Download Degeneration and Regeneration in Crustacean

AMER. ZOOL., 13:379-408(1973).
Degeneration and Regeneration in Crustacean Neuromuscular Systems
GEORGE D.
BITTNER
Department of Zoology, University of Texas, Austin, Texas 78712
SYNOPSIS. Crayfish motor neurons seem to repair damage to peripheral axons by selective fusion of outgrowing proximal slumps with severed distal processes that can survive morphologically and physiologically intact for over 200 days. Survival of isolated
motor and CNS giant axons is associated with much hypertrophy of their glial sheath.
The severed stumps of peripheral sensory neurons often degenerate within 21 days and
their glial sheath does not hypertrophy. Denervation and immobilization produce relatively little change in the morphology and physiology of the opener muscle, whereas
tenotomy produces much atrophy within 30-60 days.
Crayfish motor and CNS giant neurons show no capability for regenerating ablated
cell bodies, whereas peripheral sensory somata regenerate after limb autotomy. An entire opener muscle can be replaced after limb autotomy but the organism shows little
or no ability to redifferentiate an entire muscle in the absence of body part regeneration. However, a few opener muscle fibers can be regenerated if the bulk of the
muscle mass remains intact. The significance of all these findings axe interpreted with
respect to the developmental capabilities and environmental adaptations of the crayfish together with the evolution of regenerative abilities in anthropods and vertebrates.
Crustaceans are a favorable group in
which to study many aspects of regeneration or degeneration in nerve and muscle
because of the simple organization of their
neuromuscular systems when compared to
those of vertebrates. For example, two distal segments of die crayfish claw or walking leg each contain only two muscle masses having antagonistic actions (Fig. 1/4).
Only eight nerve axons (five excitatory,
three inhibitory) innervate these four muscles; in fact, one axon provides the sole excitatory supply of the opener and stretcher,
two separate muscle masses that are located
in different limb segments. This innervation pattern and the fact that motor axons
are much larger and have thicker glial
sheaths than sensory axons (Fig. \B-E)
make it possible to study the interaction
specificities among identified neurons. Data
obtained from Crustacea also further our
understanding of the evolution of regenerative capabilities both within the Arthropods and among different phyla. Such comparisons provide a test of the postulate that
This work was supported in part by NIH
(#26-1670-3550) and NSF (GB-30199) grants to
the author.
379
tissue or cellular regenerative capabilities
have not significantly decreased during evolution while the ability to reform lost body
parts (epimorphic regeneration) has declined; i.e., it is not regenerative capability
per se which has been selected against during phylogeny but the level of organization
at which it occurs (Goss, 1969). Finally, the
study of degenerative phenomena in Crustacea may be of interest because of the
long-term survival of certain crayfish nerve
axons and terminals following separation
from their cell bodies. Thus, the nature of
nerve-glial interactions, the longevity of
messenger RNA, or the axonal capability
for protein synthesis may be more dramatic and, therefore, more easily studied in
crustacean axons than in vertebrate neurons. Much of this data should be applicable to vertebrate neurons if cellular
mechanisms of degeneration and regeneration—or trophic interactions between
nerve, muscle, and glia—have undergone
conservative evolution similar to that observed for other basic neuronal processes
such as axonal conduction or synaptic
transmission.
Regenerative phenomena in metazoans
380
GEORGE D. BITTNER
Segmenf:
Muscle
Masses:
Meropodite
Carpopodite
Bender
Stretcher
• Propodite
Ooctyl
Opener
Closer
Thick f.
Bundle t
Thin
f
Bundle"-
E
FIG. 1. Innervation pattern and segmental arrangement of the claws or walking legs in the crayfish
Procambarus clarkii. A, Organization of nerves and
muscles in the carpopodite and propodite segments: solid line, excitatory motor neuron; dotted
line, inhibitory motor neuron. Thick bundle and
thin bundle: major divisions of main meropodite
nerve trunk in which the peripheral motor axons
are found. B, Sections of the main nerve trunk at
the level of the proximal meropodite showing motor axons (M) , sensory axons (S), and tendon
(T) of the small accessory flexor muscle. The
nerve trunk is surrounded by darker staining muscle fibers of the extensor and flexor muscle. In
this section, fifteen out of sixteen possible motor
axons are distinguishable having a large size and
thicker glial sheath than the sensory axons (arrow points to the motor axon innervating the accessory flexor muscle) . C, Section of distal meropo-
dite nerve showing the eight nerve axons diagrammed in A which innervate the muscles of the
carpopodite and propodite. D, Section of the main
nerve trunk in the midcarpopodite showing the
arrangement of the five motor neurons which innervate the muscles of the propodite; the grouping of two axons will innervate the opener muscle
and the grouping of three axons go to the closer
muscle. A blood vessel (BV) normally separates
the two groups. E, Section of the dorsal nerve
trunk in the midpropodite showing that the two
motor axons innervating opener muscle fibers to
the left are located within a few hundred microns
of the closer axons innervating closer muscle fibers
(X) . All sections fixed in cold aqueous Bouins for
24-72 hours and double embedded in paraffin.
Magnification (X40) . (Figure from Bittner and
Johnson, unpublished.)
such as Crustacea can be studied in terras
of tissue (wound healing) or epimorphic
(body part) regeneration. The former level
can be further subdivided into (1) the regeneration of lost or damaged pieces of
cytoplasm, e.g., neurons with severed axons
or severed muscle fibers need only hypertrophy in order to restore the original tissue morphology, and (2) the regeneration
of lost cells, e.g., after removal of nerve
cell bodies, muscle fibers, or muscle masses,
the original tissue morphology can be restored only by a local hyperplastic response
of the remaining tissue and/or by hyperplasia and differentiation of "reserve" cells.
Since regeneration of body parts also requires both a hyperplastic and hypertrophic response by the remaining tissues and/
or reserve cells, the distinction and relationship between tissue and epimorphic regeneration is often hard to define. One
should also keep in mind that the fate of
REGENERATION IN CRUSTACEA
lost body parts or damaged tissues is variable. For example, vertebrate and arthropod "tails" or limbs degenerate when separated from the rest of the organism but
echinoderm limbs (with CNS ganglia) and
annelid or platyhelminth "tails" do not;
in fact, an entire organism can "regenerate" from these lost parts. Furthermore,
enucleated, membrane-bound cytoplasm
from the unicellular marine alga Acetabularia or from the mature mammalian
erythrocyte degenerates much more slowly
than do enucleated pieces of cytoplasm
from other unicellular organisms or from
mammalian neurons (Harris, 1970; Young,
1942).
SURVIVAL OF SEVERED PERIPHERAL
NERVE AXONS
The degeneration of severed (distal1)
nerve axons has been extensively studied
in vertebrate neurons (Birks et al., 1960;
Ohmi, 1961; Ramon y Cajal, 1928; Ranson, 1942; Young, 1942) as evidenced by destructive cytological changes, loss of electrical excitability of the axonal membrane,
and inability to release transmitter from
synaptic terminals. These processes are
generally completed within a week, although elimination of remnants of the
myelin sheath may take as long as 50 days.
Analogous studies on invertebrates have
most often focused on insects (Bodenstein,
1957; Guthrie, 1962, 1967; Jacklet and Cohen, 1967; Usherwood, \963a,b; Usherwood et al., 1968). In this class of organisms, the time course of peripheral axonal degeneration is often longer than for
11 shall use the terms distal and proximal to
refer to those neuronal (cytoplasmic) processes
that are respectively severed from, or in direct
communication with, their nucleated perikaryon.
I shall use the terms central and peripheral to
refer to that part of a severed limb nerve that
is nearer to, or further from, the ventral nerve
cord (CNS). The distinction is important in crustacean peripheral nerves because all the sensory
cell bodies are located in the periphery. The central stump of a mixed nerve trunk severed in a
limb, therefore, contains the proximal processes of
motor neurons and the distal processes of sensory
neurons.
381
vertebrates (Boulton, 1969; Guthrie, 1967;
Tung and Pipa, 1971; Usherwood, 19636;
Usherwood et al., 1968; see also facklet and
Cohen, 1967). Some of the variation in reported degeneration times among insects
or between insects and vertebrates can be
attributed to the differences in temperature at which the organisms are maintained
(Katz and Miledi, 1959; Usherwood et al.,
1968). Two studies in vertebrates have reported long survival times for severed axonal stumps, but the interpretation of each is
questionable and both were carried out at
low temperatures (May, 1925; Kriiger and
Maxwell, 1969). Temperature differences
may also account for discrepancies in the
relatively few studies reported for organisms of other phyla. For example, Wilson
(1960) mentions persistent excitability of
severed distal motor axons in octopuses
kept at 15 C, whereas Sereni and Young
(1932) and Young (1972) find histological
evidence of degeneration within a few days
in animals kept at 25 C. (The latter two
studies did not differentiate between motor and sensory axons, an important consideration in crayfish axonal degeneration.)
However, when ectotherms or hetero therms
such as frogs (Birks et al., 1960), locusts
(Usherwood et al., 1968), cockroaches
(Guthrie, 1962, 1967; Jacklet and Cohen,
1967), and crayfish (Bittner, present report;
Hoy et al., 1967; Nordlander and Singer,
1972) are all maintained at room temperatures of 16-22 C, their motor axon degeneration times are 3-6, 14-28, 2-7, and 90 to
over 200 days, respectively.
Crustacean motor axons have been reported to survive for many months following separation from their cell body (Bittner and Atwood, Bittner and Johnson, in
preparation; Hoy et al., 1967; Nordlander
and Singer, 1972). In making this claim,
however, one must be careful to eliminate
not only the effects of temperature as discussed above, but also the possibility that
axonal fusion between outgrowing proximal stumps and surviving distal stumps has
somehow rapidly occurred to provide a
tenuous morphological connection which is
unable to transmit an action potential but
382
GEORGE D. BITTNER
REGENERATION IN CRUSTACEA
which can pass important metabolic substances (Fig. 6C). Since this possibility was
not eliminated in previous studies (Hoy et.
al., 1967; Nordlander and Singer, 1972), we
have tried to rule it out, (1) by resecting
long (1-2 cm) segments of the peripheral
nerve trunks in the meropodite and then
placing a small ball of wax in the lesion
site (15 animals), and (2) by tying off the
proximal and/or distal stumps with a fine
strand of hair or platinum wire (30 animals). No evidence of physiological reconnection or significant neuronal outgrowth
was seen even 400 days after either operation, yet the distal stumps of the motor
axons remained morphologically and
physiologically intact for many months. We
are currently resecting 0.5-2 cm segments
of peripheral nerve axons from the meropodite segment of the walking legs and
implanting them in other claw or abdominal sites to try to isolate them even more
effectively from regrowing processes of motor axons. Preliminary results indicate that
isolated motor axons in these nerve trunks
also survive for at least 20 days.
