Plasticity in the cockroach neuromuscular system

DEVELOPMENTAL

BIOLOGY

111,306-315

(1985)

Plasticity in the Cockroach JEFFREY Biology Department,

University

Neuromuscular

System

L. DENBURG of Iowa, Iowa City, Iowa 5.Z.242

Received August 14, 1984; accepted in revised

f&-r, Ap.1 15, 1985

The retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase extracellularly injected into a leg muscle was used to identify the regenerating cockroach motor neurons that have grown an axonal branch into that muscle. At least 66% of the animals with crushed nerve roots eventually reform the original innervation pattern of this muscle with no mistakes. In spite of this apparent specificity the cockroach neuromuscular system can express plasticity as evidenced by the correction of mistakes made at early stages of regeneration. These mistakes are corrected through elimination during the time interval between 40 and 60 days after nerve crush. In addition, when the distal segments of the leg are removed, thus depriving some motor neurons of their normal target muscles, many of them form stable inappropriate axonal branches in denervated as well as fully innervated muscles. These observations are discussed in terms of possible mechanisms responsible for the specificity of the cellular interactions and in terms of their relevance to understanding the development of vertebrate neuromuscular systems. o 19% Academic Press, 1~. INTRODUCTION

priate axon branches in previously denervated muscles as well as in fully innervated ones. Previous observations that the formation of connections between single identified motor neurons and muscles during embryonic development and axonal regeneration always results in the same pattern of innervation have emphasized the specificity expressed by the system (Pearson and Bradley, 1972; Young, 1972; Denburg et ah, 1977; Fourtner et al., 1978; Whitington, 1979; Denburg, 1982a). However, the results presented here demonstrate that with experimental manipulation this system can be made to express its potential to form novel, stable connections.

The cockroach neuromuscular system contains identified motor neurons and muscles, many of whose patterns of synaptic connectivity have been determined. For the six coxal depressor muscles in the leg of Periplaneta americana the innervation pattern by identified motor neurons is known (Pearson and Iles, 1971). When the axons of these motor neurons are severed they soon regenerate and eventually reform the original innervation pattern (Pearson and Bradley, 1972). In spite of this apparent specificity it has been observed, using both electrophysiological and anatomical techniques, that during early stages of regeneration motor neurons grow into and make functional connections with muscles that they do not normally innervate (Denburg et al, 19’77, Whitington, 1979; Denburg, 1982a). In a high percentage of the cockroaches undergoing this axonal regeneration these inappropriate synapses are inactivated and the inappropriate axon branches become eliminated. Similar cellular events occur during the development of the vertebrate neuromuscular system (Bennett, 1983) and of various parts of the nervous system (Purves and Lichtman, 1980). This communication reports the results of experiments using anatomical techniques to provide increased resolution of the time course of the elimination of inappropriate regenerated axon terminals. It also reports on studies done to determine the effects that removal of the normal target muscles have on the ability of regenerating motor neurons to eliminate inappropriate axon branches. An unexpected morphological plasticity was discovered when it was observed that removal of target muscles enabled motor neurons to form stable inappro0012-1606/85 $3.00 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

MATERIALS

AND

METHODS

Materials. Adult male cockroaches, P. americana, were obtained from laboratory colonies. Wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was obtained from Sigma Chemical Co. SurgicaZ procedures. Operations were performed as previously described (Denburg, 1982), but with modifications aimed at controlling parameters that might introduce variability into the experimental results. Male adults were operated on within 3 days of their final molt. Animals anesthetized with COZ were immobilized ventral surface upwards, using pieces of clay. Nerve 5 was crushed with forceps after penetration of the soft transparent region of cuticle between the thorax and the coxa, the most proximal segment of the leg. Previous histological examination with the light microscope demonstrated that this treatment severed the axons in the nerve (Denburg et al., 1977). Care was taken not to damage the trachea, other nerves, or muscles in the region. Axotomy was performed on the left metathoracic and 306

