They found that the lead axons from retinal ganglion cellsgrowing in to meet lamina neuroblasts were the only ones of the group toproduce growth cones. Furthermore, they suggested that this transient structuremay be functionally associated with the recognition of cell surfaces or spatialrelations by pioneer fibers. This idea is supported by a more recent paper(Lopresti, Macagno & Levinthal, 1974), where transiently formed gap junctionswere found at these contact points.

The question remains, if cereal afferents depend upon intact motor nerve forpathway guidance, how are they able to regenerate to their normal locationswhen motor nerves are cut? The postulated dependence on contact with motornerves could be facultative and not absolute. We can suggest two possiblemechanisms: regenerating motor axons may have reached the periphery beforecereal afferents terminated in the ganglion, thus providing pathway guidance.Alternatively, it may be that there exists a hierarchy of cues available to aregenerating nerve, so that in the absence of a primary cue (in this case, theintact motor nerve) a secondary one provides the requisite factor. Such asecondary cue might be factors diffusing from the stumps of cut nerves.

Terminal contact formation. The evidence presented above suggests that once aregenerating axon has established a contact, then the fibers following areguided into the same pathway. However, at the level of synaptic terminals, otherprocesses must be operating, for only a small proportion of cereal afferents incut 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 post-axotomy phenomenon is that of' somatic stripping', the loss of synapses on cell


bodies and proximal dendrites. This has been measured electrophysiologically(Kuno & Llinas, 1970a, b; Mendell, Munson & Scott, 1974), and observedultrastructurally in the form of glial displacement of synaptic terminals onaxotomized motoneurons (Blinzinger & Kreutzberg, 1968; Kerns & Hinsman,1973). In our experiments, the damage in the form of loss of axon volume ofgiant interneurons of the implant could have resulted in a deficit or deactivationof synaptic sites for incoming fibers.

Trophic effects in cereal regeneration. A trophic role of efferent axons has beeninferred by Drescher (1960) who reported that antennae do not regenerate inPeriplaneta americana if deprived of all neural connexions with the brain, and bySchoeller (1964) who concluded that differentiation of transplanted antennaldisks of antennae, compound eyes and palps of Calliphora erythrocephalarequired innervation from the host.

An extensive exploration of the trophic role of the central nervous system inthe regeneration of cerci in Acheta domesticus is closely relevant to the presentstudy and must be critically assessed in relation to our own findings.

Rummel (1970) reported the effects of surgical intervention on cereal re-generation in Acheta domesticus larvae. The object of the study was to determinethe influence of the nervous system in the regeneration of sensory appendages.

Separation of the cercus from its nerve supply was accomplished by a deepincision through the body wall in the middle of the angle formed by the cercusand the adjacent abdominal margin. Presumably both motor and sensory nerveswere severed by this operation, but Rummel did not distinguish between thetwo. Cerci degenerated disto-proximally until regeneration set in. The re-generated cercus was formed partly from the existing cereal base; in some casesa small regenerate was formed in the wound region, just medial to the normally-situated cercus. Rummel suggested that maintenance of a normal cercusdepended on the presence of an uninterrupted nerve supply, and that productionof a regenerate must await the arrival of regenerated nerves in the wound area.

A comparable operation, but with the insertion of barriers of mica, plasticfilm, or aluminum foil yielded more varied results. The aim was to preventnormal regeneration or induce supernumerary regenerates by diverting nervesfrom their normal course. Mortality of operated animals was higher, and cerealregeneration reduced. Various abnormalities included distal degenerativechanges in the contralateral cercus. Rummel inferred the need for a centrifugallymoving substance from the central nervous system, requiring neural connexionbetween the central ganglion and the site of regeneration following the wellestablished vertebrate model. Several questions arise from this interpretation.First, the innervation of the cereal musculature, the only possible pathway fora neurogenic stimulus to cereal regeneration since sensory fibers arise from thecercus itself, is entirely extrinsic to the cercus and has no known terminationson the epidermis in the tissue from which the sensory regenerate arises.

Secondly, the diminution of cereal regeneration obtained by interposition of


barriers could be due to the diversion of hemolymph and tracheation by thebarrier and scar tissue. The cerci lack pulsatile organs at their base which ensurehemolymph circulation in other elongate appendages such as antennae, andmay thus be sensitive to alterations of patterns of hemolymph flow which couldlead to local stagnation and thus to tissue necrosis.

A simple method for testing the importance of the motor nerve in cerealregeneration would be to remove the host terminal ganglion, thereby preventingefferent regeneration. This experiment was attempted with 7th, 8th and 9thstage larvae but none survived beyond ten days and none molted. Presumablythe loss of regulating functions such as control of water balance accounted forthe mortality. Cutting all nerves to the host terminal ganglion and leaving it insitu gave the same negative results in four animals. A decisive test of the needfor centripetal innervation by means of such simple surgical intervention thusseems to be precluded. The less drastic expedient of severing the cereal motornerve alone at its exit from the terminal ganglion had no significant effect on thetime span or quality of cereal regeneration. We conclude that firm evidence fora trophic role of central innervation in the development of cereal regenerateshas not yet been demonstrated.

This work was supported by Developmental Biology Training Grant HD-00266 fromNICHHD and by research grant NB 07778. We thank Drs John Palka, Eldon Ball andRobert Seecoff for critical reading of the manuscript and for helpful discussion.

REFERENCES

D'AJELLO, V., BETTINI, S. & CASAGLIA, O. (1972). A new efferent pathway in the cockroachcentral nervous system. Experientia 28, 40-41.

BLINZINGER, K. & KREUTZBERG, Z. (1968). Displacement of synaptic terminals from re-generating motoneurons by microglial cells. Z. Zellforsch. mikrosk. Anat. 85, 145-157.

