Friedrich Miescher Institute, CH-4002 Basel, Switzerland
Long-term functional plasticity in the nervous system can involve structural changes in terminal arborization and synaptic connections. To determine whether the differential expression of intrinsic neuronal determinants affects structural plasticity, we produced and analyzed transgenic mice overexpressing the cytosolic proteins cortical cytoskeleton-associated protein 23 (CAP-23) and growth-associated protein 43 (GAP-43) in adult neurons.
Like GAP-43, CAP-23 was downregulated in mouse motor nerves and neuromuscular junctions during the second postnatal week and reexpressed during regeneration. In transgenic mice, the expression of either protein in adult motoneurons induced spontaneous and greatly potentiated stimulus-induced nerve sprouting at the neuromuscular junction. This sprouting had transgene-specific features, with CAP-23 inducing longer, but less numerous sprouts than GAP-43. Crossing of the transgenic mice led to dramatic potentiation of the sprout-inducing activities of GAP-43 and CAP-23, indicating that these related proteins have complementary and synergistic activities. In addition to ultraterminal sprouting, substantial growth of synaptic structures was induced. Experiments with pre- and postsynaptic toxins revealed that in the presence of GAP-43 or CAP-23, sprouting was stimulated by a mechanism that responds to reduced transmitter release and may be independent of postsynaptic activation.
These results demonstrate the importance of intrinsic determinants in structural plasticity and provide an experimental approach to study its role in nervous system function.
The formation of neuronal connections involves a sequence of distinct stages in axonal growth, i.e., longdistance elongation, growth of collaterals to reach
the target region, and innervation of target cells. Terminal
arborization initiates the final phase of the innervation
process. This dynamic process provides the presynaptic
substrate for the formation and activity-sensitive refinement of synaptic connections during development. In the adult , it can be reactivated by local deafferentation, leading to sprouting and reinnervation (e.g., Brown, 1984 One factor that affects nerve sprouting and terminal arborization is the expression of competence conferring components in responding neurons (Skene, 1989 One protein that may be functionally related to GAP-43
is the cortical cytoskeleton-associated protein CAP-23 (Widmer and Caroni, 1990
To determine whether CAP-23 has sprout-promoting
activity and to explore the possibility that neurons may express growth-promoting proteins with overlapping and/or
synergistic activities, we generated transgenic mice that
constitutively expressed CAP-23 in adult neurons. Like
GAP-43-overexpressing mice, these mice displayed spontaneous and greatly potentiated induced nerve sprouting
at the neuromuscular junction. However, the sprouting
patterns in the presence of GAP-43 or CAP-23 were different, and double-transgenic mice expressing both proteins in motoneurons displayed dramatic quantitative and
qualitative potentiation of sprouting. We then carried out
experiments aimed at defining the properties and regulation of the structural plasticity promoted by these growthassociated proteins. These experiments revealed that: 1) Alterations in both nerve growth and postsynaptic structures
were induced at the neuromuscular junction of the transgenic mice; 2) growth-promoting pathways are rapidly activated by reduced transmitter release, possibly independent of muscle nicotinic acetylcholine receptor (AChR) activation; and 3) paralysis promotes sprout elongation.
Generation of CAP-23 Transgenic Mice
The Thy1.2-based expression cassette was as described (Aigner et al.,
1995
Detection of mRNAs and Proteins
In situ hybridization for chick CAP-23 was carried out on frozen sections with a digoxigenin-labeled cRNA probe, as described (SchaerenWiemers and Gerfin-Moser, 1993; Arber and Caroni, 1995 Lesion Protocols
For most experiments, local paralysis was induced by a single subcutaneous injection of 1 pg of purified Botulinum toxin A (kind gift of C. Montecucco, Pavia, Italy, and S. Catsicas, Geneva, Switzerland) over the right
gluteus maximus or gastrocnemius of 4-6-wk-old mice. For 3-wk paralysis
experiments, a second injection of 0.8 pg Botulinum toxin-A (Bot-A) was
applied at 10 d. In an alternative protocol, paralysis was induced by local
applications of Detection and Analysis of Sprouting
Intramuscular nerves and neuromuscular junctions were visualized on
50-µm cryostat sections of gastrocnemius and gluteus maximus muscle, as
described (Pestronk and Drachman, 1978a Parallel Regulation of GAP-43 and CAP-23 in
Motoneurons and at the Neuromuscular Junction
In 2-3-d chick embryos, CAP-23 is expressed at high levels
in most cells (Widmer and Caroni, 1990 CAP-23-overexpressing Mice Exhibit
Spontaneous and Potentiated Induced Nerve Sprouting
at the Neuromuscular Junction
To determine whether the presence of CAP-23 in adult
nerves affects nerve sprouting, we produced CAP-23-
overexpressing transgenic mice. We used the same mouse
Thy1.2 expression cassette that had been used to generate
GAP-43-overexpressing mice, and that leads to neuronspecific expression of transgene, starting around P5-10 (Aigner et al., 1995 Analysis of neuromuscular innervation patterns with a
combined silver-esterase reaction revealed prominent ultraterminal sprouting in CAP-23-overexpressing mice
(Fig. 3, A and C). Quantitative analysis revealed that the
values for the fraction of neuromuscular junctions with ultraterminal sprouts and the total length of ultraterminal sprouts per muscle were comparable to those detected in
GAP-43-overexpressing mice (see Fig. 5 B) and higher
than those induced in nontransgenic mice by paralysis
with Bot-A (Brown, 1984
To determine whether the presence of CAP-23 potentiates induced nerve sprouting, thus producing a gain-offunction phenotype with respect to this local nerve growth
reaction, we induced local paralysis with the presynaptically acting toxin-A from Clostridium botulinum (Brown,
1984
In an independent test for the effects of CAP-23 on induced nerve sprouting, we analyzed short-term nerve
growth induced by sciatic nerve crush. In this experimental
paradigm, sprouting of axons into the distal section of the
lesioned nerve is assayed at a time (64 h after lesion) when
neurofilament-positive material from degenerating nerves
has been largely removed, and lesion-induced CAP-23 and
GAP-43 have not yet reached the crush site. As shown in
Fig. 4 B, and in further analogy to GAP-43-overexpressing mice, the presence of CAP-23 significantly promoted
this form of regenerative sprouting. In contrast, reinnervation of gastrocnemius muscle 8-11 d after the crush was
not obviously faster in transgenic mice (data not shown,
but see Aigner et al., 1995 Synergism between CAP-23 and GAP-43 in the
Induction of Local Nerve Growth
As shown in the previous sections, the patterns of ultraterminal sprouting at the neuromuscular junction of GAP43- and CAP-23-overexpressing mice displayed consistent
differences, and both proteins are expressed in motor
nerves during development and regeneration. To determine whether GAP-43 and CAP-23 may have synergistic
activities on nerve sprouting, we analyzed offsprings of
transgenic mice crossings. Mice transgenic for GAP-43 and
CAP-23 expressed both transgenes at the neuromuscular
junction (data not shown). As shown in Fig. 5, A and B,
this resulted in dramatic potentiation of nerve sprouting in
untreated animals, with long and numerous sprouts that
were detected at essentially all neuromuscular junctions.