JEFFREY L. DENBURC

Plasticity

the right mesothoracic nerves 5 in the same cockroach. Earlier experiments demonstrated that identical results were obtained whether two nerves or one was crushed in the same animal. After the operation cockroaches were maintained in a 30°C incubator with a 12-12 hr light-dark cycle, with food (Lab chow) and water available continuously. In order to eliminate normal target muscles of some of the motor neurons, distal segments of the leg were removed by clasping the femur with forceps and allowing the escape behavior of th.e cockroach to break the joint between the femur and the trochanter. This joint contains a natural autotomy plane which is constructed so as to produce minimum d.amage when leg segments distal to the coxa are removed. This procedure removes all leg muscles distal to the joint between the trochanter and the femur (Fig. 2). In young nymphs autotomy at this point is followed by rapid regeneration of the distal leg segments (Bodenstein, 1955). No such leg regeneration occurred in the adult cockroaches used in this study. Injection and detection of enzyme. Injection of WGAHRP extracellularly into the muscle was done as previously described (Denburg, 1982a). Briefly, stock solution of 33 mg/ml was prepared by dissolving 0.5 mg of enzyme in 15 yl of 0.1 M KCl, 10 mM phosphate buffer at pH 7.2. A Hamilton 1.0 ~1 syringe was used to transfer 0.1 ~1 of enzyme solution to a piece of Parafilm. The solution was then transferred by capillary action into a glass microcapillary tube whose tip had been machinepulled and broken to a diameter of approximately 50 pm. After injection of this 0.1 ~1 through a hole made in the cuticle, the cockroaches were maintained at 30°C for 24 hr. The nerve cord was then removed and peroxidase activity detected lby treatment with the HankerYates reagent (Hanker et al., 197’7) and 0.01% hydrogen peroxide in 0.1 Mcacodylate buffer, pH 5.2. The ganglia were washed in buffer, fixed in Carnoy’s solution, dehydrated in ethanol, cleared in methyl benzoate, and examined as whole mounts with the light microscope; camera lucida drawings were made of all stained neuronal cell bodies. There was great variation in the background among ganglia from different specimens. Neurons were considered positively stained only when the granular nature of the stain could be detected upon examination at a high magnification. Can neuwns take up WGA-HRP at locations other than axon terminals in muscle 1?‘8? The validity of the results presented in this commu:nication depends on the observation that only neurons with axonal branches in muscle 178 will endocytose and retrogradely transport detectable amounts of WGA-HRP to their cell bodies in the ganglia. Previous results demonstrated that the injection technique employed exposed axon terminals only in muscle 178 to the enzyme (Denburg, 1982). No motor

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neurons (defined as cells with somata greater than 20 pm in diameter) innervating neighboring muscles were ever stained. In rats and cats WGA-HRP transported in the retrograde direction can cross synapses to interneurons innervating motor neurons (Harrison et al., 1984). After injection of WGA-HRP into muscle 178 of unoperated cockroaches small cell bodies of neurons other than Df were occasionally stained (Denburg, 1982). These cells were not considered to be motor neurons because of their small size and location in the ganglion. However, this raised the possibility that neurons stained after injection of WGA-HRP into muscle 178 did not necessarily have an axon branch in the muscle, but may have formed transient synapses with other motor neurons that had grown into the muscle. However, when nerve 5 was crushed in cockroaches with intact legs the cell bodies of identifiable neurons with axons in other nerves were not stained. This suggests that regenerating axons do not form synapses with nongrowing neurons through which detectable amounts of WGA-HRP could be transported. Cobalt backjills. Cobalt backfills of the three nerves containing axons of motor neurons innervating leg muscles were performed in vitro as previously described, but using 40 mM CoC& and incubating 16 hr at 4°C (Denburg et al., 1977). RESULTS

Anatomical

Background

The thoracic ganglia of the ventral nerve cord of the cockroach contain the cell bodies of the motor neurons innervating the leg muscles. Three separate nerves (numbered 3b, 5, and 6) carry the axons of these neurons into each leg. Neurons in nerve 6 exclusively innervate the flexor muscles in the coxa, the most proximal segment of the leg. None of the experimental manipulations used in this study affect the axons of these neurons so they will not be further considered and they will be omitted from all figures. Neurons in nerve 3b also innervate muscles in the femur. When the leg is autotomized at the trochanter-femur joint this nerve is severed and some of the axons of its neurons extending beyond the joint are axotomized. Nerve 5 contains the axons of the six identified neurons that innervate a set of extensor muscles called the coxal depressor muscles. This nerve also contains about 30 unidentified motor axons innervating muscles in the more distal segments of the leg (femur and tibia). Nerve 5 is crushed in all experiments and is additionally severed a few millimeters distal to the crush when distal segments of the leg are removed. The motor neurons with axons in each of these nerves were visualized by cobalt backfills. The cell bodies