The distal stumps of motor axons isolated by tying off the nerve trunks or by
resecting long segments of nerve in the meropodite are sometimes able to conduct action potentials and to release transmitter
for over 200 days (347 days in one axon)
when stimulated with hook or suction electrodes at 1-2 Hz (Bittner and Johnson, Bittner and Atwood, unpublished). Beyond
383
200 days, however, most distal stumps fail
to conduct action potentials or to release
transmitter. In general, there is little difference between the ability of severed stumps
and intact motor axons to fire repetively
or to release transmitter for about 60 days;
from 60 to about 120 days, severed axons
become more refractory to repetive stimuli
and transmitter release defacilitates more
rapidly than normal; beyond 120 days, the
ability of the nerve axon to conduct repetitive action potentials is rather limited and
transmitter release is often impossible to
elicit after 2-5 min of 10 Hz stimulation.
Recently severed axons usually conduct action potentials and release transmitter
without defacilitating for 5-12 hr at this
stimulus frequency. This eventual failure
to release transmitter in normal or severed
axons during continuous stimulation appears to result from an inability of the
axon to conduct action potentials for the
following reasons: (1) Failures are usually
"all or nothing" to a given impulse and
occur simultaneously in different muscle
fibers of the same muscle body or in fibers
located in different muscles (in the case of
the opener-stretcher excitor). (2) The quantal content of the ejp's is usually well facilitated before and after such "missed" releases. (3) Once they begin, all or nothing
failures occur more and more frequently
during a stimulus train, and within a few
minutes the axon no longer fires even after
a long rest period. This evidence that axon-
FIG. 2. Electron micrographs of the two motor Typical motor nerve terminal on the opener musaxons which innervate the opener muscle. A, cle from an animal in which the distal stump of
Branches of nonlesioned (control) axons taken
the severed excitatory axon showed normal nerve
from the midpropodite. Both axons are surrounded
function at 34 days. Such functioning terminals
by a 1-2 JJ. layer of glial cells and are enclosed in are almost indistinguishable from those of nonseva connective tissue capsule. Mitochondria are typi- ered axons. D, Nerve terminal on the opener muscally clustered around the periphery of the axon. cle from an animal in which the distal stumps of
B, Cross-section through the same two motor axons
the opener axons showed no ability to conduct
severed 142 days previously and for which no func- action potentials or to release transmitter 203 days
tional connection to the CNS was noted. Stimula- after lesioning. Such terminals are typically swollen
tion of the axons distal to the lesion site did not
and contain few, if any, synaptic vesicles. All secevoke transmitter release from the nerve terminals. tions fixed in 5% glutaraldehyde, post-fixed with
Both Axon 1 and 2 are of smaller diameter than
1% osmium, and embedded in epon. Magnification
the control axons in A and have thicker glial
(X4.100) for A and B. (X21.000) for C, and
sheaths. Satellite axons lie between what appears
(X 9,200) for D. (Micrographs from Kennedy and
to be the membrane of the original distal stump Bittner, Atwood and Bittner, unpublished.)
of Axon 1 and its surrounding glial sheath. C,
384
GEORGE D. BITTNER
al conduction is more labile than synaptic
transmission in normal or severed crayfish
axons contrasts with most data obtained
from vertebrate or insect systems (Birks et
al., I960; Del Castillo and Katz, 1954; Hubbard and L0yning, 1966; Usherwood,
1963&; Usherwood et al., 1968). Atwood
has examined the synaptic morphology of
these severed nerve axons and has observed
that those terminals which continue to release transmitter in a normal fashion generally maintain a fairly normal synaptic
ultrastructure (Fig. 2C). These crustacean
synapses appear to undergo degenerative
changes similar to those seen in vertebrates
and insects (mitochondria! swelling, vesicle
agglutination, loss of vesicles, etc., Fig. 2D)
except that the crustacean time scale seems
FIC>. 3. Electron micrographs of sensory (A-C)
and motor (D-£) axons in the meropodite nerve
trunk. A, Nonlcsioned (control) sensory axons.
Note dark-staining glial nuclei but lack of glial
sheath layers surrounding these axons. B, Proximal
stumps of sensory axons severed 49 days previously. C, Distal stumps of sensory axons severed for
49 days. D, Section through one edge of two adjacent, nonlfsioned, motor axons (AX) surround-
ed by an adaxonal glial layer (AG) and a glial
sheath-connective tissue layer (GS) . E, Section
through the distal stumps of two adjacent motor
axons lesioned 34 days previously showing an increase in the thickness of the adaxonal (AG) and
sheathing (GS) layers. Fixation and embedding
as in Figure 2. Magnification (X4.200) for A-C and
(X 10,880) tor D-E. (Micrographs from Kennedy
and Bittner, unpublished.)
REGENERATION IN CRUSTACEA
to be calibrated in months instead of hours
or days (Bittner and Atwood, unpublished).
Distal stumps of peripheral sensory nerve
axons in crustacean limbs appear to degenerate much more rapidly than those of motor axons (Fig. 3). Action potentials from
sensory fibers can be identified by recording from medial branches of the meropodite thick bundle that contain at most one
or two motor axons whose large spikes are
clearly separable from those of the smaller
sensory axons in extracellular recordings.
It is possible to elicit sensory action potentials for about 10 days after lesioning when
stimulating and recording central to the lesion site; after 10 days, these potentials are
more and more difficult to obtain, and
those few which are recorded sometimes
appear to come from axons that have regenerated through the cut region, since
they generally respond to stimulation of
carapace hairs located peripheral to the lesion site. Morphological studies (both light
and EM) show that distal stumps of some
sensory axons begin to degenerate within a
few days after sectioning, many are obviously wasted within 15-20 days, and most
are lysed within 30 days (Fig. 3).
SURVIVAL OF SEVERED CENTRAL AXONS
Wine (1971) has reported that the distal
stumps of medial giant fibers (cell bodies
located in the supra-esophageal ganglia, see
Fig. 4) are also able to conduct action potentials for up to 230 days and retain synaptic efficacy for up to 160 days after cutting of the ventral nerve cord. The diameQ f
V o f
FIG. 4. Location o£ the cell bodies which form the
medial and lateral giant axons in the crayfish ventral nerve cord. Diagonal lines show the amount
of tissue resected during operations to remove the
third abdominal ganglion or the 3-4 abdominal
connective. (Figure from Bittner, Larimer and
Ballinger, unpublished.)
385
ter of the distal stumps is unchanged for
about 50-60 days and then decreases gradually over the next 200 days until little axonal material remains. Hoy (1968) and Wine
(1971) also report no physiological regeneration of the medial or lateral giants for up
to one year after cutting these axons. We
(Bittner, Larimer, and Ballinger) have examined these axons both morphologically
and physiologically 2-3 weeks and 9-14
months after removing most of the connective between the 3rd and 4th abdominal
ganglia or the 3rd abdominal ganglion
(Fig. 4). As reported by Wine (1971), we
find that the posterior segment of the medial giants have indeed degenerated morphologically and physiologically by 9 months
after lesioning, while the segment anterior
to the lesion seems to maintain normal
structure and function (Fig. 5). Even
though their cross sectional diameters are
normal, we also find that the lateral giants
posterior to the operative site are essentially non-functional in their ability to
di'ive peripheral motor neurons nine
months after sectioning. In fact, most CNS
synapses posterior to the lesion (but not
anterior) operate in a very abnormal fashion. This result is particularly interesting
upon considering several reports by Fernandez et al. (1970, 1971) that the transport of labelled proteins injected into crayfish abdominal ganglia occurs only in the
caudal direction. This result is unexpected
on anatomical grounds because many—perhaps a majority—of the cell bodies located
in these abdominal segments have axonal
processes directed cephalad; the lateral
giants, in fact, offer an excellent example
of this morphological arrangement (Fig. 4).
We have noted that "isolated" lateral giant
axonal segments caudal to a septate junction (Fig. 4, Fig. bD)~segments that have
been isolated from their cell body in the
caudal ganglion, but connected to a cell
body in the more cephalad ganglion aooss
a tight junction—survive noticeably better
than do distal stumps of the medial giants
(Fig. 5F). If the results of Fernandez et al.
(1970, 1971) reliably indicate the direction
of transport in the lateral giants, then the
GEORGE D. BITTNER
REGENERATION IN CRUSTACEA
survival of the "isolated" lateral giant segments might be explained by the caudal
transport of trophic substances across a septate junction which is known to allow the
passage of procion yellow molecules of at
least 500 mol wt (Payton et al., 1969). The
abnormal physiology of the caudal segments of the lateral giants might then result from the lack of transport of such
trophic substances. However Pappas et al.
(1971) have also shown that an increase in
coupling resistance occurs within several
hours after cutting the lateral giants 1.5
mm anterior to the system; unless reversed
with time, such a response would make the
passage of substances across the septate
junction more difficult.
MECHANISMS FOR DISTAL STUMP SURVIVAL
Many of the above observations on severed CNS or motor axons raise questions
about possible mechanism(s) underlying
the long-term survival of these isolated
pieces of neuronal cytoplasm. Three major
possibilities are:
1) The motor and CNS giant axons
have significant amounts of the molecular
machinery necessary for protein synthesis
(messenger, ribosomal, and transfer RNA;
mitochondria; enzymes; etc.). These mole-
FIG. 5. Cross-sections through the abdominal nerve
cord in crayfish (Procambarus) , %\/r%" in length.
A, Section through the 1-2 connective in a control
animal. The medial (M) and lateral (L) are
uniquely identifiable by their size and position.