Analysis of activity-sensitive muscle mRNAs (Fig. 5 C)
and of nerve muscle explants (data not shown) established
that this remarkable sprouting activity was not accompanied by detectable signs of neuromuscular inactivation. In
control experiments, in mice homozygous for the GAP-43
(Fig. 5 B) or CAP-23 (data not shown) transgene, nerve sprouting was comparable to that detected in corresponding heterozygous mice, indicating that in these mice elevating transgene levels did not lead to potentiation of
sprouting. We then determined whether the presence of
both transgenes also potentiated induced sprouting. Double-transgenic mice displayed detectable, but comparatively modest potentiation of crush-induced sprouting
(data not shown). In contrast, the extent and pattern of
Bot-A-induced growth at the neuromuscular junction
were dramatically affected by the presence of both transgenes (Fig. 5 D). In addition, double-transgenic mice displayed a dramatic increase in the density of nerve processes at and in the immediate vicinity of paralyzed
neuromuscular junctions. Therefore, the presence of both
GAP-43 and CAP-23 at the neuromuscular junction leads
to quantitative potentiation and qualitative alterations in
paralysis-induced sprouting.
Synaptic and Extrasynaptic Growth in GAP-43
(or CAP-23)-overexpressing Mice
The demonstration of similar and synergistic activities of
GAP-43 and CAP-23 on nerve sprouting at the adult neuromuscular junction strongly supports the notion that local
nerve growth is affected by the presence of intrinsic
growth-promoting components in neuronal processes.
These findings lead to questions about the range of the
structural alterations induced by the presence of these
growth-promoting proteins and the signaling mechanisms that promote this type of growth. To begin to address the
question of whether these proteins also promote alterations in synaptic structures, we visualized the distribution
of acetylcholine receptors (AChRs) in RITC-
Multiple Activity-sensitive Mechanisms Promote Nerve
Sprouting at the Neuromuscular Junction
What are the types of growth-inducing signals to which
GAP-43- or CAP-23-expressing terminals respond? To
begin to define relevant local mechanisms, we carried out
a detailed analysis of Bot-A-induced sprouting. The purified Bot-A used for these studies induced detectable local
signs of paralysis upon subcutaneous injection of 1 pg of
toxin over the gluteus maximus or gastrocnemius muscle
(toe-spreading reflex). A dose of 0.5 pg of toxin induced detectable paralysis signs in about one-fifth of the mice,
and these signs were only detectable between 1 and 3 d after injection. If anything, transgenic mice were less sensitive to Bot-A-induced paralysis than nontransgenic ones.
Finally, 0.3 pg of Bot-A induced no detectable signs of paralysis, and direct stimulation of the sciatic nerve 2 d after
application of toxin doses below 0.5 pg elicited gastocnemius twitching undistinguishable from control (untreated
animals). Therefore, although we cannot exclude that even
very low toxin levels did induce paralysis at a subpopulation of neuromuscular synapses, these combined observations argue that local application of toxin doses lower than
0.3 pg failed to block nerve-induced muscle activation at
most neuromuscular junctions. In agreement with these
behavioral assessments, toxin doses lower than 1 pg failed
to induce muscle wasting or activity-sensitive mRNAs in
muscle (data not shown).
A dose-response analysis of Bot-A-induced sprouting
revealed that in GAP-43- or CAP-23-overexpressing mice
local growth reactions were already induced with 0.1 or
even 0.05 pg of toxin, i.e., with doses substantially lower than
those required to induce any signs of muscle paralysis (Fig.
7 A). Subparalyzing doses induced no detectable growth
reactions in nontransgenic mice (Fig. 7 A). In transgenic
mice, growth reactions in the absence of paralysis included
an increase in endplate branching points and the appearance of numerous short ultraterminal sprouts. These reactions were already detectable 24 h after toxin application
and developed to a maximal extent within 4-5 d (Fig. 7 B;
similar results were obtained with CAP-23[C11] mice).
Paralyzing doses of toxin induced similar reactions during
the first 3 d, followed by pronounced extension of ultraterminal sprouts, starting about 4 d after toxin application
(Fig. 7 B). Similar morphological responses were induced when neuromuscular transmission was abolished by blocking nerve action potentials with a tetrodotoxin cuff placed
onto the sciatic nerve (Fig. 7 C), indicating that they were
due to impairment of evoked transmitter release and not
to effects more specifically linked to the activity of Bot-A
(i.e., cleavage of nerve terminal SNAP-25). These results
suggested that a mechanism linked to reduced calcium-
induced transmitter release from motor nerves can induce local nerve growth in GAP-43- or CAP-23-expressing nerve
terminals. To explore this possibility, we analyzed nerve
sprouting upon local blockade of postsynaptic AChR activation with
We have shown that at mouse motor nerve synapses, both
GAP-43 and the related protein CAP-23 are downregulated during the second postnatal week and are reinduced
by nerve lesion. Like corresponding GAP-43-overexpressing mice (Aigner et al., 1995 Analysis of AChR distribution in whole-mount preparations of transgenic and nontransgenic mice with and without Bot-A treatment indicated that these growth mechanisms affect both extrasynaptic and synaptic structures.