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stained after backfills of nerves 3b and 5 are shown schematically in Fig. 1 and these results agree with those previously published (Pearson and Fourtner, 1973; Denburg et al., 197’7; Brlunig, 1982). The neurons with axons in nerve 3b, excluding a medial inhibitory cell, are observed to be in three distinct locations. These include a large anterior medial neuron, an extreme lateral group of lo-13 motor neurons, and a more medial group of small neurons that are probably sensory. The large anterior medial neuron occupies a similar position in the ganglion and sends its axon out the same nerve root as a neuron shown to innervate the extensor tibiae muscle in the femur of the locust (Burrows and Hoyle, 1973). Assuming homology, this neuron in the cockroach is here labeled SETi although its target muscle has not been identified. This cell could readily be distinguished from the other neurons with axons in nerves 3b or 5. The major branching of nerves 3b and 5 in the metathoracic leg are shown schematically in Fig. 2 along with the sites of the experimental manipulations used in this study. These include crushing nerve 5 (Fig. 2a) and crushing this nerve and additionally removing distal leg segments (Fig. 2b). There are six metathoracic coxal depressor muscles: muscles 178 and 179, innervated by only one identified motor neuron, Df; muscles 177d and 177e, innervated by motor neuron D,, 3 inhibitory neurons and at least one octopaminergic dorsal unpaired median cell; and muscles 177d’ and 177e’, with all fibers innervated by both DI and D, (Pearson and Iles, 1971; Denburg and Barker, 1982). The muscles are numbered according to the system of Carbonell (1947). When nerve 5 is crushed the axons of the neurons innervating these muscles as well as those

VENTRAL

DORSAL anterior

FIG. 1. Schematic drawing of the metathoracic ganglion showing cell body positions of the motor neurons examined in this study. Dr innervates muscle 178 while D., I (inhibitory neurons), and DUM (dorsal unpaired median) cells innervate the other coxal depressor muscles. Unlabeled and unfilled cells on the ventral surface are neurons with axons in nerve 5 that innervate muscles in the segments of the leg distal to the coxa. Unlabeled and shaded cells are neurons with axons in nerve 3b. SETi is a motor neuron with its axon in nerve 3b and which innervates a muscle in the femur.

VOLLJME 111, 1985 nrdb nr5

COXA& TROCHANTER TIBIA

a

nr3b

TROCHANTER b

FIG. 2. Schematic representation of the state of the neuromuscular system in the two experimental paradigms used in this study. The final innervation of muscle 178, after axonal regeneration and synaptic elimination are completed, is also shown with arrowheads indicating the origin of the axons. (a) Nerve 5 is crushed just proximal to the coxa and the leg is left intact. The normal target muscles of the unidentified neurons with axons in nerves 3b and 5 are present. Muscle 178 is finally reinnervated by only Dr. (b) Nerve 5 is again crushed proximal to the coxa and the distal segments of the leg are removed. This autotomy eliminates the normal target muscles of the unidentified motor neurons and also cuts nerves 3b and 5. After regeneration is completed muscle 178 is reinnervated by Dr and the unidentified motor neurons. Scale bar is 2 mm.

of the other 30 unidentified motor neurons with axons in this nerve are severed. Muscle 178 is particularly suitable for following the reinnervation by regenerating axons because of its relatively large size, the ease of access to its muscle fibers and its simple normal innervation. It was previously demonstrated that during early stages of axonal regeneration nearly all of these neurons incorrectly grew into muscle 178 and some formed inappropriate functional connections (Denburg et ak, 1977; Whitington, 1979; Denburg, 1982). These mistakes were gradually corrected until in most cases muscle 178 was again innervated only by Df. In this study we address the question of whether the presence of their normal target muscle influences the elimination of inappropriate axonal branches made by regenerating motor neurons. Distal segments of the leg were removed, thus eliminating the normal target muscles of the 30 unidentified motor neurons (Fig. 2b). The stability of the inappropriate axonal branches these regenerating neurons send into muscle 178 was then assessed, using the retrograde

JEFFREY L. DENBURG

Plasticity

transport of wheat germ agglutinin-conjugated horseradish peroxidase extracellularly injected into the muscle, and compared with those of neurons regenerating in intact legs. Elimination of Inappropriate Axonal Branches in Cockroaches with Intact Legs Our earlier study demlonstrated the applicability of the retrograde transport of WGA-HRP extracellularly injected into leg muscle 1178for determining which regenerating motor neurons have axon terminals in that muscle (Denburg, 1982a). Additional data were collected to include more time points after nerve crush as well as many more experimental animals per time point. This was necessary in order to have a more extensive and higher resolution base with which to compare the effects of experimental manipulations on the time course and extent of elimination of inappropriate axonal branches. WGA-HRP was injected into muscle 178 at various times after crushing nerve 5. In agreement with previous results every muscle 1’78 (n = 89) examined at, or later than, 10 days postnerve crush was innervated by the appropriate motor neuron Df. This neuron is easily recognized as the largest cell body on the ventral surface of the ganglion (Figs. 1 and 3). In addition, by 10 days after nerve crush the muscle was also innervated by D,, inhibitory neurons, and other unidentified motor neurons. At 20 days after nerve crush every muscle examined (n = 10) received an inappropriate axonal branch from D,. It was relatively easy to identify D, in the ganglia because it is the only leg motor neuron whose cell body is present on the dorsal surface (Fig. 1). During the time