B-F, Sections through the nerve cord of crayfish in
which the third abdominal ganglion was removed
at least nine months previously. B, Section through
the 1-2 connective showing that medial (M) and
lateral (L) axons appear relatively normal as does
the entire nerve cord. C, Cross-section through the
2-3 connective just posterior to the second ganglion of an animal in which the segments of lateral giant axons arising from the third ganglion
have degenerated. A glial cap has almost completely sealed off the medial giant axon (M) on the
right. A similar cap is beginning to seal off the
left medial giant axon. This axon was completely
sealed off within 30 ji. D, Cross-section through
the second ganglion showing medial giant axons
about 150 fi anterior to the formation of a glial
cap. The lateral giant septate junction (L2-L3)
387
cules are either very stable or can be renewed by cytoplasmic processes that do not
require additional information from the
perikaryon; only small molecules like glucose or amino acids need be taken up from
the hemolymph or supplied by (say) glial
cells.
Edstrom and Sjostrand (1969) and Koenig (1970), among others (for review, see
Lasek, 1970), have reported that vertebrate
nerve axons possess considerable ability
for protein synthesis and need not depend
entirely upon axoplasmic transport from
nucleated cell bodies for all of their metabolic requirements. Experiments on eukaryotic organisms such as Acetabularia
have demonstrated that non-nucleated
pieces of cytoplasm can survive for at least
90 days as a result of their ability to synthesize new protein (Hammerling, 1963).
Harris (1970) postulates that the difference
in mechanisms of protein synthesis between eukaryotic systems demonstrating
long cytoplasmic survival times (such as
Acetabularia or mammalian erythrocytes)
and prokaryotes probably lies in the greater
stability of some forms of eukaryotic messenger RNA. Therefore, it could be that
the difference in survival times between
severed vertebrate and crustacean motor
axons—or between severed crustacean
on the left appears reasonably normal, i.e., the
lateral giant segment from the resected third ganglion (L3) has remained intact. The same axon
(L3) on the right has degenerated and is not in
obvious contact with the segment contributed by
the cell body located in the second ganglion (L2) .
E, Cross-section through area of resected tissue at
the level of the third ganglion. No neuronal cell
bodies are present, but a few axons had regrown
to reconnect the separated cord halves (insert) in
this animal. In most (10/12) animals, no axons
were seen to have regrown across the resected segment. F, Section through the 4-5 connective showing lateral giant axons (L) of normal diameter
and medial giant axons (M) with greatly decreased diameter and a thickened glial sheath. All
ventral nerve cords fixed in 5% glutaraldehyde,
embedded in paraffin, and cut in 10 ^ sections.
Magnification for A-F (XI10), insert for E (X
275) . (Data from Bittner, Larimer and Ballinger,
unpublished.)
388
GEORGE D. BITTNER
motor and CNS axons as opposed to crustacean sensory axons—lies in the greater
stability of some of their messenger RNA.
2) The axons and terminals of the motor
and giant inter-neurons contain very large
stores of the metabolites and precursors
needed to maintain normal function for
many months.
Nitzberg (unpublished) has shown that
the excitor motor neuron which innervates
the opener muscle continues to release
transmitter normally when its severed distal stump is continuously stimulated for
up to 12 hr at 20-50 Hz. If electrodes are
implanted over the distal stumps of severed opener axons in chronically maintained crayfish, those axons will respond
normally when stimulated distal to the
lesion site for 1 hr/day at 20-50 Hz for at
least 30 days. After this time period, implanted electrodes often fail to stimulate
the opener excitor, but this failure appears
to result from an intense glial-connective
tissue reaction around the stimulating
electrodes. If the claw is removed from the
animal and electrodes are moved to a more
distal location, the motor axons can again
be stimulated for many more hours. This
chronic stimulation rate (20-50 Hz/hr) of
the opener excitor is significantly higher
than that normally maintained in freely
moving (1-5 Hz/hr; Bittner, personal observations) or deliberately agitated animals
(10-20 Hz/hr; Bittner and Kennedy, 1970).
Extrapolating from the data of Bittner and
Kennedy (1970), in one month these axons
should turn over at least 1500 mm2 of
synaptic vesicle membrane. This calculation would, therefore, seem to provide further evidence that synaptic vesicles are
both reusable and refillable (Bittner and
Kennedy, 1970), if indeed, they are involved in the quantal packaging and release of transmitter. In any case, the ability of severed axons to fire and release
transmitter without defacilitation or fatigue when chronically stimulated for over
a month cannot be easily explained by postulating a large, non-renewable, metabolic
reserve.
3) Metabolic
precursors,
messenger
RNA, or other substances are supplied to
the motor or giant axons from other cells.
Unlike Acetabularia, crayfish are metazoans in which a severed cytoplasmic process could be maintained by other somatic
cells that possess the same complement of
genetic information. The most likely candidates for such trophic maintenance of
severed crustacean motor neurons would
be the glial sheath cells, sensory axons
whose cell bodies are more peripheral to
the cut, the outgrowing fingers of the motor axons themselves, and the muscle fibers
upon which the severed axon makes synaptic contact. The cells of the glial sheath
which surround severed motor axons start
to hypertrophy and to fill with rough endoplasmic reticulum within a few days after nerve section (Nordlander and Singer,
1972; Bittner and Kennedy, personal observations). The glial response seems to
increase in magnitude for some weeks
thereafter and to continue as long as the
distal stump remains functionally and/or
morphologically intact (Fig. IB, 3£). Glial
hypertrophy does not occur to any significant degree around the distal slumps of
sensory axons (Fig. 3C) which remain functionally competent for "only" 7-14 days
(Bittner and Johnson, personal observations). In fact, glial cells often seem to
phagocytize sensory axons, especially after
14 days. Crustacean sensory axons, therefore, appear to degenerate in a manner
similar to that found in vertebrates
(Ramon y Cajal, 1928) and insects
(Boeckh et al., 1969; Lamporter et al.,
1967). We also have light microscopic evidence of glial sheath hypertrophy around
distal stumps of CNS giant axons (Bittner,
Larimer, and Ballinger, unpublished).
However, it is conceivable that these distal stumps could also be maintained by
substances diffusing from the proximal
stumps of nearby sensory axons or from
post-synaptic muscle fibers. Furthermore,
"satellite axons" (see below) seen inside the
sheaths of severed motor neurons could be
rapidly outgrowing processes of sensory
or motor axons that are in at least as favorable an anatomical position as are the
glial cells to supply the severed distal
stumps with appropriate trophic sub-
REGENERATION IN CRUSTACEA
stances. Therefore, Nordlander and Singer
(1972) and we have performed operations
to isolate peripheral nerve axons by making two simple cuts on either side of a
segment of meropodite nerve. This operation may truly "isolate" the nerve segments for a few weeks at most, but during
this time the isolated motor neuronal segments remain intact, the sensory neurons
degenerate, and significant glial hypertrophy occurs only around the motor axons
(Bittner and Johnson, personal observations). We have found that rentoval of
2-4 mm of nerve on either side of the segment "isolates" it for a somewhat greater
time period and gives results similar to
simple cut operations. These data from
"isolated" crustacean axons contrast with
that of Boulton and Fraser-Rowell (1969)
who reported that only 2% of singly cut
axons on either side of the lesion site degenerated within 23 days after severing insect subesophageal connectives, whereas
100% degenerated within two days if isolated by two separate cuts. Nevertheless,
the finding of Tung and Pipa (1971) of
12% axonal survival for 9 days after isolation of waxmoth interganglionic connectives may reflect processes seen in crustacean nerve segments isolated by simple
cuts.
Our observation that motor axons survive for at least 20 days if a piece of meropodite nerve is transplanted to a different limb or to the ventral abdomen is
consistent with the hypotheses that either
the axon has its own synthetic machinery
or the glial cells are supplying trophic
substances necessary for their proper functioning (Singer and Salpeter, 1966). However, the observation that glial hypertrophy also occurs around an as yet unknown length of the proximal stumps of
severed motor axons may indicate that the
glial hypertrophy is a generalized response
to axonal injury and is not necessarily involved in the trophic maintenance of the
distal stump. Furthermore, neither Boulton (1969) nor Tung and Pipa (1971) report glial hypertrophy around severed or
isolated insect interneurons which survive
for at least 9-23 days. The presence of glial
389
cells is also not essential for the maintenance of intact insect neurons since cockroach neurons grown in glial-free cultures
remain in excellent condition for many
months (Chen and Levi-Montalcini, 1969).
Glial hypertrophy has been observed
around central nerve processes in insects
(Hess, 1960; Lamporter et al., 1967) and
vertebrates (Nathaniel and Pease, 1963;
Singer and Steinberg, 1972), but in each
case these axons degenerate within a few
days. Thus, an apparent increase in glial
synthetic activity can signal the start of a
sequence of degenerative processes which,
in vertebrates, includes the production of
lysosomes (Holtman and Novikoff, 1965).
Finally, Boulton and Fraser-Rowell (1969)
have speculated that the persistence of severed axons in singly cut subesophageal connectives in locusts or mantids is due to
an inhibition of glial phagocytosis by a
product released from secretory vesicles of
axons still connected to their cell bodies.
Therefore, double cutting a CNS connective to isolate a segment severs all axons
from their cell bodies and might disinhibit
glial phagocytic responses. In crayfish
axons, phagocytes appear in large numbers
only adjacent to the operative site (Nordlander and Singer, 1972; Bittner and Johnson, unpublished), and would not appear
to be responsible even for the degeneration of sensory neurons.
In considering the three general mechanisms for distal stump longevity outlined
in (l)-(3) above, it seems unlikely on a priori grounds that other cells could provide
all the metabolites and precursors needed
for maintaining the severed stump of CNS
giant neurons or motor axons. That is, stability of axonal synthetic processes (including long-lived messenger RNA) would
be a necessary, if not sufficient, prerequisite
for long-term survival of any cytoplasmic
process. This hypothesis certainly does not
rule out the possibility that glial cells may
contribute important trophic substances—
perhaps even RNA as suggested by the results of Pevzner (1965) and Singer and
Green (1968) for vertebrate interneurons
or by Anderson et al. (1970) for crayfish
giant interneurons.