Finally, experiments with paralyzing and nonparalyzing
doses of Bot-A, tetrodotoxin, and Characteristics and Possible Mechanisms of GAP-43/
CAP-23-potentiated Sprouting
A main finding of this study is that the patterns of sprouting induced by GAP-43 or CAP-23 were different and that
expression of both proteins in double-transgenic mice
greatly potentiated nerve sprouting. The significance of
these double-transgenic results is strengthened by the fact
that, in analogous experiments, crossing of transgenic mice
expressing different phosphorylation-site mutants of
GAP-43, which did display subtle differences in nerve sprouting patterns (Aigner et al., 1995 What may be the basis for the distinct and synergistic effects of GAP-43 and CAP-23 on local nerve growth? The
molecular mechanisms affected by GAP-43 are still not clear.
This cell membrane and cortical cytoskeleton-associated
protein appears to be involved in a variety of dynamic processes at the cell periphery, including growth cone activity
(Aigner and Caroni, 1995 Mechanisms Regulating
GAP-43/CAP-23-mediated Plasticity
What local signaling mechanisms induce GAP-43/CAP-23-
promoted growth? As shown by the results with the pre-
and postsynaptically acting toxins, one of them appears to
be mediated by reduced transmitter release and may not
require postsynaptic nicotinic AChR activation. Although
there is evidence suggesting that reduced transmitter release could signal back to the motor nerve (Hory-Lee and
Frank, 1995 Functional Implications of
GAP-43/CAP-23-mediated Plasticity
Structural changes at the neuromuscular junction of GAP43- and CAP-23-overexpressing mice were not restricted
to presynaptic structures. In the presence of either transgene, RITC- In spite of these marked differences in overall synaptic
structure, we found no evidence for major functional differences at the neuromuscular junction of transgenic and
double-transgenic mice. Functional parameters not obviously different from control included the degree of muscle
fiber polyinnervation and the amplitude and frequency of
spontaneous miniature endplate potentials (data not shown).
Although twofold and smaller changes in the frequency of
spontaneous discharges may have gone undetected in our
analysis, the results suggest that major alterations in overall synaptic structure were not accompanied by obvious
changes in synaptic function. This finding is consistent
with the evidence from other studies indicating that at
the neuromuscular junction, structural plasticity does not
necessarily lead to corresponding functional plasticity
(Tsujimoto et al., 1990 Finally, because of the ease and resolution of the histological analysis and the possibilities for targeted experimental manipulations, we focussed our analysis on the neuromuscular junction. However, because of the restricted and
distinct expression of endogenous CAP-23 and GAP-43 in
the adult nervous system and the widespread expression of
CAP-23 and GAP-43 in the transgenic mice, one may predict similar effects on local nerve sprouting also in the central nervous system. In several transgenic lines overexpressing GAP-43, we detected pronounced sprouting of
zinc-containing mossy fiber terminals into the pyramidal
cell layer of CA3 (Aigner et al., 1995 In conclusion, a substantial amount of experimental evidence supports the view that terminal arborization and alterations in synaptic structures are involved in activitysensitive plasticity during development and in the adult.
The results of this study now suggest that differential expression of intrinsic neuronal components affects the extent and pattern of activity-sensitive structural plasticity in
the nervous system. The availability of transgenic mice with
greatly elevated potential for structural plasticity should
provide a valuable experimental system to study the regulation and roles of structural alterations for the normal function of the adult nervous system.
). In
addition, there is now substantial evidence supporting the
view that the dramatic extent of learning-induced functional plasticity in the adult vertebrate nervous system also
involves local changes in terminal arborization and synaptic connections (see e.g., Bailey et al., 1994
; Darian-Smith
and Gilbert, 1994
; Das and Gilbert, 1995
). Because of the
fact that terminal arborization plays such a central role in
nervous system plasticity, it is of great interest to define
the mechanisms that promote and regulate this process.
; Woolf et al.,
1992
; Aigner et al., 1995
). These include specific receptors
for extracellular signals, as well as corresponding signal
transduction and growth machinery to translate defined
signals into local growth. The neural protein kinase C
(PKC)1 substrate growth-associated protein 43 (GAP-43)
is a cytosolic growth cone and nerve terminal protein that
promotes local nerve growth and appears to belong to the
latter category (for reviews see Benowitz and Routtenberg, 1987
; Skene, 1989
; Liu and Storm, 1990
). This axonal
protein is expressed during nerve elongation and terminal
arborization and is usually downregulated when synaptic rearrangements are completed, but its expression is selectively maintained in defined types of neurons in the adult.
Although GAP-43 is not required for nerve growth (Strittmatter et al., 1995
), the existence of a strong correlation
between its expression in the developing and adult nervous system and competence for nerve sprouting suggests
that GAP-43 may potentiate local nerve growth. This view
received support from the recent demonstration that overexpression of GAP-43 in the neurons of adult transgenic mice leads to spontaneous and greatly potentiates induced
nerve sprouting (Aigner et al., 1995
). The results with
GAP-43 have raised the possibility that the expression of
growth- and plasticity-associated proteins in neurons may
promote their growth responses to local signals. This hypothesis predicts that intrinsic growth-promoting properties are not restricted to GAP-43 and that neurons may express diverse sets of plasticity-promoting proteins. Such
proteins would act downstream of signal transduction
pathways initiated by extracellular ligands to promote the
translation of receptor activation into cytoskeletal rearrangements and local growth. Expression of particular combinations of these proteins may affect the intrinsic
competence of neurons for local structural plasticity.
). Although GAP-43 and CAP-23 do
not have homologous sequences, they share several characteristic biochemical properties. These include: 1) Both
proteins bind calmodulin and are phosphorylated by PKC
in a mutually exclusive manner (Liu and Storm, 1990
;
Maekawa et al., 1993
, 1994); 2) they have highly hydrophilic sequences, the same type of unusual amino acid
composition rich in Ala, Pro, Lys, and Glu, and very little
secondary structure (Skene, 1989
; Widmer and Caroni,
1990
); 3) both are acylated proteins (GAP-43 is palmitoylated [Skene and Virag, 1989
], whereas CAP-23 is myristoylated [Maekawa et al., 1994
]) that colocalize at unique
punctate structures at the cell membrane (Wiederkehr, A.,
and P. Caroni, manuscript submitted for publication) and
interact with the cortical cytoskeleton (Meiri and GordonWeeks, 1990; Moss et al., 1990
; Widmer and Caroni, 1990
);
and 4) their expression is highly regulated and peaks during development of the nervous system, when they can represent up to 0.2% (GAP-43) and 0.8% (CAP-23) of total
brain protein (Skene, 1989
; Widmer and Caroni, 1990
;
Maekawa et al., 1993
). In addition, GAP-43 (Caroni and
Becker, 1992
) and CAP-23 (see Fig. 1) are downregulated and reinduced with comparable kinetics in spinal motoneurons, and they induce the same type of characteristic
surface activities in transfected cells (Wiederkehr, A., and
P. Caroni, manuscript submitted for publication).
Fig. 1.