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interval between 40 and 60 days after nerve crush this inappropriate axonal branch became eliminated (Fig. 4). From 60 days onward no muscles (n = 52) were observed to be innervated by D,. The regeneration of the three inhibitory neurons could easily be followed because of the position of their cell bodies in the medial region of the ventral surface of the ganglia (Figs. 1 and 3). Although in unoperated cockroaches muscle 1’78never receives innervation from these neurons, by 20 days after nerve crush every muscle (n = 10) received at least one inhibitor. The elimination of these inappropriate axonal branches required a longer time interval than those of D, (Fig. 5). Although most of the inappropriate axon branches were eliminated during the same interval of 40 to 60 days after nerve crush, 25% of the muscles (n = 12) at 60 days still had axon terminals from an inhibitor. From 80 days onward no muscles (n = 34) were observed to be innervated by any inhibitory neurons. Unidentified motor neurons were also observed to grow inappropriately into muscle 178 so that at 20 days after nerve crush there were on the average 33 (n = 10) such neurons innervating this muscle (Fig. 6). The number of cell bodies and their positions within the ganglia coincide with those of neurons with axons in nerve 5 (Figs. 1 and 3a). Although nerve 3b had not been crushed, it is possible that axons in it may sprout into the denervated muscle 178. However, the extreme lateral motor neurons and SETi with axons in nerve 3b were never observed to send an axon into this muscle when nerve 5 alone was crushed. On the basis of this observation we infer that no neurons with axons in nerve 3b sprout into muscle 178 although they cannot all be definitively iden-

a FIG. 3. Metathoracic ganglia stained for HRP in order to detect those neurons which have an axonal branch in muscle 178. WGA-HRP had been injected in the muscle. Nerve 5 had been crushed 20 days previous and axonal regeneration occurred in an intact leg (a) and in one in which distal segments were removed (b). In both ganglia Dr (black arrowhead) and inhibitory neurons (asterisk) were stained. Additional neurons, including SETi (white arrowhead) with axons in nerve 3 were stained in the ganglion from the animal in which distal leg segments were removed.

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1

A 20

40 Time

60

80

After

Axotomy

100

w-7

(days)

FIG. 4. The frequency with which muscle 178 became inappropriately innervated by D, is plotted against time after nerve 5 was crushed. Data are presented for cockroaches with intact legs (0) and with distal leg segments removed (A). The number of animals analyzed at each time point is in Fig. 6.

tified because of the partial overlap in position of some of their cell bodies with those of neurons whose axons are in nerve 5. The unidentified neurons were, therefore, probably neurons with axons in nerve 5 that had been axotomized and were undergoing axonal regeneration. Like the identified neurons, most of the inappropriate

I------

Time After

Axotomy

(days)

FIG. 5. The frequency with which muscle 178 became inappropriately innervated by at least one inhibitory neuron is plotted against time after nerve 5 was crushed. Data are presented for cockroaches with intact legs (0) and with distal leg segments removed (A). The number of animals analyzed at each time point is in Fig. 6.

20

40 Time

60 After

80 Axotomy

(days)

FIG. 6. The total number of unidentified motor neurons inappropriately sending an axonal branch into muscle 178 is plotted against time after axotomy. Data are presented for cockroaches with intact legs (0) and with distal leg segments removed (A). The numbers in parentheses are the number of experimental ganglia examined at each time. Each experimental point is the mean and the standard deviation is shown.