390
GEORGE D. BITTNER
Post-synaptic
Cell
a)
0
W
0 ©:
FIG. 6. Some possible mechanisms of axonal regeneration, a, Distal stump degenerates while a
new axon grows out from the proximal slump to
reform nerve terminals, b, Distal stump survives
for a long time while the proximal axon regrows
to form new terminals. The original distal segment eventually degenerates, c, Distal stump survives intact and cytoplasmic projections from the
proximal stump fuse with it and/or electrotonically activate it.
MECHANISMS FOR AXONAL REGENERATION
Regeneration of •'he axonal and synaptic apparatus by outgrowth from the
proximal stump coupled with degeneration
of the severed distal stump (Fig. 6a) is not
the only possible way to restore the
morphological and physiological integrity
of damaged nerve axons. Neurons could
regenerate function by rather limited outgrowths from proximal stumps which fuse
with surviving distal stumps (Fig. 6c), a
mechanism postulated by May (1925) for
regeneration of sensory nerves in catfish
barbels. Hoy et al. (1967) also postulated
that crustacean motor neurons use this
regeneration mechanism on the basis of
the following evidence obtained from the
opener and stretcher muscles of the crayfish claw:
1) Stimulation of severed, non-regenerated axons central to a cut or pinch of the
meropodite nerve trunk did not produce
excitatory junctional potentials (ejp's) or
muscle contractions in the opener or
stretcher muscles. In contrast, stimulation
of the severed stumps peripheral to the
lesion site almost always evoked an electrical or contractile response in these muscles
for at least 90 days after the operation.
Distal stumps of peripheral inhibitory
axons also seemed to have long survival
times.
2) Using methylene blue staining, only
two axons were seen to innervate the
opener muscle at any time after severing
the meropodite nerve whether or not a
functional CNS connection has been reestablished. Since two axons—an excitor
and an inhibitor (Fig. \A, £)—normally innervate this muscle and both axons showed
evidence of long-term survival, we would
have expected to find more than two axons
if regeneration of either motor neuron occurred by an outgrowth mechanism analogous to that found in insects or vertebrates
(also see Hoy, 1970, for similar results in
abdominal flexor motor neurons).
3) Behavioral and physiological function returned simultaneously to all fibers
of the opener and stretcher muscles. Since
both muscles are solely innervated by the
same excitatory motor neuron, are located
in different limb segments, and each is innervated from proximal to distal by that
nerve axon, on the basis of the outgrowth
model one would have expected a temporal sequence of proximal to distal reinnervation both of the fibers of each muscle
and of the two muscle masses.
Nordlander and Singer (1972) have also
cut or pinched nerve trunks in the proximal meropodite (lesion width 1 mm or less)
and examined the operative site and/or
sites only up to 1-1.5 cm peripheral to the
lesion. They interpret their results as evidence for the outgrowth hypothesis as follows:
"In our material outgrowth from the [central]
stump was seen by 1 week after nerve section. By the
fourth week each [motor] axon had sent out many
growing sprouts some of which found their way
to the [peripheral] cut edge where they infiltrated
the glial sheaths of the distal segments of [motor]
axons, becoming the satellite axons. Fusion with
the transected [motoij axons could not be estab-
REGENERATION IN CRUSTACEA
lished and the growth pattern of the satellite
axons suggest that it did not not occur. Satellite
axons appeared first close to the lesion and later
more and more distally, growing out along [motor]
axon branches to the muscles. In cross sections of
the [motor] fibers the proportion of the intrasheath
area represented by the satellite axons increased
with time and was greatest close to the lesion and
progressively decreased [peripherally]."
In the last 24 months we (Bittner and
Kennedy, unpublished) also have independently obtained additional data on the
separate, but interrelated phenomena of
(1) long-term morphological and physiological survival of severed crustacean axons,
and (2) refusion of proximal and distal
axon stumps. We have seen a pattern and
time course of outgrowth from proximal
stumps of these motor neurons after pinching or cutting the meropodite nerve trunk
similar to that described by Nordlander
and Singer (1972) and have attempted to
trace uniquely identified motor axons
which have regenerated within the previous
3-5 days as determined by behavioral and
physiological criteria. While we usually
cannot morphologically identify a particular axon solely by its position in one section of the nerve trunk, we have generally been able to narrow the morphological
possibilities to two to three axons by sampling at many sites starting from several
millimeters proximal to the lesion and proceeding distally to the motor terminals.
Data from our current studies, from
Table 1 of Nordlander and Singer (1972),
and from Table 1 of Hoy et al. (1967) or
from Tables 1 and 2 of Hoy (1970) show
that some neuron terminals are physiologically reconnected to the CNS within
20 days after cutting or pinching and that
many more have done so by 30 days. However, both we and Nordlander and Singer
(1972) have found outgrowing fingers 11.5 cm distal to the operative site only
after about four weeks; none of these
fingers has a diameter approaching that of
the large distal process which can be followed to the target muscle. When axons
are examined a few days after behavioral
and physiological reconnection is noted,
we do not observe the predictions (Nordlander and Singer, 3972) for the outgrowth
391
model; that is, we do not see the small
diameter "satellite" axons approach the
nerve-muscle synaptic regions nor do we
see evidence of degeneration of what was
once a motor axon of large (40-80 /x) diameter. (In fact, Nordlander and Singer,
1972, report seeing only four cases of distal
axonal degeneration out of more than 300
motor axons examined at various times after nerve section up to 180 days, and these
four cases were seen in axons separated
for less than four weeks.) After physiological function has returned in a given
neuron, our light- and electronmicroscopic
studies also show that the axonal diameters
are large proximal and distal to the lesion
site, but no large axons completely
traverse the region of the cut or pinch.
This finding also holds for those cases in
which function returns several months after 5-20 mm of axon has been resected.
Finally, Hoy et al. (1967) reported that
function returned simultaneously to the
opener and stretcher muscle after cutting or
pinching. Such "regenerated" axons show
all the complex facilitation properties and
excitor-inhibitor relationships as do normal axons (Atwood and Bittner, 1971;
Bittner, 1968a; Bittner and Harrison,
1970). Our more recent results show that
behavioral evidence of regeneration after
axonal damage is not as easily obtained or
interpreted as once thought (particularly
in animals that take more than 80-90 days
to regenerate); however, there is no obvious proximal-distal sequence of behavioral or electrophysiological return of function as would be expected from the vertebrate outgrowth model. In addition, we
note that the pattern of satellite axon outgrowth described by Nordlander and
Singer (1972) as decreasing in number from
0 to 1.5 cm distal to the lesion site and
increasing with time in this axonal region
is also consistent with the fusion hypothesis for regeneration as long as no fingers regrow to the synaptic region and form new
terminal sites. These satellite fingers could
be the sites of functional connection between the proximal and distal segments at
which electrical activity in the former can
spread to the latter. The actual sites of
392
GEORGE D. BITTNER
fusion might be as difficult to detect as in
fusing muscle cells (Church, 1970; Lipton
and Konigsburg, 1972), even by serial sectioning for electronmicroscopy (an arduous
task not yet attempted). Our current data
could also support the hypothesis that these
motor axons regenerate by some combination of outgrowth and fusion. For example, if the outgrowing fingers all came
from one axon (instead of from many axons
as postulated by Nordlander and Singer),
they might be able to activate the surviving distal stump without membrane fusion
by (1) generating electrical currents that
depolarize the distal stump due to the large
area of opposed membranes or (2) through
the activity of the glial sheath which surrounds both proximal and distal processes
and which could act to insulate the membrane currents from the extracellular fluid.
This phenomenon would resemble the
mechanism of electrical coupling of certain
cells in the chick ciliary ganglion (Martin
and Pilar, 1963) and would result in "functional," but not morphological fusion of
proximal and distal processes. The proximal fingers might then continue to grow
out and eventually form new sy nap tic connections on the appropriate muscle fibers.
If these axons use such a regeneration
mechanism, it should be clear that such
outgrowth models are not directly comparable to those observed in mammals.
Finally, these axons might be "facultative"
fusers in that a proximal stump might
morphologically fuse with a distal stump
that is functionally intact, but still retain
the ability to regrow and form new synapses if die distal stump is non-functional
(Fig. 6/J). In fact, we have some morphological, physiological, and behavioral evidence that such outgrowth can occur in
crayfish which first return function 6-16
months after resection of 0.5-2 cm of nerve
from the meropodite. For example, long
recovery times in these or otlier operations
are often initially associated with very weak
tension development as might be expected
on the outgrowth hypothesis (although
very few axons, < 15%, ever demonstrate
functional reconnection after resecting 1
cm of nerve) and satellite axons are now
seen in very distal regions of the axons
(Fig. 2B).
The functional importance and adaptive
significance of distal stump survival is obvious should some variant of the fusion
hypothesis prove to be correct; that is, fusion would shorten reinnervation time.
Long-term survival of distal stumps might
also play a useful role in increasing the
probability of selective reinnervation by
releasing substances which attract the original motor neuron no matter which of the
above regenerative mechanisms are used.
Since Crustacea use so few excitor motor
neurons to innervate skeletal muscles,
most reconnection errors would certainly
lead to grossly inappropriate behavior patterns (unless one postulates a reordering of
synaptic inputs to the motor neurons). We
have observed almost 300 examples of reinnervation that have occurred within 45
days after lesioning and have rarely noted
inappropriate CNS activation of a muscle
mass; in contrast, inappropriate or inconsistent activation is rather common in those
animals which return function after 90
days. Furthermore, the single excitor and
inhibitor axons to the opener muscle can
be selectively cut in the carpopodite-propodite joint without damage to a major
branch of the two closer excitor and single
inhibitor axons. Although these latter three
axons are within a few hundred microns
of the severed opener axons and of the
opener muscle (Fig. IE), there was no
physiological evidence of collateral innervation of the opener muscle by these "inappropriate" axons in seven animals which
had denervated opener muscles for over
one year. The specificity of these axons in
this experiment is emphasized by noting
that the inhibitor axon to the closer muscle
and the opener excitor axon both innervate the stretcher muscle (Fig. 1/4). The
specificity of crustacean axons reinnervating a muscle within 45 clays after cutting
may even be determined at the sub-cellular level since opener muscles reinnervated by excitor and/or inhibitor axons
seem to have the same correlation of ejp
and ijp amplitudes and facilitation ratios
in single muscle cells as do cells of unop-
REGENERATION IN CRUSTACEA
erated controls (Atwood and Bittner, 1971).