BODENSTEIN, D. (1955). Contributions to the problem of regeneration in insects. / . exp. Zool.129, 209-214.

BODENSTEIN, D. (1957). Studies on nerve regeneration in Periplaneta americana. J. exp. Zool.136, 89-116.

CERF, J. A. & CHACKO, L. W. (1968). Retrograde reaction in motor neuron dendrites follow-ing ventral root section in the frog. / . comp. Neurol. 109, 205-216.

DRESCHER, W. (1960). Regenerationsversuche am Gehirn von Periplaneta americana unterBeriicksichtigung von Verhaltensanderung und Neurosekretion. Z. Morph. Okol. Tiere 48,576-649.

EDWARDS, J. S. (1971). The composition of an insect sensory nerve, the cereal nerve of thehouse cricket, Acheta domesticus. Proc. Electron. Microsc. Soc. Am. 17, 248-249.

EDWARDS, J. S. & PALKA, J. (1974). The cerci and abdominal giant fibers of the housecricket, Acheta domesticus. I. Anatomy and physiology of normal adults. Proc. R. Soc. B.185, 83-103.

EDWARDS, J. S. & SAHOTA, T. S. (1967). Regeneration of a sensory system: the formation ofcentral connections by normal and transplanted cerci of the house cricket, Acheta domes-ticus. J. exp. Zool. 166, 387-396.

GURR, E. (1953). A Practical Manual of Medical and Biological Staining Techniques, p. 159.London: Leonard Hill Ltd.

GUTHRIE, D. M. (1966). Physiological competition between host and implanted ganglia in aninsect {Periplaneta americana). Nature, Lond. 210, 312-313.

Regenerating insect sensory nerve 39GUTHRIE, D. M. & BANKS, J. R. (1969). The development of patterned activity by implanted

ganglia and their peripheral connections mPeriplaneta americana. J. exp. Biol. 50, 255-273.HAMBURGER, V. (1929). Die Entwicklung experimentell erzeugter nervloser und schwach

innervierter Extremitaten von Anuren. Arch. EntwMech. Org. 114, 272-363.JACKLET, J. W. & COHEN, M. J. (1967). Synaptic connections between a transplanted insect

ganglion and muscles of the host. Science, N. Y. 156, 1638-1640.KERNS, J. M. & HINSMAN, E. J. (1973). Neuroglial response to sciatic neurectomy. II. Electron

microscopy. / . comp. Neurol. 151, 255-280.KUNO, M. & LLINAS, R. (1970a). Enhancement of synaptic transmission by dendritic poten-

tials in chromatolysed motoneurones of the cat. / . Physiol., Lond. 210, 807-821.KUNO, M. & LLINAS, R. (19706). Alterations of synaptic action in chromatolysed moto-

neurones of the cat. / . Physiol., Lond. 210, 823-838.LEVINE, L. (1966). Tonicity of Acheta hemolymph. Physiol. Zool. 39, 253-258.LOPRESTI, V., MACAGNO, E. R. & LEVINTHAL, C. (1973). Structure and development of

neuronal connections in isogenetic organisms: cellular interactions in the development ofthe optic lamina of Daphnia. Proc. natn. Acad. Sci., U.S.A. 70, 433-437.

LOPRESTI, V., MACAGNO, E. R. & LEVINTHAL, C. (1974). Structure and development ofneuronal connections in isogenic organisms: transient gap junctions between growingoptic axons and lamine neuroblasts. Proc. natn. Acad. Sci., U.S.A. 71, 1098-1102.

MENDELL, L. M., MUNSON, J. B. & SCOTT, J. G. (1974). Connectivity changes of la afferentson axotomized motoneurons. Brain Research 73, 338-342.

MURPHEY, R. K., MENDENHALL, B., PALKA, J. & EDWARDS, J. S. (1975). Deafferentationslows the growth of specific dendrites of identified giant interneurons. / . comp. Neurol. 159,407-418.

PALKA, J. & EDWARDS, J. S. (1974). The cerci and abdominal giant fibres of the house cricket,Acheta domesticus. II. Regeneration and effects of chronic deprivation. Proc. R. Soc. B185, 105-121.

PEARSON, K. G. & BRADLEY, A. B. (1972). Specific regeneration of excitatory motoneuronesto leg muscles in the cockroach. Brain Research 47, 492-496.

RUMMEL, H. (1970). Die nerveninduzierte Regeneration der Cerci bei Acheta domesticus L.Deutsche Entomologische Zeitschrift 17, 357-409.

SAHOTA, T. S. & EDWARDS, J. S. (1969). Development of grafted supernumerary legs in thehouse cricket, Acheta domesticus. J. Insect Physiol. 15, 1367-1373.

SCHOELLER, J. (1964). Recherches descriptives et experimentales sur la cephalogenese deCalliphora erythrocephala (Meigen). Archs Zool. exp. gen. 103, 1-216.

SUMNER, B. E. H. & WATSON, W. E. (1971). Retraction and expansion of the dendritic tree ofmotor neurones of adult rats induced in vivo. Nature, Lond. 233, 273-275.

TYRER, N. M. & BELL, E. M. (1974). The intensification of cobalt filled neuron profiles usinga modification of the Timm's sulphide-silver method. Brain Research 73, 151-155.

WIGGLESWORTH, V. B. (1953). The origin of sensory neurons in an insect. Q. Jlmicrosc. Sci.94, 93-112.

YOUNG, D. (1972). Specific re-innervation of limbs transplanted between segments in thecockroach, Periplaneta americana. J. exp. Biol. 57, 305-316.

(Received 3 November 1975; revised 19 February 1976)

target discriminatio in regeneratinn g insect sensory nerve · embryo!, exp. morph. vol. 36, 1, pp....

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