axon branches of these unidentified neurons were eliminated in the interval of 40 to 60 days after nerve crush. However, when the number of unidentified neurons innervating muscle 178 was counted, it appeared as though these mistakes were not all corrected (Fig. 6). In the interval between 60 and 200 days after nerve crush there remained on the average between 10 and 6 inappropriate, unidentified motor neurons innervating muscle 1’78. It should be noted that the standard deviation around the mean for these later time points was very large (Fig. 6). Further examination of the data reveals that there probably are two populations of experimental cockroaches, one which does not undergo axonal elimination and one which does. In 12 different preparations at 100 days after nerve crush the numbers of unidentified neurons with inappropriate axons in muscle 178 were 19,0, 24,13,0, 5, 0,13,0,0, 23,22. Similar data for 12 preparations at 200 days after nerve crush were 0, 0, 29, 15, 0, 0,15,0,0,0,17,0. The presence of a group of animals that did not undergo axonal elimination was made more apparent by calculating the percentage of muscles which have no mistakes (inappropriate axon branches from unidentified motor neurons) at each time point after nerve crush (Fig. 7). At the latest time examined, 200 days after nerve crush, there remained a group, containing at least 33% of the preparations, which did not undergo axonal elimination of unidentified motor neurons. The use of cockroaches of the same age, standardized surgical procedures, and standardized conditions of

JEFFREY L. DENBURG

Plasticity

80 t

Time

After

Axotomy

(days)

FIG. 7. Frequency with which muscle 178 became reinnervated just by Df with no inappropriate neurons is plotted against time after axotomy. Data are presented for cockroaches with intact legs (0) and with distal leg segments removed (A). Muscles with no mistakes were never found in animals with distal leg segments removed.

postsurgical incubation failed to eliminate this population of animals. It is not likely that these results were caused by variability in the injection of WGA-HRP because injection of enzyme into unoperated animals never resulted in staining of thle unidentified motor neurons. In addition, at the later t.imes after nerve crush (later than 60 days), D, never innervated muscle 1’78 even in preparations in which many unidentified motor neurons were stained. Hormonal differences are not likely to account for this variability among the cockroaches because in some animals in which nerve 5 was crushed in both the metathoracic and the mesothoracic ganglia it was observed that one ganglion exhibited good axonal elimination while the other di#d not. It is not known why, in such a significant number of cockroaches, the unidentified motor neurons innervating more distal leg muscles fail to eliminate inappropriate axon branches from the more proximal muscle 178. Elimination of Inappropriate Axonal Branches in Cockroaches with Distal Segments of Leg Removed The extracellular injection of WGA-HRP into muscle 178 was again used to fol.low the axonal branching of motor neurons in cockroaches whose nerve 5 was crushed immediately after removal of the distal segments of the leg. Although this operation, when performed on nymphs, results in replacement of the missing leg segments (Bodenstein, 1955), there was no evidence of such leg regeneration in the adult cockroaches used here. This additional surgery did not affect the ability of the motor

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neurons to regenerate after the axotomy. In every muscle 178 examined at 10 to 200 days after nerve crush (n = 95) the appropriate motor neuron Df was observed to have reinnervated the muscle successfully. The removal of distal segments of the leg also did not affect the ability of motor neurons, whose normal target muscles were still present in the remaining parts of the leg, to eliminate inappropriate axonal branches from muscle 178. In particular, D,, whose normal target muscles are other coxal depressor muscles which are still present, eliminated its inappropriate axonal branches in muscle 178 at nearly an identical rate in cockroaches with distal leg segments removed as in those with intact legs (Fig. 4). The inhibitory neurons innervate the still present coxal depressor muscles but also have other target muscles in the distal segments of the leg. Their inappropriate axonal branches in muscle 178 were eliminated at a slower rate in the animals with distal leg segments removed than in those with intact legs. Even at long times after nerve crush there remained between 30 and 10% of the muscles containing inappropriate axonal branches of an inhibitory neuron. However, it was the unidentified motor neurons that responded most to the removal of the distal segments of the leg. At early stages of regeneration muscle 178 became reinnervated by an average of 43 unidentified motor neurons (Figs. 3b and 6). This was a greater number than the 33 that reinnervated the muscle during axonal regeneration in intact legs. This discrepancy resulted from the fact that when the distal leg segments were removed, several neurons with axons originating in nerve 3b were additionally axotomized. Nerve 3b fused with nerve 5 just proximal to the femur. Of these neurons the large anterior, medial SETi and the extreme lateral ones could be readily identified. During the interval between 40 and 80 days after nerve crush there was some elimination of inappropriate axonal branches of the unidentified motor neurons. However, from 80 days until the latest time examined, which was 200 days after nerve crush, there remained on the average 25 unidentified motor neurons with axons in muscle 178. The removal of their normal target muscles in distal leg segments enabled them to form stable, inappropriate axonal branches in muscle 178. The question remains as to which of the 43 unidentified motor neurons with axons in nerves 3b or 5 had their inappropriate axonal branches eliminated during this 40-day interval. From this set of neurons the only one which could reliably be identified in ganglia from different individual cockroaches was SETi which is indicated in Fig. 3b. The inappropriate axonal branches of SETi were eliminated during the interval of 40 to 80 days after nerve crush (Fig. 8).