Finally, the distribution of high frequency and low frequency facilitating terminals
of the excitor axon is the same immediately after regeneration as it is in normal animals in those cases in which CNS reconnection occurs within 45 days. Such results are not surprising (indeed are expected) on a fusion hypothesis; however,
if a variant of the outgrowth hypothesis is
correct, then these results indicate precisely determined connection specificities
at the cellular level during regeneration.
The apparent specificity of motor neuronal reinnervation in Crustacea contrasts
with most data obtained from mammals
(Bernstein and Guth, 1961; Edds, 1953;
Weiss and Hoag, 1946), although regenerating motor neurons in the lower vertebrates may show somewhat greater selectivity (Grimm, 1971; Mark, 1965; Sperry
and Aurora, .1965). Insect motor neurons
have generally been reported to show relatively little reconnection specificity (Bodenstein, 1957; Guthrie, 1964, 1967; Jacklet
and Cohen, 1967; see also Pearson and
Bradley, 1972). In contrast, regenerating
393
sensory or CNS axons in vertebrates (see
Gaze, 1970; Jacobson, 1970, for reviews)
and insects (Baylor and Nichols, 1971; Edwards, 1969; Edwards and Sahota, 1968;
Horridge, 1968) do rein nervate a well-defined set of postsynaptic interneurons, perhaps even uniquely specified interneurons
(Edwards and Sahota, 1968). Regenerating
sensory axons in crayfish may also reconnect to specific interneurons (Kennedy, personal communication). The reconnection
specificity of crustacean interneurons is very
difficult to examine because so few axons
regrow more than 1-2 mm into a lesion site
even after one year (Bittner, Larimer, and
Ballinger, unpublished), possibly because
of the formation of a glial cap (Fig. 5C)
which could act to prevent outgrowth in a
manner similar to that seen in mammalian
spinal cords (Guth and Windle, 1970). It
seems that the significantly greater regenerative capability observed for CNS interneurons of fish (Guth and Windle, 1970)
and insects (Boulton, 1968) than for mammals or Crustacea, is associated with a
smaller glial response at the lesion site in
the former two cases.
TABLE 1. Some hypotheses for trophic regulation of muscle properties.
1) Neuronal trophic substances
Nerves might release trophic substances that can affect various muscle fiber properties (outlined in Table 3) via processes that are not explainable by impulse transmission and subsequent
muscle contraction. This theory has three major refinements.
(la) Such substajices might be continuously secreted by nerve terminals or (lb) only during the evoked release of the neurotransmitter substance; in fact, (lc) the substance could be
the neurotransmitter.
(Buller et al., 1960o,6; Draehman, 1967; see Guth, 1968, 1969, or Gutmann and Hnik, 1963 for
reviews)
2) Frequency or pattern of muscle activity
Muscle activity in itself might determine the properties of the muscle fibers and nerve impulses simply control the frequency and pattern of that activity. This theory has several major
refinements.
The relevant "muscle activity" might be the pattern, frequency, and/or magnitude (2a)
of muscle action potentials, (2b) of tension generation by muscle fibers, (2c) of length changes
by muscle fibers, or (2d) of changes in factors external to muscle tissue, such as the blood
supply.
(One or more possibilities suggested by Buller et al., 1960a,Z>; Eccles, 1944; Fischbach and
Robbins, 1969; Tower, 1937.)
3) Muscle resting length
The resting length at which the muscle is held during an experimental procedure might affect muscle fiber properties independent of (1) or (2) above; passive muscle tension, in turn,
should be directly determined by fiber length.
(Fischbaeh and Robbins, 1969; Solandt et al., 1943)
394
GEORGE D. BITTNER
muscles also produces biochemical, morphological, and physiological changes in the
muscle fibers; some of these changes are
Nerve cell bodies are often considered believed to result from the cessation of
to have a trophic effect not only on their trophic input from the motor neuron (see
peripheral axons as described above but Guth, 1968, 1969; Jacobson, 1970, for realso on other nerves or muscles with which cent reviews). The identification of trophic
they make pre- or post-synaptic contact influences on muscle is a multi-factorial
(Guth, 1968, 1969). One possible media- problem of which many of the complicanism for the inability of the posterior seg- tions have not been explicitly discussed;
ments of lateral giant fibers sectioned at Tables 1-5 attempt to outline some conthe 3-4 connective (Fig. 4) to respond to siderations that need be kept in mind when
appropriate sensory input is that the op- reviewing or interpreting any results aceration has interrupted an anterior to pos- cording to one or more theories of possible
terior transport of trophic substances neces- trophic dependencies of muscle (Table 1).
sary to maintain normal synaptic contacts. First of all, the type of muscle fiber (Table
These effects may be analogous to trans- 2) may determine the nature of a certain
neuronal trophic changes seen in mam- trophic response (Table 3) to a given opmalian striate cortex after severing fibers eration (Table 4) at a given developmental
in the optic nerve as well as in many other stage (Table 5). For example, denervation
systems. Denervation of mammalian results in a more rapid and extensive deTROPHIC INTERACTIONS OF CRUSTACEAN
NERVES AND MUSCLES
TABLE 2. General classes of vertebrate and arthropod muscle.
1) Vertebrate twitch muscle
Innervation: single ' ' en plaque'' eiidplate from one motor neuron.
Muscle fiber characteristics: " a l l or nothing" membrane responses; 2-3 y. sarcomere
length; fibrillenstrukture (punctate) myofibrils; abundant sarcoplasmie retieulum; regular
transverse tubular system. Muscles comprised of these fibers may be divided into phasic, tonic,
or mixed on the basis of the contraction times or tetanus/twitch tension ratios of individual
fibers.
a) Phasic: posterior latissimus dorsi (birds).
b) Tonic: soleus (amphibians, mammals).
c) Mixed: gastrocnemius (mammals).
2) Vertebrate " s l o w " or " t o n u s " muscle
Innervation: multdterminal " e n grappe" endings from Eeveral motor neurons (polyneuronal).
Muscle fiber characteristics: phasic may have active membrane response, tonic generally
do not; felderstruktur (punctate) myofibril arrangement; little sarcoplasmic retieulum, less
extensive transverse tubular system than for twitch fibers.
a) Phasic: some fish muscle?
b) Tonic: anterior latissimus dorsi (birds).
e) Mixed tonic " s l o w " and twitch: ileofibulario (amphibians), biventer centralis (birds).
Trophic phenomena observed in these "mixed" muscles need be interpreted with care (Hikida
and Bock, 1970).
3) Arthropod muscle
Innervation: multiterminal, polynouronal.
Muscle characteristics: one can find examples of most any combination of tonicity, phasieity, muscle structure, and membrane properties (Atwood, 1967); however, one generally finds
muscle combinations of:
a) Phasic: active membrane responses, short (2—6 p.) sarcomeres, fibrillenstrukture; example: crayfish " f a s t " abdominal muscles, some coxal muscles of cockroach.
b) Tonic: passive membrane responses, long (8—14 fi sarcomeres), felderstruktur; example:
slow abdominal muscles, crayfish opener or stretcher muscles.
c) Mixed: most crustacean or insect limb and body musculature.
(References: Atwood, 1967; Bittner, 1968a,6; Hess, 1970; Kennedy, 19G7; Ubhenvood, 1962,
1907.)
REGENERATION IN CRUSTACEA
395
TABLE 3. Some measures of trophic states of whole muscle and/or individual fibers.
1) Structure: muscle weight, muscle volume, relative amounts of connective or fatty tissue;
fiber diameter, fiber weight, nuclear number/eytoplasmic volume; ultrastructure of fiber membranes, contractile filaments, etc.
2) Contraetilo properties: speed of shortening, speed of relaxation, tetanus/twitch ratio, etc.
3) Bio-electric properties: resting potential, input resistance, ion permeabilities, sensitivity
to neurotransmitter molecules, etc.
4) Biochemical properties: distribution and sensitivity of receptor molecules, enzyme activity or concentrations, protein turnover, carbohydrate metabolism, etc.
crease of fiber diameter in phasic than in
tonic muscle, as seen for (1) mammalian
twitch muscle (Bajusz, 1964; McMinn and
Vrbova, 1964; Nelson, 1969); (2) avian
"slow" and twitch muscle (Feng and Yang,
1962; Hikida and Bock, 1972; Jirmanova
and Zelena, 1970)—in fact, a "tonic" slow
muscle may even undergo hypertrophy due
to fiber prolifieration; and (3) arthropod
(cockroach) phasic or mixed muscle and
crustacean slow muscle (Fig. 7A) (Guthrie,
1962, 1964; Jacklet and Cohen, 1967). Tenotomy seems to have a greater effect than
denervation on the more tonic muscle fibers
in each case as paired above: (1) mammalian twitch, tonic vs. phasic (McMinn
and Vrbova, 1964; Nelson, 1969); (2) avian
"slow" vs. avian twitch (Hikida, 1972;
Hikida and Bock, 1970, 1972); and (3)
crustacean slow muscle atrophies very rapidly after tenotomy but not after denervation (Fig. 1CJD). However, no comparison can yet be made to phasic crustacean
muscle. A final observation is that multiterminal, tonic muscle shows very little denervation atrophy in crayfish (Fig. 1A) after 200 days.
One study in vertebrates to date that
seems to provide a rather firm interpretation of the trophic dependence of a muscle
property according to the possibilities outlined in Tables (1) and (2) for vertebrate
twitch muscle is that by Loma and Rosenthai (1972). In this study, the spread of
Ach receptor sensitivity that normally follows denervation of all vertebrate twitch
muscle still occurred after nerve blockage
with an anaesthetic cuff, was not seen after
tenotomy, and was greatly reduced by direct stimulation of muscle fibers after denervation or indirect stimulation after
blocking nerve impulses with an anesthetic
cuff. Lomo and Rosenthal's (1972) interpretation of these results is that "muscle
fiber activity" determines the trophic
spread of Ach sensitivity. If these results
are inserted into Table 4, one can see that
"muscle fiber activity" is defined in this
case as the continued generation of either
muscle fiber action potentials (Table 1: 2a),
active shortening by muscle fibers (Table
1: 2b), or the maintenance of a minimum
length or (passive) tension (Table 1: 2c).