Expression of CAP-23 in mouse motor nerves and at the neuromuscular junction. (A) At the neuromuscular junction, CAP-23
immunoreactivity is downregulated during the second postnatal week. The figure shows double-labeling immunocytochemistry of cryostat sections from formaldehyde-fixed gluteus maximus muscle (equivalent photographic exposures). To visualize neuromuscular junctions, the sections were counterstained with RITC--bungarotoxin. CAP-23 immunoreactivity was well detectable at P8 and nearly undetectable at P14. In parallel experiments, a combination of
-bungarotoxin and antibody to neurofilament-160 labeled all CAP-23-
positive structures (data not shown), suggesting that in postnatal muscles, CAP-23 expression was restricted to intramuscular nerves.
(B) CAP-23 is reexpressed in regenerating intramuscular nerves in the adult. Double-labeling immunocytochemistry of gastrocnemius
sections from a control (CON) and a mouse 10 d after mid-thigh level crush of the sciatic nerve (REG). Note that neurofilament-160
(NF-160)-positive nerves do not express CAP-23 in the adult but reexpress this protein during regeneration. These findings indicate
that CAP-23 is a growth-associated protein of motor nerves. Bar, 23 µm.
[View Larger Version of this Image (58K GIF file)]
Materials and Methods
; see also Vidal et al., 1990
). It drives transgene expression specifically
in postnatal mouse neurons, including spinal motoneurons. A description
of its expression properties can be found elsewhere (Caroni, 1996). cDNA
coding for chick CAP-23 (Widmer and Caroni, 1990
) was cloned into the
Thy1 expression cassette. To avoid possible regulation of CAP-23 mRNA
stability or translation due to regulatory sequences in the untranslated regions, the cDNA sequences only contained 7 nucleotides of 5
-, and 10 nucleotides of 3
-untranslated region. Seven independent transgenic lines were
generated. Of these, three lines were excluded from further analysis because of low expression of the transgene, as assayed by immunoblots of
brain homogenates and by immunocytochemistry on skeletal muscle sections (motor nerve expression). To minimize possible effects due to differences in genetic background, all lines of GAP-43- and CAP-23-overexpressing mice were bred into C57Bl6 mice for at least five generations.
Inbreeding did not appear to affect spontaneous nerve sprouting in the
transgenic mice. However, because of better breeding results, most experiments were carried out in outbred Balb/C × C57Bl6 mice. A survey of mouse tissues revealed no further expression outside the nervous system.
In particular, we found no transgene expression in innervated or denervated muscle, nor in intramuscular Schwann cells (data not shown, but see
Aigner et al., 1995
). In the nervous system, we detected prominent expression in a diverse range of neuronal types in the peripheral nervous system
and central nervous system (see Fig. 2 A), but no expression in GFAPpositive astrocytes, peripheral nerve Schwann cells, terminal Schwann
cells at the neuromuscular junction, or GalC-positive oligodendrocytes
(data not shown). The transgenic lines were designated as follows: CAP23(2, 11, 13, 17), mice expressing chick CAP-23; and GAP-43(wt2, wt3), mice expressing chick GAP-43 (Aigner et al., 1995
).
Fig. 2.
A mouse Thy1.2-based expression cassette drives expression of transgenic chick CAP-23 in adult mouse neurons, including
spinal motoneurons. (A) Neuronal expression of chick CAP-23 transgene. In situ hybridization of adult mouse (line CAP-23[C11]) brain and spinal cord cryostat sections with digoxigenin-labeled chick CAP-23 cRNA. (Left) Strong transgene expression was detected in several neuronal types in the hippocampal formation (e.g., dentate gyrus granule cells, hilar cells, CA1; low in CA3) and in thalamic nuclei.
No signal was detected with a corresponding sense probe (center). (Right) Strong transgene expression in spinal cord neurons (lumbar
level), including large ventral horn motoneurons (arrows). Note absence of signal in the white matter (oligodendrocytes, astrocytes). (B)
Transgene expression levels in adult mouse brain were comparable to those detected for endogenous CAP-23 in E17 chick brain, when
levels of this protein are maximal. The immunoblot of brain homogenate fractions (40 µg of protein) was probed with monoclonal antibody 15C1, which specifically detects chick, but not mouse (nontransgenic sample) CAP-23. The transgenic lines were CAP-23(C11),
CAP-23(C13), and CAP-23(C17). (C) Detection of transgenic, but not endogenous CAP-23 at the neuromuscular junction of a CAP23(C11) transgenic mouse. Double-labeling immunocytochemistry for CAP-23 and -bungarotoxin. (Left) Section reacted with monoclonal antibody 15C1. (Right) Section reacted with antiserum against carboxyl-terminal sequence from mouse CAP-23 (no crossreactivity with chick CAP-23). Bar, 45 µm.