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nervous systems. Three examples of morphological plasticity have been revealed in the cockroach neuromuscular system by the retrograde transport of WGAHRP extracellularly injected into the muscle in order to detect neurons sending axon branches into that muscle. These examples are:

6 2

60 I

:

4.

\ \ \ \

A \

i?

--__

:

r’ ‘ij

--

-q+.

--

& 20. I

!i l-LLuA+J G a

20

40 Time

60 After

60 Axotomy

100 (days)

200

FIG. 8. Frequency with which muscle 1’78 became inappropriately innervated by SETi is plotted against time after axotomy. Data are presented for cockroaches with intact legs (0) and with distal leg segments removed (A).

Can Regenerating Motor Neurons Whose Normal Target Muscles Have Been Removed Grow into Fully Innervated Muscles? The possible growth of inappropriate axon branches into fully innervated muscle 178 could be examined because of the absence of a detectable transfer of WGAHRP between regenerating and nonregenerating motor neurons. In animals in which the distal segments of the leg were removed but nerve 5 was not crushed, Df was intact but the unidentified neurons in nerves 5 and 3b were axotomized. It was observed that these unidentified neurons were stained after injection of WGA-HRP into muscle 178 even though this muscle’s normal innervation was unperturbed. The staining of the unidentified neurons probably resulted from the growth of inappropriate axonal branches into the muscle. It was also interesting to note that in spite of the existing intact and extensive innervation of the muscle by Df these inappropriate axonal branches remained in muscle 178 for at least 200 days after the removal of their normal target muscles. This formation of stable, inappropriate axonal branches in normally innervated muscles was an additional and unexpected example of plasticity in the cockroach neuromuscular system. DISCUSSION

Plasticity

in the Cockroach Neuromuscular

System

The results presented in this paper are consistent with the recent view that invertebrate nervous systems are able to express morphological and synaptic plasticity in a manner analogous to that long attributed to vertebrate

(1) The formation and elimination of inappropriate axon branches innervating muscle 178 during axonal regeneration in animals with intact legs. (2) The decrease in rate of elimination and the formation of stable inappropriate axon branches innervating muscle 178 when the normal target muscles of these neurons are removed prior to crush of nerve 5. (3) The formation of stable, inappropriate axon branches growing into an intact, fully innervated muscle 178 when the normal target muscles of these neurons are removed. Other observations on the cockroach neuromuscular system previously made using electrophysiological techniques are consistent with these observations. It was first reported by Whitington (1977) that Df had the ability to form an inappropriate synapse on muscles when its normal target within the coxal depressor muscles was removed. In addition, he demonstrated that the coxal depressor muscle becomes functionally innervated by inappropriate motor neurons after crushing nerve 5 (Whitington, 1979). Although many of these inappropriate synapses became inactivated at later stages of regeneration, it was noted that several still persisted for long periods of time. These probably correspond to the 33% of the cockroaches that we have observed to not undergo elimination of inappropriate axonal branches. The anatomical technique used in this communication validly detects only neurons with axon terminals in muscle 178. Its major advantage over electrophysiological techniques is that it enables the visualization in one preparation of perhaps all the neurons with axon terminals in muscle 178. However, many of these axon terminals may not be forming functional synapses. This was most likely to be true for the inhibitory neurons which grew into muscle 178, which probably does not have a receptor for the putative neurotransmitter, y-aminobutyric acid. Therefore, electrophysiological techniques by themselves cannot give a total picture of the plasticity of the system. It was pleasing to find that the two approaches give a consistent picture of the events occurring during axonal regeneration in the cockroach neuromuscular system. It certainly will be necessary to confirm with electrophysiological techniques the new results obtained here, i.e., that removal of their normal target muscles enables motor neurons to make stable, inappropriate axonal branches in both previously denervated and fully innervated muscle 178.

JEFFREY

L.