Again from Table 4, experiments in which
the muscle is immobilized (Fischbach and
Robbins, 1969) coupled with indirect nerve
stimulation should distinguish between
these three possibilities. Thus, the terms
"use and disuse" or "muscle fiber activity"
as used previously by most authors have
several, currently testable, refinements for
their mechanism of action. Finallv, the data
of Lomo and Rosenthal (1972) indicate that
if muscle atrophy (decrease in muscle fiber
diameter) were used instead of receptor
spread as a measure of trophic dependence,
then a different underlying mechanism
must be postulated since fiber atrophy, but
not receptor spread, is seen after tenotomy
(Lomo and Rosenthal, 1972: Nelson, 1969);
that is, the mechanism for the trophic
maintenance of one parameter (Table 2)
should not be expected to hold for all
parameters.
We have attempted to isolate the factors responsible for fiber atrophy in the
crayfish opener muscle (a multiterminally
innervated, tonic muscle—Table 2). Crayfish opener muscles denervated for up to
200 davs appear to have normal range of
fiber diameters viewed under a dissecting
microscope (Bittner, personal observations)
or when studied by conventional histological techniques (Fig. 7/4). EM studies of
X
X
(N,-)
(N,-)
X
N, -|
X
0
0
X
0
0
X
(-)
0
0
0
0
0
N
0
0
0
(—)
(?)
0
0
(—)
(X)
0
0
(X)
0
(-)
(N)
0
0
X
X
X
N
X
X
X
X
X
N
0
X
X
X
X
X
N
X
X
X
X
Joint
Anaesimmobili- thetic
Denervacuff
zation
tion
Teuotomy
Postsynaptic
receptor
block
Anaesthetic
cuff
0
DenervaTenotomy
tion
Joint
immobilization
X
X
X
0
X
N
N
Postsy nap tic
receptor
block
Theoretical effects of operative procedures when
combined with direct or indirect stimulation of
the muscle
B
Assumptions for each operation
Muscle tlenervation: eliminates all dependencies on trophic substances or on muscle activity.
Tenotomy: prevents muscle fibers from developing tension.
Joint immobilization: prevents muscle fibers from shortening. Muscle resting length is assumed to be at a normal, half-open position after each operation except tenotomy, unless the joint is immobilized in an abnormally flexed or extended position.
Anaesthetic cuff: prevents nerve spiking, but may not prevent continuous release of trophic substances since activity seems to have no effect on axoiiiil transport rates (Oehs, 1972). Xouronal activity can also be greatly reduced (but no eliminated) by CXS damage such as isolation of spinal
cord segments (Eccles, 1944).
Post-synaptic. receptor block: assumed to prevent the effects of the neurotransmitter but not of some other trophic substance. Appropriate blocking agents for vertebrate skeletal muscle might be curare, or a-bungarotoxin. Local application of the latter compound to vertebrate skeletal muscle
might well produce a long lasting receptor block.
Direct or indirect muscle stimulation: electrical stimulation of a denervated muscle mass will cause many fibers to generate action potentials and
thereby initiate the excitation-contraction process. Direct muscle stimulation also causes intact nerve endings to release transmitter. The pattern of
stimulation is assumed to be chosen to duplicate the normal as closely as possible, unless deliberately altered.
Key
X, normal; 0, essentially eliminated; + , — , somewhat increased or decreased; + + , = , dramatically increased or decreased; ( + , X,—), theoretically, no change nred occur but indicated change has been observed in most vertebrate muscles for that operation (tenotomy: Xelson, 1969; immobilization: Fishbach and Bobbins, 1969).
la) Continuous release of neuronal
trophic substance
lb) Evoked release of neuronal trophic
substance (not the neurotransmitter)
lc) Evoked release of neurotransmitter
2a) Generation of muscle action potentials
2b) Tension generation by muscle
2c) Active shortening by muscle
3) Resting length
Possible trophic dependency
of muscle (see Table 1)
] Dxperiinen
Theoretical effects of various operative procedures
A
TABLE i. Effects of some operative procedures.
SO
W
O
W
o
§
—
—
+
—
—
+
—
—
+
+
+
—
Crustacea
(crayfish)
—
+
+
—
Fish
(goldfish)
+
+
+
+
+
+
+
+
paper.)
—
+
+
+
Urodeles
(newt)
Regeneration capabilities:
5) Peripheral body parts, pre-adult
+
+
+
+
6) Peripheral body parts, adults
—
—
+
+
7) CNS somata, pre-adult
+
+
—
+
S) CNS somata, adult
—
—
—
+
9) Sensory somata, pre-adult
-f
+
+
+
10) Sensory somata, adult
+
+
+
+
11) Muscle tissue, pre-adult
?
?
—
+
12) Muscle tissue, adult
?
?
—
+
(Bliss, 1960; Edwards, 1969; Goss, 1969; Needham, 1965; Niiesch, 1968: and data presented in this
KEY: -)-, potential demonstrated; —, no potential observed; ?, no data available.
Developmental capacities:
1) Neuronal hyperplasia, adult
2) Major nerve and muscle reorganization,
pre-adult, i.e., metamorphosis
3) Minor nerve and muscle reorganization,
pre-adult, i.e., molting
4) Minor nerve and muscle reorganization,
adult, i.e., molting
Holometab. Hemimetab.
insects
insects
(butterfly) (cockroach)
+
—
+
—*
+
—1
+
+
—
4"
+
4"
Anurans
(frog)
TABLE 5. Developmental and regenerative capability in arthropods and vertebrates.
—
—
—
—
—
—
+
+
—
—
—
—
Birds
(chicken)
~
—
—
—
+
+
—
—
—
—
—
—
Mammals
(rat)
pi
w
§
>
H
9,
2.
~s.
_
J2
12
~ n
£
GEORGE D. BITTNER
FIG. 7. Cross-sections through the middle portion
of opener muscles in propodites 15-20 mm long.
A, Opener muscle denervated for 200 days. Fibers
are of almost normal diameter when compared to
unoperated fibers such as shown in Fig. 8A. B,
Opener muscle immobilized in fully open position
for 130 days. C-D, Central tendon of the opener
muscle cut in the midpropodite region 130 days
previously. Muscle fibers proximal to the cut have
a tenotoinized insertion and have atrophied (C);
muscle fibers distal to the cut insert upon an in^
tact tendon and have not atrophied. All tissues
fixed for 3-5 days in cold, aqueous Bouins and
double embedded in paraffin. Magnification (X
30) for A-B; (X20) for C-D.
such denervated muscles have shown some
atrophy of Z line structure, particularly after axonal and terminal degeneration have
occurred (Bittner and Atwood, personal observations). Immobilization of the opener
muscle in a stretched position by banding
the dactyl shut does not lead to gross morphological or histological changes in muscle structure, even after one year; opener
muscles held at normal or shortened length
by joint fixation at the half open or full
open position for several months also show
no dramatic atrophy (Fig. IB). Tenotomized opener muscles are significantly
shorter than muscles immobilized in the
fully opened position and atrophy dramatically within 30-60 days (Fig. 1C). Denervation does not dramatically alter the time
course or extent of the trophic changes after tenotomy. However, if a cut tendon reattaches to the exoskeleton proximal to
the joint—an event that often occurs during a molt—then the fibers show much less
atrophy. Although these muscles with a reattached tendon are functionally useless,
their fibers can probably generate some
tension upon neuronal stimulation, and fiber resting lengths are somewhat longer
than those fibers in tenotomized muscles
whose tendons have not reattached.
The most consistent and direct interpretation of our crustacean data (see Table
6A) is that fiber atrophy is dependent upon
the resting muscle length. In contrast, the
trophic dependence of adult vertebrate
twitch (slow or fast) muscle cannot as yet
be determined (Table 6B), without additional data from procedures such as nerve
block (anaesthetic cuff) and muscle stimulation (Table 4B). One way to interpret
the data from mammals and crustaceans is
that fiber diameter in mammalian twitch
muscle is dependent upon the muscle remaining completely functional and useful
(i.e., to remain essentially normal in structure, the muscle must be neurally driven
REGENERATION INT CRUSTACEA
and be able to shorten and generate tension); the crayfish opener muscle, in contrast, need only maintain a resting length
(tension) within a certain "normal" range.
The difference between the two strategies
for trophic interaction in mammals and
crayfish could lie in differences in the type
of innervation or muscle fiber structure
(Table 1). It could also be that the long
term survival of severed crustacean motor
axons allows for the continued release of
trophic substances which maintain the muscle fibers in good functional condition. In
contrast, the rapid degeneration of vertebrate motor axons might result in severe
trophic changes in denervated muscle fibers
(Guth, 1968). However, we have not observed significant atrophic changes (other
than Z line changes mentioned above) in
several muscles which had nonfunctional
and morphologically degenerated nerve
terminals (Bittner and Atwood, personal
observations). Therefore, it currently appears that both spread of receptor sensitivity (in vertebrate twitch muscle) and fiber diameter (in vertebrate twitch muscle
or crustacean tonic muscle) may not be
heavily dependent upon a neural trophic
substance. These data certainly do not
eliminate the possible existence of neural
trophic substances; in fact cholinesterase
activity in mammalian twitch muscle seems
to have a neuronal trophic dependence
since denervation has a much greater effect on cholinesterase spread than does immobilization or tenotomy (Guth, 1969;
Guth et al., 1967), and electrotherapy fails
to retard the loss of cholinesterase activity
in denervated muscle fibers (Guth et al.,
1967).