[View Larger Versions of these Images (60 + 23K GIF file)]
). Total RNA was
isolated and analyzed on Northern blots with digoxigenin-labeled riboprobes,
as described (Arber et al., 1994
). A rat cDNA coding for the
-subunit
of the acetylcholine receptor was a kind gift from A. Buonanno (National
Institutes of Health, Bethesda, MD). For immunoblots, brains were homogenized in SDS-PAGE sample buffer, protein contents were determined, and 40 µg of protein were loaded per slot. CAP-23 was visualized
with either monoclonal antibody 15C1 (Widmer and Caroni, 1990
), which
specifically detects chick but not mouse CAP-23, or with a rabbit antiserum to the carboxyl-terminal residues VASSEQSVAVKE of rat and mouse
neuronal acidic protein 22 (NAP-22). (This antiserum does not crossreact with chick CAP-23.) NAP-22 (Maekawa et al., 1993
) is the rat homologue of CAP-23. (The correct carboxyl-terminal sequence of chick
CAP-23 is 171-AETKSEVAPASDSKPSSPSSKETVAATAAPSSTAKASDPSAPPEEAKPSEAPATNSDTTIAVQD-234; due to two frameshift errors in the 3
-end sequence of the cDNA, the original sequence
[Widmer and Caroni, 1990
] contained two translation errors.) GAP-43
was visualized as described (Aigner et al., 1995
). Immunocytochemistry
was performed on 12-µm cryostat sections of 4%-formaldehyde-fixed tissues. Antibodies were applied in PBS with 0.5% NP-40 (Sigma Chemical
Co., St. Louis, MO) and 5% BSA as described (Widmer and Caroni,
1990
). Monoclonal antibody supernatants were diluted 1:100. Neurofilament-160 was detected with a specific monoclonal antibody (Sigma Chemical Co.). Rhodamine-
-bungarotoxin was from Molecular Probes (Eugene, OR). Bound antibodies were visualized with Biotin-conjugated
second antibodies, followed by Lucifer yellow-conjugated streptavidin,
and with rhodamine-conjugated second antibodies. (All secondary antibody reagents were from Molecular Probes.) Postsynaptic neuromuscular
junction configurations were visualized by labeling whole-mount preparations of gluteus muscle with RITC-
-bungarotoxin. All procedures were
carried out at 4°C. Briefly, muscles were incubated for 5 h in L15/PBS (1:2) with 1 µg/ml of RITC-
-bungarotoxin, rinsed with PBS, and fixed overnight with 2% paraformaldehyde in phosphate buffer (pH 7.4). Groups of
one to five muscle fibers were then dissected in water and mounted in airvol.
-bungarotoxin (Sigma Chemical Co.; in PBS, with 0.1%
BSA; 1 µg on the first day, and then 0.5 µg every second day) onto the
gastrocnemius. Because of the potential difficulties in inducing persistent
paralysis with
-bungarotoxin, we also carried out a series of experiments in
which a priming injection (1 µg) was followed by daily applications of 0.35 µg
of the toxin. In these experiments, absence of the toe-spreading reflex
was also verified daily. A third procedure consisted in applying tetrodotoxin (Sigma Chemical Co.; 1.2 µg per day in DME with 0.1% BSA) through
a sciatic nerve cuff supplied by an osmotic minipump (ALZET model
1007D; Alza Corp., Palo Alto, CA), according to a published procedure (Witzemann et al., 1991
). Depending on the paralysis protocol, paralysis
was obvious 5-24 h after the beginning of the treatment and was verified
by the missing toe-spreading reflex (gastrocnemius), as described (Witzemann et al., 1991
). Possibly because of technical difficulties in obtaining a
tight seal at the cuff site, in most tetrodotoxin-treated animals, paralysis
signs became less obvious after 3-4 d. For crush experiments, mice were
anesthetized and the right sciatic nerve was exposed at mid-thigh level and
crushed, as described (Aigner et al., 1995
).
; Caroni et al., 1994
). For the
unambiguous identification of sprouts, only nerve profiles longer than 5 µm
that clearly extended beyond the endplate area (ultraterminal sprouts)
were included in the analysis. At least 300 endplates per animal were analyzed as described (Aigner et al., 1995
). Values are averages ±SEM. For
statistical analysis, the Mann-Whitney U test was applied. To provide a
measure of the total length of the sprouts in a given muscle, the lengths of all ultraterminal sprouts and the total number of neuromuscular synapses
(including all neuromuscular junctions in a given field, i.e., those with and
without sprouts) in randomly selected fields were summed and normalized to 500 neuromuscular junctions (i.e., total length of sprouts in all randomly selected fields, divided by total number of neuromuscular synapses
in the same fields, times 500). Branch-points per endplate included every
silver (neurofilament)-positive side-branch of >4 µm within the esterasepositive synaptic region. These therefore reflect both ultraterminal sprouting and the extent of terminal branching at the synapse. Not included
were branch-points from the preterminal region of the motor nerve and
from ultraterminal sprouts. Regenerative nerve growth 64 h after sciatic
nerve crush was analyzed on longitudinal 12-µm cryostat sections by
counting the number of neurofilament-160-positive profiles 1-2 and 5-6 mm
distal from the crush site, as described (Aigner et al., 1995
). Synaptic areas
were estimated on RITC-
-bungarotoxin-labeled whole-mount preparations of 5-6-wk gluteus maximus muscles with NIH Image 1.4 software.
Only endplates with most of their apparent overall outlines in the plane of
focus were included in the analysis. The values are averages ±SEM of 75 endplates each (3 animals, 25 endplates each).
Results
). Subsequently,
expression becomes restricted to the nervous system,
where it peaks around embryonic day 17 (Widmer and Caroni, 1990
). In the adult, expression is lower and is mainly
restricted to certain types of neurons. To better define the
time course of CAP-23 downregulation and to determine
whether its expression is induced upon nerve lesion, we
carried out appropriate experiments in the developing and
adult mouse neuromuscular system. Intramuscular nerve
and neuromuscular junction CAP-23 immunoreactivity
did not change significantly between birth and postnatal
day (P) 8 (data not shown). Subsequently, as shown in Fig.
1 A, it was downregulated between postnatal days 8 and
14, when no CAP-23 immunoreactivity could be detected
at most neuromuscular junctions. Thus, like GAP-43 (Caroni and Becker, 1992
), downregulation of CAP-23 in this
system coincides with the time when muscle stops growing
by the addition of new fibers and with the final phase of
synapse elimination. Fig. 1 B shows nerve-associated
CAP-23 immunoreactivity in adult gastrocnemius muscle
before and 10 d after sciatic nerve crush. CAP-23 was not detectable in adult intramuscular nerves, where it was reinduced during regeneration. Therefore, like GAP-43
(Skene, 1989
), CAP-23 is reexpressed in regenerating peripheral nerves.