DENBURG

Plasticity

Similar types of synaptic plasticity have previously been observed during axo:nal regeneration after removal of muscles in the crayfish neuromuscular system (Hunt and V&lez, 1982; Clement et al,, 1983). Removal of the limb during embryonic development of the locust enables an identified motor neuron to send axon branches into and to form functional synapses with muscles it normally never innervates (Whitington and Seifert, 1984; Whitington, 1985). In addition, collateral sprouting of intact cricket motor neurons was induced by partial denervation of the extensor tibiae muscle (Donaldson and Josephson, 1981a). During the subsequent axonal regeneration inappropriate motor neurons innervated the muscle, but there was no displacement of sprouted axons and when the muscle was totally denervated reinnervation did not produce the original innervation pattern (Donaldson and Josephson, 1981b). This apparent absence of specificity may rseflect the relatively short time intervals between axotomy and examination, the longest being 90 days, and the fact that the nerves were cut, not crushed. The above examples further demonstrate the plasticity of invertebrate neuromuscular systems. It is of interest to compare these observa.tions with those made on vertebrate neuromuscular systems experimentally manipulated in a similar manner. In the frog (Lamb, 1981) and the chick (Whitelaw and Hollyday, 1983) the removal of hindlimb segments at ea.rly stages of embryonic development results in a selective increase in cell death in those pools of motor neurons whose target muscles were removed. In spite of an initial axon growth into the remaining muscles these motor neurons do not have the ability to form stable synapses with inappropriate muscles. After the period of cell death there is extensive elimination of synapses in the vertebrate neuromuscular system until each muscle fiber is innervated by a single motor neuron (reviewed by Bennett, 1983). This synaptic elimination does not occur in a random manner but ensures that within a pool of motor neurons innervating a single muscle there is a topographical projection from different segmental nerves across a dimension of the muscle (Brown and Boot.h, 1983; Bennett and Lavidis, 1984). The effect of removing part of the target muscle on the formation of this pattern of innervation has not been examined. With few exceptions, axonal regeneration in the vertebrate neuromuscular system does not result in the reinnervation of muscles by neurons only from the original motor pool. Stable, inappropriate synapses are formed with neurons from other motor pools (reviewed by Mark, 1980). Transplantation of a foreign nerve into a denervated muscle (Wigston, 1980) as well as a fully innervated muscle (Bixby and Van Essen, 1979) leads to the formation of stable, inappropriate synapses. These experiments are similar to those in which target

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muscles are removed since the transplanted nerve never has the opportunity to reinnervate its normal muscles. From the examples in the preceding discussion it appears that vertebrate motor neurons in the embryonic state exhibit great specificity for their target muscles with little potential for synaptic plasticity. In the mature state these neurons have acquired this plasticity but lost the ability to specifically recognize and reinnervate their target muscles. In comparison, the regenerating motor neurons from the adult cockroach retain the ability to selectively reinnervate their target muscles while also having the ability to express morphological plasticity when the system is experimentally manipulated. In this paper the term “inappropriate” was used to describe the connections formed between motor neurons and muscles that are not observed in the intact, normal cockroach. The use of this term also signifies that any contraction of muscles by these connections produced movement that was behaviorally incorrect. However, with the removal of its distal leg segments the leg does not contribute to behavior. This change in state of the system enabled the stabilization of these connections and allows them to now be considered as “novel” instead of “inappropriate.” The use of the term “novel” emphasizes the plasticity of the system and more closely draws the analogy to similar phenomena occurring in vertebrate nervous systems. SpeciJicity of the Interactions and Muscles

between Motor Neurons

The observation was made that at least 66% of the adult cockroaches with intact legs undergoing axonal regeneration ultimately restore the original innervation pattern. This means that in muscle 178 of these animals axon branches and synapses of Df are maintained while those of other neurons are eliminated. Apparently, some intercellular interaction of great specificity is occurring. However, this specificity is not absolute, since transient mistakes are made, and when the normal target muscles of motor neurons are removed axons of these cells make permanent, inappropriate connections with muscle 178. This potential plasticity of the system is regulated by intercellular interactions of sufficient specificity to ensure that the original innervation pattern reforms when all target muscles are present. Our observations of the extensive branching of the motor neurons within the muscles (Denburg, 1982b) and of the specific regeneration of the dorsal unpaired medial neurons (Denburg and Barker, 1982) led us to suggest that the specificity arises from selective enhancement of axonal growth and sprouting in the appropriate muscle. Each neuron has the potential to grow into and make a small number of functional connections with any muscle. However, only