In addition to atrophic structural
changes in insect phasic or mixed muscle
(Guthrie, 1962; Jacklet and Cohen, 1967),
there are reports of changes in electrical
excitability (Bodenstein, 1957; Jacklet and
Cohen, 1967; Usherwood, 1963b) and resting potential (Jacklet and Cohen, 1967;
Usherwood, 1963a). Although changes reported in vertebrate twitch muscle for resting potential (Albuquerque and Thesleff,
1968; Guth, 1968; Levine, 1961; Locke and
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GEORGE D. BITTNER
Solomon, 1967; Ware and Bennett, 1954)
or for membrane excitability (Gutmann
and Hink, 1963) seem to vary somewhat
between species, nevertheless most data
from vertebrates are similar to those reported for insects. In contrast, denervated
crustacean muscles do not undergo significant changes in resting potential or input
resistance, even after one year (Bittner and
Boone, personal observation; see Girardier
et al., 1962). Therefore, insect fast or mixed
muscle seems to resemble mammalian
twitch muscle more closely in its trophic
relations more than it resembles crustacean
tonic muscle. Other trophic or regenerative
phenomena shared by insect and mammalian—but not crustacean—motor neurons
are a relative lack of reinnervation specificity (see Pearson and Bradley, 1972),
much capability for axonal regrowth, and
fairly rapid degeneration of severed axonal
slumps (Bodenstein, 1957; Guthrie, 1962,
1964, 1967; Jacklet and Cohen, 1967). Although hemi-metabolous insects and crustaceans are phylogenetically closer than insects and mammals, hemi-metabolous adult
insects and adult mammals have several
developmental and regenerative strategies
in common (such as items 1, 4, and 6 of
Table 5) that are not shared with crustaceans. These common developmental and
regenerative capabilities may well be the
most important determinant of trophic interactions between nerve and muscle.
REGENERATION OF NEURAL SOMATA
Tissue regeneration of nerve and muscle
without concomitant body part regeneration has been less extensively studied in
mammals than has axonal regeneration,
perhaps because man and his nearest relatives seem to possess little capability for
nerve and muscle hyperplasia (Table 5).
While regeneration of lost neurons may
not occur in mammals, lower vertebrates
are able to regenerate cei^ebral hemispheres
or spinal cords (Butler and Ward, 1965;
Jordan, 1958; Piatt, 1955; also see Table
5). Sense organs, including the compound
eye, can regenerate in insects but there is
no good evidence that CNS cell bodies can
ever be replaced (Needham, 1965). Insects
and Crustacea redifferentiate sensory cell
bodies during regeneration of body parts
(Bliss, 1960) and apparently remake specific CNS connections (Edwards and Sahota,
1968). There have been several reports
(Herland-Meewis, 1964; Herland-Meewis
and Deligne, 1965) of regeneration of annelid ganglia after selective removal of a segment of the ventral nerve cord. We (Bittner, Larimer, and Ballinger, unpublished)
have found no evidence for regeneration
of cell bodies in the 3rd abdominal ganglia in small (5-8 cm) crayfish even up to
400 days and three molts after ganglion
removal (Fig. bE). Very few inter-neurons
regrow across the lesion site after this procedure or after remioval of a segment of
the 3-4 connective. After ganglion removal, (but not connective removal) many sensory fibers from the denervated segment regrow to enter die surviving segments of
the cord at points where they would not
have entered in a normal animal, e.g., the
1st or 2nd roots of the (missing) 3rd abdominal ganglion. This result therefore
demonstrates (1) that sensory nerve axons
whose cell bodies are located in the periphery have greater regenerative ability than
axons of inter-neurons whose cell bodies
are located in the CNS; this result is consistent with our studies of limb nerves reported earlier in which sensory axons appear to glow much faster than m'otor axons
(whose cell bodies are also located in the
CNS); and (2) that regenerating sensory
axons need not enter the ventral cord at
their previously specified site; however, we
do not know the specificity of the CNS
connections made by these regenerating
neurons.
REGENERATION OF SKELETAL MUSCLE
The regenerative ability of vertebrate
muscle from amphibia to mammals has
been dramatically illustrated by Studitsky
(1952) and Carlson (1968, 1970), who have
shown that entire muscles can reform within two months from minced fragments im-
REGENERATION IN CRUSTACEA
planted into the bed of the removed muscle. The original fragments of minced muscle lose their original identity, tendinous
connections are re-established at the origin
and insertion of the regenerating muscle,
and differentiating myoblasts appear within the first two weeks. After 60-90 days,
minced muscle regenerates usually contain
a distinct central tendon upon which are
attached reasonably normal fibers. The total mass of the regenerated muscle is considerably less than normal and often contains much more connective tissue which
apparently renders the muscle non-fninctional, even though individual fibers may
be able to respond to indirect (neural)
stimulation (Carlson, 1970). This tissue regenerative response differs from body part
regeneration (in all those vertebrates that
retain both abilities) in that: (1) a blastema
of dedifferentiated cells does not form, (2)
interactions between epidermis, nerve and
muscle is not necessary for the initiation
of the regenerative response, (3) the appearance of differentiated muscle fibers is more
rapid, (4) the degree of mjorphological perfection in the size, arrangement, and composition of the regenerated mass is abnormal, and (5) the functional capabilities of
the tissue regenerate are usually minimal.
Crush lesions to mammalian skeletal muscle in which no miuscle fibers are removed
are often completely repaired within 1-3
months (Church, 1970).
With the results of these vertebrate studies in mind, we have performed similar operations on the opener muscle of adult
crayfish, an arthropod species that can regenerate limbs upon molting (Table 5) but
which requires several molts before those
limbs attain the size of other "normal"
limbs (Bittner and Muse, personal observations). If pieces of muscle fibers are removed from the middle of the crayfish
opener muscle, the fragments which still
remain attached to the exoskeleton or the
central tendon degenerate within a few
weeks (Fig. 8/3). New fibers are recognizable within 50-60 days and seem to cluster
in distinct bundles (Fig. 8D). The diameter of these "regenerating" fibers increases
401
with time although they are smaller than
any normal fibers even up to 150 days after the operation. There is some difficulty
in defining an appropriate set of control
fibers against which the diameter of these
regenerating fibers can be compared because of the large range of fiber diameters
in these and other crustacean muscles (Atwood, 1967; Bittner, 19686) which depends
upon claw size (Bittner, 19686). Furthermore, compensatory hypertrophy could occur in "control" fibers of the same muscle
located on either side of the regenerating
fibers. Since Bittner (19686) has shown that
opener muscles from very small (2 inches
overall length) and very large (8 inches
overall length) crayfish contain about 250
muscle fibers, it is possible that these muscles will eventually attain their original fiber number by fusion of the small regenerating fibers. We have also noted that the
number of molts does not have a dramatic
effect on the size of these regenerating muscle fibers from limbs which are not themselves regenerating. We have performed
other operations to remove the central tendon of the opener muscle plus the central
two-thirds of each muscle fiber from the
propodite segmient by pulling the tendon
and attached fibers out through a small
opening in the exoskeleton (about onethird of the length each fiber remains attached to the exoskeleton—Fig. 8B). After
muscle and tendon removal, no obviously
regenerating fibers are seen, even after 150
days and three molts. This result is not altered by immediately stuffing the muscle
and tendon back into the propodite segment after removing it intact or after mincing it into 1 mm2 fragments (Fig. 8C).
However, if we remove the propodite segment containing the opener and closer
muscles, within five to six molts the animals can regenerate a functional propodite
of almost identical size and muscle mass as
their non-operated propodite in the contralateral limb.
The extent and rate of regeneration of
damaged muscle fibers reported for vertebrates (particularly for mammals) as described above is significantly greater than
4Q&
GEORGE D. BITI'NER
FIG. 8. Cross-sections through the dorsal, midportion of 15-20 mm propodites to show opener muscles in situ. A, Normal, unoperated, muscle. B,
Pieces of fibers remaining attached to the exoskeleton one day after removing the opener muscle
by pulling out its central tendon. About 1-2 mm
of each fiber remained attached to the exoskeleton; opener fibers were 5-8 mm in total length.
C, Opener tendon and muscle fibers removed 174
days previously. Animal has since molted two
times. No muscle fibers were seen in this propodite region that normally would contain fibers as
shown in A above. D, Propodite in which fibers
were removed from the middle region of the opener muscle 119 days previously. Animal has since
molted one time. Small muscle fibers seen in the
vicinity of the central tendon were traceable from
the central tendon to the exoskeleton and were
arranged in small clusters. The larger muscle fibers
seen in cross-section adjacent to the exoskeleton
altached to the tendon much more distally than the
smaller diameter fibers and were not removed at
the time of the original operation. Magnification
(X30) for A-B; (X20) for CD.
that which we have found for crayfish.
There are several possible reasons why this
result might not hold for all muscles in
the crayfish or in other Crustacea:
1) The opener muscle is located in the
next-most distal limb segment and Crustacea often show a proximo-distal decrease
in ability to regenerate body parts (Bliss,
I960). We are controlling for this possibility by removing meropodite extensor muscles but would note that these crayfish can
regenerate a full sized propodite segment
in five to six molts.
2) The crayfish is ontogenetically programmed to regenerate a small muscle in
a small segment after loss of a body part
and cannot regenerate a full sized muscle
in the "large" exoskeletal shell which remains after removal of the opener muscle.
If this explanation is correct, then Crusta-
cea like the land crab Gecarcinus lateralis,
which can regenerate an entire full-sized
limb in one molt (Bliss, 1960), might be
expected to regenerate an opener muscle
in one molt.
Conversely, the data from the opener
muscle may apply to most crustacean muscles. For example, in cases in which body
part regeneration is not operative, Crustacea might be able to regenerate muscle fibers only when the central tendon is left
attached. Since tenotomy rapidly leads to
fiber atrophy (Fig. 7C), it could also be
that Crustacea can regenerate muscle fibers
only if intact fibers from the same muscle
are available to provide a source of dedifferentiated myoblasts. An entire muscle
may be able to regenerate only during body
part regeneration when dedifferentiating
muscle fibers and/or other cells can inter-
REGENERATION IN CRUSTACEA
act to form a limb bud; that is, crayfish
may have "specialized" their regenerative
processes for the repair of entire body parts
to the detriment of tissue regenerative capabilities of peripheral nerve and muscle.