). Chick and mouse CAP-23 are highly
homologous but can be distinguished with specific antibodies. Therefore, to allow for independent detection of
endogenous and transgenic CAP-23, we used chick CAP-23
as a transgene. Four independent transgenic lines with substantial expression levels in spinal motoneurons were selected for further analysis. These will be designated in the
following as CAP-23(C11), CAP-23(C2), CAP-23(C17), and CAP-23(C13). As shown in Fig. 2 A, transgenic CAP-23
was expressed in several types of adult neurons, including
spinal motoneurons. The data shown in the figure are from
a CAP-23(C11) mouse. Although the other transgenic
lines displayed similar widespread expression, with strong
signals in spinal motoneurons, specific differences were
noticed. However, due to the focus of this study, these
differences will not be discussed here. Fig. 2 B shows that
transgene levels in homogenates of adult mouse brain
were comparable to maximal levels reached for endogenous chick CAP-23. The figure also shows that monoclonal antibody 15C1 does not crossreact with mouse
CAP-23. Finally, Fig. 2 C demonstrates that prominent
levels of chick CAP-23 immunoreactivity could be detected at the adult neuromuscular junction of CAP-23(C11)
mice, whereas from P15 on endogenous CAP-23 (Fig. 2 C)
and GAP-43 (not shown) were undetectable. Comparable results were obtained for the other transgenic lines included in the analysis, with highest signals in CAP-23(C2)
and lowest signals in CAP-23(C13) mice. In situ hybridization and Northern blot analysis failed to reveal any transgene expression in muscle (data not shown). In addition,
intramuscular nerve and neuromuscular junction transgene immunoreactivity was abolished 3 d after denervation. Taken together, these results show that, as already
described for the corresponding GAP-43-overexpressing
mice (Aigner et al., 1995
), transgene expression was restricted to neurons and did not activate the expression of
endogenous CAP-23 or GAP-43.
) (Fig. 3 C). Like in GAP-43-
overexpressing mice (Aigner et al., 1995
), sprouting extents and apparent local expression levels as detected by
CAP-23 immunocytochemistry were correlated. Accordingly, CAP-23(C11) and CAP-23(C2) mice exhibited substantially more sprouting than CAP-23(C17) and CAP23(C13) mice (data not shown). Although sprouting patterns
were different, substantial sprouting was detected in all
analyzed muscles, including lateral and medial gastrocnemius, extensor digitorum longus, gluteus maximus, soleus,
and diaphragm. Strongest sprouting was detected in gluteus maximus and lateral gastrocnemius, and most of the analysis was carried out in the gluteus muscle. Detailed
comparison with GAP-43-overexpressing mice revealed
the following differences: (a) In the presence of CAP-23,
sprouts were on average about three to four times longer
(Fig. 3 D; P < 0.05); (b) CAP-23-overexpressing mice had
fewer sprouts per endplate (about half; Fig. 3 D; P < 0.05);
and (c) growth cone-like enlargements were detected in ~30% of GAP-43-induced sprouts but in <5% of those
induced in the presence of CAP-23. These different qualitative features of the sprouting patterns in GAP-43- and
CAP-23-overexpressing mice were consistently detected
in several independent transgenic lines. As for sprouting in
the presence of GAP-43, no signs of functional denervation could be detected in the muscles of CAP-23-overexpressing mice. Thus, mRNAs that are induced in skeletal
muscle by denervation or paralysis in the presence of
Bot-A were not induced in any of the CAP-23-overexpressing mice (Fig. 3 B; further denervation-sensitive mRNAs that were not induced in CAP-23-overexpressing
mice included those coding for MLP [Arber et al., 1994
]
and N-CAM). In addition, intracellular recordings of
spontaneous and nerve-induced postsynaptic potentials in
nerve-muscle explants failed to reveal abnormalities in
evoked potentials, or in the amplitude of miniature endplate potentials (data not shown). Therefore, the presence
of CAP-23 in motor nerves and at their synaptic endings is
sufficient to induce a substantial ultraterminal sprouting
reaction in the absence of any signs of functional denervation, indicating that, like GAP-43, CAP-23 promotes spontaneous nerve sprouting at the neuromuscular junction.
Fig. 3.
Neuromuscular
junctions of CAP-23-overexpressing mice exhibit spontaneous nerve sprouting, with
features distinct from those induced by GAP-43. Data
are from 4-6-wk-old mice;
gluteus maximus muscle. (A)
Ultraterminal nerve sprouting in a CAP-23(C11) and a
GAP-43(wt3) mouse. The
combined silver-esterase reaction visualizes nerves
(black) and acetylcholine esterase reaction product (blue; delimiting synaptic area).
Note long ultraterminal
sprouts in the presence of
CAP-23 (arrows) and shorter
sprouts (arrows; sprout at the
bottom with growth cone
structures) in the presence
of GAP-43. (B) Contents of
denervation-sensitive mRNA
(-subunit of AChR) in skeletal muscle of nontransgenic
and CAP-23(C11) mouse. As
in nontransgenic animals, the
gluteus of CAP-23(C11) mice
did not express genes induced by the absence of electrical activation (e.g., BotA-induced paralysis [8 d;
paralyzed]). (C) Quantitative analysis of ultraterminal
sprouting in nontransgenic
and CAP-23(C11) mice;
comparison with the sprouting reaction induced in the
same type of muscle by local
paralysis (8 d) with Bot-A.
N = 8. (D) Quantitative
analysis of nerve sprouting
patterns at the neuromuscular junction of nontransgenic (Con), CAP-23(C11), CAP-23(C2), GAP-43(wt3), and GAP-43(wt2) mice. Branch points per endplate:
silver-stained processes; sprouting and nonsprouting endplates included. For a description of the sampling and analysis procedures, see
Materials and Methods. Note distinct features of sprouting in the presence of CAP-23 (longer, less numerous sprouts) and GAP-43
(high number of branching points per endplate). Also note that "% of endplates with sprouts," "total length of sprouts," "mean sprout
length," and "number of sprouts per sprouting endplate" refer to extrasynaptic branches, whereas "branch points per endplate" is a measure for branching within the endplate region, thus including intra- and ultraterminal growth. N = 5. Bar, 57 µm.
[View Larger Versions of these Images (158 + 22 + 23K GIF file)]
Fig. 5.
Synergism between
CAP-23 and GAP-43 in nerve
sprouting induction. All data
from 4-6-wk-old mouse gluteus muscle. (A) Spontaneous ultraterminal nerve sprouting
in GAP-43(wt3)-plus-CAP23(C11) double-transgenic mice.