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the axon of the appropriate motor neuron will continue to grow and sprout profusely. The concomitant elimination of inappropriate connections may result from: (a) intrinsic control over the maximum number of axon terminals a motor neuron may maintain, and/or (b) insufficient amount of a muscle-derived trophic factor that is removed by the eventually successful competing motor neuron that has a greater number of axon terminals in the muscle (Purves and Lichtman, 1980). The results presented in this communication are consistent with these hypotheses. The elimination of inappropriate axon branches occurs between 40 and 60 days after axotomy. This represents a relatively late occurring phenomenon in light of the observation that muscle 1’78 is fully innervated by 20 days after nerve crush. During this long interval between initial innervation of the muscle and the elimination of inappropriate axon branches, the motor neurons are reinnervating their normal target muscles within which they are sprouting profusely. The profuse branching of a regenerating motor neuron within its target muscle may therefore be a prerequisite for its elimination of axonal branches in inappropriate muscles. In addition, the absence of the normal target muscles, in which the regenerating motor neurons normally would sprout profusely, somehow enables the maintenance of axonal branches in an inappropriate muscle. These results were not expected when this study was initiated. They are inconsistent with those hypotheses involving absolute specificity or a strict competitive interaction between appropriate and inappropriate axons in which, no matter how the system is perturbed, the appropriate axon always prevails. An alternative hypothesis often invoked to explain observations of the formation of stable inappropriate synapses during axonal regeneration is that there has been a change in the macromolecular markers distinguishing the different postsynaptic cells (Schmidt, 1978). This probably did not occur in muscle 178 because the inappropriate axon branches of D,, the inhibitory neurons, and SETi were always eliminated. The hypothesis of a selective sprouting of appropriate axons within muscles implies a selective adhesion of axonal growth cones to the muscle or its basal lamina and/ or a selective response to growth or sprouting factors. We already have evidence that the various coxal depressor muscles can be characterized by their cell surface glycoproteins (Denburg et al, 1983). Each of the muscles, therefore, offers a chemically different substratum to regenerating axons. The observation that the intercellular interactions involved in the reformation of the original innervation pattern of the cockroach muscles are selective rather than absolutely specific suggests that there are quantitative differences in the strengths of the interactions between the cells. There may exist a

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hierarchical arrangement of the interactions between the different motor neurons and muscle 178. The rules for selectivity might involve the identity of the nerve containing the axon, whether the motor neuron is fast or slow, or the position along the proximal-distal axis of the normal target muscle. Further examination of the selective sprouting hypothesis and determination of the hierarchical rules of selectivity will require the observation of the behavior of axon terminals of various regenerating, identified neurons in appropriate and inappropriate muscles at various times after nerve crush. Toward this goal we are attempting to obtain monoclonal antibodies that will recognize various identified neurons and that could be used to observe their regenerating axon terminal immunohistochemically (Denburg et aZ., submitted for publication). In addition, since most elimination of inappropriate axons occurs in the relatively short interval of 40-60 days after nerve crush, it may be feasible to perturb this process with specific antibodies and other chemicals. Although a single hypothesis was proposed here, it is realized that there may exist a multitude of intercellular interactions which operate during the formation and elimination of nerve-muscle synapses. In conclusion, it appears that the plasticity exhibited by the regenerating cockroach neuromuscular system is similar to that expressed by many parts of the vertebrate nervous system. Further studies on this experimentally accessible “simple” system may give insight into the mechanisms invoked to explain the constraints imposed on this plasticity by specificity generating intercellular interactions. I thank Robert Caldwell for preparing the figures, Mark Clymer for photographic assistance, Ana Garner for typing the manuscript, and Dr. Jerry Kollros and Dr. Stanley B. Kater for critically reading the manuscript. This research was funded by NIH Grant NS14295 and by Research Career Development Award NS00422. REFERENCES BENNETT, M. R. (1983). Development of neuromuscular synapses. Physiol. Rev. 63, 915-1048. BENNETT, M. R., and LAVIDIS, N. A. (1984). Segmental motor projections to rat muscles during the loss of polyneuronal innervation. Dev. Brain Res. 13, l-7. BIXBY, J. L., and VAN ESSEN, D. C. (1979). Competition between foreign and original nerves in adult mammalian skeletal muscle. Nature (Lmdon) 282,726-728. BODENSTEIN, D. (1955). Contribution to the problem of regeneration in insects. J. Exp, Zoo1 129,209-224. BRKUNIG, P. (1982). Strand receptors with central cell bodies in the proximal leg joints of orthopterous insects. Cell TissueRes. 222,647654. BROWN, M. C., and BOOTH, C. M. (1983). Postnatal development of the adult pattern of motor axon distribution in rat muscle. Nature (Lxdon) 304,741-742. BURROWS, M., and HOYLE, G. (1973). Neural mechanisms underlying

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