EVOLUTION OF REGENERATIVE CAPABILITIES
Consideration of the regenerative abilities of neuromuscular systems in mammals
and Crustacea at various levels of organization (axonal hypertrophy, nerve and
muscle hyperplasia, body part regeneration) indicates that mammals may demonstrate significantly better tissue regenerative abilities than phylogenetically lower
Crustacea which possess significant ability
for body part regeneration. Goss (1969, p.
275-276) has postulated that "at molecular
and ultrastructural levels, regeneration is
equally efficient for all organisms throughout the phylogenetic scale. It is at the higher levels of organization, where histologically complex body parts are involved, that
the ability to grow replacements has been
curtailed in the course of evolution. . . .
Therefore it is not regeneration per se
which has been selected against during evolution, but the level of organization at
which it occurs." As an alternative to this
theory, one might postulate that the ability of an organism to repair a given type
of tissue damage (say axonal severance)
need be considered with regard to the organism's ability to repair other types of
tissue damage (say loss of neuronal somata) or body part loss. These regenerative
abilities, in turn, need be considered in
conjunction with the organisms developmental capabilities (as outlined in part for
nerve and muscle tissue in Table 5). According to this theory, the total regenerative capabilities of the organism need not
decrease with increased tissue complexity
or with phylogenetic ascent. In fact, the
ability to regenerate a limb muscle during
body part regeneration might lead to a decreased capability for its repair in the absence of regeneration of the limb segments.
For example, repair of severed motor
axons in crayfish results in a rather slow
403
outgrowth from proximal stumps probably
followed by fusion with surviving distal
stumps (Fig. 6C); if the distal stump has
degenerated, then reinnervation may occur
by very slow regrowth from, the proximal
stumps. Thus, a lesser ability for neuronal
hypertrophy (when compared to mammals)
is usually compensated by a greater ability
for distal stump survival and the evolution
of a different mechanism for repairing
axonal damage. The observation of a slow
rate of motor axonal outgrowth is compatible with the fusion hypothesis as well
as with the reinnervation that occurs during body part regeneration since in each
case the motor axon need grow out for only
a few millimeters before contacting its target muscle or axon. This ability to make
contact with a developing muscle or surviving axon after regrowing only a few millimeters is associated with a high degree of
reinnervation specificity whereas motor
neuronal outgrowth over several centimeters or more before making contact with a
target tissue seems to lead to many inappropriate connections as has been observed
in mammals (Gaze, 1970; Jacobson, 1970).
The main advantage of distal stump survival may, therefore, be to insure precise
connection specificity in an organism with
very few motor neurons. The postulated
ability of an outgrowing proximal stump
of a crustacean motor neuron to fuse with
its surviving distal stump would not only
restore functional competency by a morphological mechanism different from, that
used by vertebrates or insects, but also different from the mechanism used by Crustacea to innervate limb muscles during
their embryological development or during body part regeneration.
Like motor axons, severed axons of the
CNS giant interneurons survive morphologically and physiologically intact for
many months. Unlike motor axons, we
have never observed return of function after cutting these interneurons. Axonal outgrowth in crustacean giant neurons may be
prevented by glial hypertrophy similar to
that which is thought to prevent axonal
outgrowth in mammals (Guth and Windle,
404
GEORGE D. BITTNER
1970). However, this mechanistic explanation does not account for why such an "inappropriate" glial response should occur
in either class of organisms. It might be argued that mammals have excellent hypertrophic (axonal outgrowth) capabilities for
neuronal repair, but have almost no ability for neuronal hyperplasia during the
adult stage; hence, they might be expected
to show little ability to repair CNS damage since almost any CNS lesion destroys
many cell bodies. Lower vertebrates (Table
5), in contrast, do have the ability to add
new CNS neurons during the adult stage,
in which they grow continuously in body
size, and, hence can repair CNS damage by
a hyperplastic response (Guth and Windle,
1970). Although crayfish increase in size
during each adult molt, they do not increase the number of CNS giant interneurons and, therefore, really possess a CNS
developmental capability for these cells
like that found in mammals. Unlike in
mammals, cutting a connective of a crustacean ventral nerve cord does not directly
damage CNS cell bodies. Furthermore, the
ventral nerve cord of most Crustacea lies
in a very vulnerable position just beneath
a thin abdominal exoskeleton, and these
animals can survive for very long periods
(more than 14 months) in isolation after
the abdominal nerve cord has been severed.
However, when these operated animals are
kept in community tanks, the chelipeds
need be removed from all inhabitants to
prevent fatal attacks on those animals with
denervated posterior segments (Eggleston,
Johnson, and Bittner, personal observations). That is, in addition to considering
tissue regeneration in the context of both
body part regeneration and ontogenetic
capabilities of die organism, one must also
recall that the goal of any regenerative
process is to restore functional ability to a
lost or damaged body part not absolutely
essential for survival (Goss, 1969). Damage
to both mammalian and crustacean CNS
may affect a body part that is essential for
short-term survival outside the experimental laboratory. Similarly, mammals may
not regenerate entire peripheral limbs be-
cause these structures may be too important for the survival of warm-blooded animals with high metabolic rates (Goss, 1969).
In contrast to mammals, crustacean peripheral limbs are probably not absolutely essential for the Survival in an aquatic environment, of a rather sluggish ectotherm
which has eight paired appendages and a
relatively low metabolic rate.
Since appendages contain mlany sensory
cell bodies, new sensory somata must differentiate and grow their axons toward the
CNS after limb autotomy. Given that the
number of sensory fibers in a regenerating
limib is much less than that of the original
limb (Bittner, personal observations), a
smaller number of regrowing sensory axons
would have a much larger number of surviving distal stumps with which to fuse if
sensor)' regeneration mechanisms were like
that in peripheral motor neurons. Furthermore, none of the distal stumps of the
original sensory neurons might have appropriate CNS connections for new, ingrowing axons. Finally, since the diameter
of recently differentiated sensory axons is
much less than older, non-regenerating
axons (Kennedy, personal communication),
fusion of the two processes would lead to
problems in the safety factor for axonal
transmission. For all these reasons, it seems
appropriate that the rate of growth of peripheral sensory axons is greater than that
of motor neurons and that the severed distal stumps of most sensory neurons degenerate much more rapidly than severed motor stumps (Fig. 3). Thus, it appears that
differences in the tissue regenerative mechanisms and rates of motor, sensory, and CNS
neurons in crayfish are not dependent upon
their complexity of tissue organization, but
rather upon the developmental capabilities
and body part regenerative strategies
evolved by this organism!.
Crustacea demonstrate some ability to
regenerate damaged muscle fibers in the
absence of body part regeneration (Fig.
8D), but their capability for repair of muscle tissue seems less extensive and much
slower than that reported for mammals by
Church (1970) and Carlson (1970). Once
405
REGENERATION IN CRUSTACEA
again, die relative inability of crayfish
muscle fibers to repair themselves rapidly
(as compared to mammalian rates) or to
regenerate an entire muscle mass independent of body part regeneration may result
from the fact that such damage in nature
is generally associated with body part loss;
that is, it is hard to imagine a naturally
occurring injury that removed muscle fibers or an entire muscle with little exoskeletal damage. Therefore, crayfish—and
possibly other Crustacea—may not have
had much selection pressure for repairing
muscle tissue independent of body part regeneration. In fact, there is also some evidence (Carlson, 1968, 1970) that the extent
and rate of muscle tissue repair is greater
in mammals (which cannot regenerate peripheral limbs). These data, if confirmed,
would be more easily explained by the
present hypothesis for the evolution of regeneration strategies.
From an examination of tissue repair capabilities in peripheral nerve and muscle,
it seems that mammals generally repair
damage to peripheral structures by significant structural remodeling (axonal outgrowth, muscle hyperplasia), whereas Crustacea either make minor repairs (axonal
fusion, regeneration of a few muscle fibers
if the central tendon is intact) or rebuild
an entire structure after autotomy of the
peripheral limb. It has often been observed
that damage to peripheral nerve and muscle immediately leads to limb autotomy in
Crustacea (Bliss, 1960), and we have usually performed our operations to damage
nerve and muscle tissue in chelipeds after
cutting the autotomy muscles. If the autotomy muscles are not cut and the
limb is not autotomiized during the initial
operative procedure, we have noticed that
crayfish tend to autotomize or otherwise remove their claws (if not walking legs)
about 3-4 weeks after severe nerve damage.
This suggests that when a rapid repair
cannot be made to neuronal tissue by fusion, the behavioral response of the crayfish is to remove the body part and regrow
the entire structure. The regrowth of body
parts, in turn, is dependent upon molting
frequency (Bliss, I960). It is therefore of
interest to note that severing peripheral
limb nerves and damage to limb muscles
or tendons significantly increases molting
frequency (Bittner and Kopanda, 1973).
Damage to nerve and muscle tissue in
these crayfish is often associated with loss
of appendages at the autotomy plane or
elsewhere. Therefore, the ability of such
nerve and muscle damage to increase molt
frequency has obvious adaptive advantages
since molting increases the rate at which
these appendages regenerate.
In summary, the regenerative capabilities for each level of tissue organization
in Crustacea, Amphibia, and Mammalia
appear to interact in a manner determined
by the developmental capabilities and phylogenetic adaptations of each class of organisms. Adult mammals seem to have sacrificed body part regenerative capabilities
for enhanced tissue regenerative abilities of
nerve and muscle, whereas adult crustaceans may have selected the opposite strategy
for repairing lost or damaged body parts.
Reasoning along these same lines, it would
be instructive to examine such processes in
adult insects and compare the regenerative
abilities of their nerve and muscle tissue
to that found in adult stages of mammals
and crustaceans, since adult insects seem to
resemble the former more closely in their
developmental capabilities (Table 5) but
are phylogenetically closer to crustaceans.
One might also speculate that the tissue
regenerative capabilities of pre-adult insects might more closely resemble that of
Crustacea since many immature insects molt
and can regenerate body parts. It is already
clear that insect nerve-muscle trophic relations and body part regenerative capabilities differ during ontogeny (Bliss, 1960;
Edwards, 1969; Teusch, quoted by Nuesch,
1968; Randall, 1970) as would be predicted
from the different developmental capabilities in the immature and adult stages.
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