Combined silver-esterase reaction. Arrows point to neuromuscular junctions (thick) and
sprouts (thin). (B) Quantitative analysis of spontaneous
ultraterminal sprouting in
heterozygous GAP-43(wt3) and CAP-23(C11), homozygous GAP-43(wt3), and double-transgenic GAP-43(wt3)-
plus-CAP-23(C11) mice. N = 5. (C) Contents of denervationsensitive -AChR mRNA in
gluteus muscle of doubletransgenic mice. Note absence of denervation signs, in spite of
extensive nerve sprouting. (D) Comparison of Bot-A-induced
nerve sprouting in nontransgenic, transgenic, and doubletransgenic mice. Combined silver-esterase reaction. Note
dramatic growth at and behind
neuromuscular junctions in
double-transgenic mice. In nontransgenic mice, Bot-A induced elongation of most neuromuscular junctions and detectable nerve sprouting only at a subset of them. Some of the sprouts are indicated by arrows. Bar, 50 µm.
[View Larger Versions of these Images (158 + 22 + 146K GIF file)]
). In preliminary experiments we verified that, as for
GAP-43, muscle paralysis in the presence of Bot-A does
not induce significant CAP-23 immunoreactivity in motor
nerves or at the neuromuscular junction (data not shown),
indicating that neither GAP-43 nor CAP-23 are required
for nerve sprouting to occur. In further control experiments with decreasing doses of Bot-A, we determined that
the sensitivity to Bot-A-induced paralysis was, if anything,
higher in nontransgenic than in transgenic mice. We then
analyzed nerve sprouting in 8- and 21-d-paralyzed muscles
of nontransgenic and CAP-23-overexpressing mice. As
shown in Fig. 4, A and C, toxin-induced sprouting was
greatly potentiated in the presence of CAP-23. Significantly, instead of reducing the difference between nontransgenic and CAP-23-overexpressing mice, 3-wk paralysis augmented it, leading to dramatic sprouting extents and
lengths in the presence of CAP-23 (Fig. 4 C). These results
indicate that the presence of CAP-23 can promote sustained sprouting under favorable local conditions. They also
indicate that CAP-23 must be operating through an intrinsic mechanism distinct from reduced trasmitter release.
Fig. 4.
Potentiation of induced nerve sprouting in
CAP-23-transgenic mice.
All data from 5-8-wk-old
mice; gluteus muscle or sciatic nerve. (A) Sprouting in
untreated and Bot-A-paralyzed (8 d) muscle. Analysis
as in Fig. 3 C (from which
part of the data were replotted). N = 5. (B) Regenerative sprouting (at 64 h) distal
from sciatic nerve crush. N = 4. (C) Ultraterminal nerve
sprouting in chronically paralyzed (3 wk) muscle. Combined silver-esterase reaction; the plane of focus for
the transgenic sample was selected to maximize visualization of sprouts (arrows, position of neuromuscular
junctions; in part out of focus). Note dramatic extent of
the sprouting reaction in the
CAP-23(C11) mouse and
very limited reaction in the
nontransgenic mouse. These
findings further support the
conclusion that the presence
of CAP-23 in motor nerves
produces a true gain-of-function phenotype, leading to
sustained potentiation of intramuscular nerve sprouting.
Bar, 70 µm.
[View Larger Versions of these Images (23 + 153K GIF file)]
, for reinnervation data in GAP43-overexpressing mice).
-bungarotoxin-labeled whole-mount muscle preparations. This revealed a substantial increase in the density of synaptic structures at the neuromuscular junction of CAP-23- (Fig.
6 A) and GAP-43-overexpressing (Fig. 6 B) mice. Quantitative analysis of such preparations (see also Materials and
Methods) yielded the following values: estimated areas
within the outer synaptic boundaries were 1021 ± 125 µm2
(control) and 962 ± 106 µm2 (CAP-23[C11]); inside these
outer synaptic regions, RITC-
-bungarotoxin-positive areas were 447 ± 86 µm2 (control) and 668 ± 52 µm2 (CAP23[C11]). Significantly, no ectopic AChR clusters were detected in these experiments. Since the total muscle surface
area contained within the outer outlines of the neuromuscular junction was not significantly different from control,
and average muscle fiber diameters were also not altered
in the transgenic mice (data not shown), these results suggest that the presence of the transgenes led to growth of
nerve processes within the synaptic region, which then in
turn induced postsynaptic structures underneath them. When
AChR configurations and silver-stained nerve processes
were compared, it became clear that although the nerve
branching index at the synapse was elevated in the transgenic mice (Fig. 3 D; number of branch points per endplate), it failed to reveal the degree of complexity detected
with RITC-
-bungarotoxin. This pointed to potential limitations in the effectiveness of the silver stain method to reveal nerve terminal structures at the neuromuscular junction. Because the silver precipitation method mainly
reveals the presence of neurofilaments, a possible explanation for these discrepancies is that fine synaptic endings
may have low contents of these neuronal cytoskeletal elements. To identify possible relations between the expansion in synaptic structure density detected in GAP-43- and
CAP-23-overexpressing mice and synaptic activity, we analyzed AChR configurations in Bot-A-treated muscles. 8 d
after treatment, in nontransgenic mice, paralyzing doses of Bot-A had a very modest effect on the length of AChRpositive synaptic branches, whereas an apparent expansion in total synaptic area (from 668 ± 52 µm2 to 918 ± 78 µm2) was detected in transgenic mice (Fig. 6 B). Significantly, Bot-A-induced paralysis did not induce a transgenic-type postsynaptic configuration in nontransgenic mice
(Fig. 6 B; with respect to this last point, similar results were
obtained upon 3-wk paralysis [data not shown]).
Fig. 6.
Increased neuromuscular synaptic areas in GAP43- and CAP-23-overexpressing
mice. RITC--bungarotoxin
labeling of gluteus muscle fiber
whole mounts (4-6-wk-old
mice). (A) Representative examples of neuromuscular junction configurations in nontransgenic and CAP-23(C11)
mice. Part of the nontransgenic endplate is out of focus,
but the entire long-axis is in focus. Note dramatic increase in
branching index and synaptic
area in the transgenic mouse. In contrast, the external dimensions of the synaptic region did not differ significantly
between transgenic and nontransgenic mice. (B) Representative examples of neuromuscular junction configurations in nontransgenic and
GAP-43(wt3) mice, with and
without Bot-A-induced paralysis. Note that paralysis did
not induce features of transgenic endplates (e.g., high
branching index) in nontransgenic mice. Bar: (A) 15 µm;
(B) 29 µm.
[View Larger Version of this Image (58K GIF file)]
-bungarotoxin. This paralysis-inducing paradigm led to stimulation of ultraterminal sprout elongation,
starting 4-5 d after toxin application (Fig. 7 C). Significantly, however, stimulation of nerve branching at the
neuromuscular junction (Fig. 7 C) and increased ultraterminal sprout formation (data not shown) were absent.
When 0.5 µg of
-bungarotoxin were applied daily (for 3 d),
thus subjecting the muscle to a more effective paralyzing
protocol, these early sprouting responses were also not induced (data not shown). Finally, subparalyzing doses of
-bungarotoxin were without detectable effect. As discussed below, these results are consistent with the interpretation that reduced transmitter release induces local
nerve growth through a direct mechanism that may be independent of the activation of muscle AChRs and that
muscle paralysis leads to local conditions promoting the
elongation of ultraterminal sprouts.
Fig. 7.
GAP-43- and CAP-23-transgenic mice reveal a sproutinducing mechanism sensitive to reduced transmitter release that
may be independent of postsynaptic activation. Data from gluteus (A and B) or gastrocnemius (C) muscle of 4-7-wk-old mice.
(A) Subparalyzing doses of Bot-A induce ultraterminal nerve
sprouting and elevated contents of neurofilament-positive branch
points at the neuromuscular junction. Nontransgenic (Con),
GAP-43(wt3), and CAP-23(C11) mice were analyzed. 1 pg of
Bot-A induced local paralysis in all mice, 0.5 pg failed to induce
detectable paralysis signs in >80% of the mice, and 0.1 pg induced no detectable paralysis. Note that 0.05 pg of toxin was already highly effective in inducing sprouting and endplate branching (these two parameters were correlated) in GAP-43- and
CAP-23-overexpressing mice. N = 6. (B) Time-course of Bot-A-
induced nerve growth at the neuromuscular junction of GAP43(wt3) mice. Sprout length: average length of the longest sprout
per sprouting endplate. Note that subparalyzing doses of the
toxin rapidly induced endplate branching (and the emergence of
short ultraterminal sprouts; data not shown), whereas paralysis promoted sprout elongation with comparatively slow kinetics.
N = 4. (C) Endplate nerve branching is induced by blockade of
transmitter release in the presence of either Bot-A (1 pg) or tetrodotoxin (TTX), whereas paralyzing doses of -bungarotoxin
(
Bgtx) failed to induce this reaction. In contrast, sprout elongation was induced by all paralysis-inducing protocols. These experiments were carried out in GAP-43(wt3) mice. N = 4.
[View Larger Version of this Image (37K GIF file)]
Discussion
), CAP-23-overexpressing mice
displayed spontaneous and greatly potentiated induced
nerve sprouting at the adult neuromuscular junction. While GAP-43-induced sprouts were numerous, with prominent
growth cone structures, in the presence of CAP-23 neuromuscular junctions displayed fewer, but longer sprouts
with barely detectable growth cones. Sprouting and its
transgene-specific features were greatly potentiated by
treatments with Bot-A, and double-transgenic mice displayed profound potentiation of sprouting, with structural features reminiscent of both transgenes. These results
strongly support the hypothesis that mechanisms promoted by intrinsic neuronal components affect the potential for and pattern of local nerve growth.
-bungarotoxin revealed that at the neuromuscular junction, growth is activated by mechanisms that may directly sense reduced
transmitter release independent of postsynaptic activity.
Muscle paralysis, in addition, greatly promotes ultraterminal sprout elongation. As discussed below, a possible interpretation of these findings is that at the adult neuromuscular junction, intrinsic neuronal components determine
competence and types of nerve growth, mechanisms sensitive to nerve activity, possibly involving terminal Schwann
cells, promote nerve growth, and absence of postsynaptic activation leads to local conditions favoring sprout
elongation.
), did not produce potentiation of nerve sprouting (data not shown). The specific
features of the sprouting detected in the presence of the
different transgenes may have interesting biological implications. However, their interpretation will require a more
detailed analysis of sprouting in these mice, in particular
with respect to its dynamic and ultrastructural properties.
) and guidance (Strittmatter et al.,
1995
), and regulated secretion (Dekker et al., 1989
; Imaizumi et al., 1995
; Gambi et al., 1996
). Potentially significant clues are its local abundance (up to 1% of growth
cone and synaptosomal protein) and its interactions with
calmodulin, PKC, and actin filaments (Benowitz and
Routtenberg, 1987
; Liu and Storm, 1990
; Hens et al.,
1993
). In a recent study, we found that GAP-43, CAP-23, and a further abundant calmodulin- and actin-binding
PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS) (Aderem, 1995
), share a number of
unique properties, including accumulation at the same
subplasmalemmal punctate structures and induction of the
same characteristic spectrum of cell surface activities, including blebbing and filopodia formation (Wiederkehr,
A., and P. Caroni, manuscript submitted for publication).
Like GAP-43, MARCKS has been implicated in several
processes involving cell surface dynamics, including cell
migration, phagocytosis, and exocytosis (Aderem, 1995
).
Although they share certain biochemical and functional
properties, GAP-43, CAP-23, and MARCKS are clearly regulated and targeted in very distinct manners (Skene
and Virag, 1989
; Allen and Aderem, 1995
). GAP-43 and
CAP-23 may thus affect similar local dynamic processes at
the cell surface, and their differential targeting and regulatory properties may lead to complementary and synergistic effects (Wiederkehr, A., and P. Caroni, manuscript submitted for publication). A difficulty in defining the
processes affected by these proteins is that very little is
known about the mechanisms that translate local signaling
into neurite outgrowth. However, when combined with
the fact that both GAP-43 and MARCKS have been implicated in multiple motility processes at the cell surface, the
results of this study are consistent with a role for GAP-43
and CAP-23 in the processes that link cortical cytoskeleton dynamics to membrane fusion. One possibility is that
these proteins may shift a balance between stable membrane incorporation and rapidly reversible exo-/endocytosis (see e.g., Catsicas et al., 1994
), thus favoring growth.
), it seems very likely that the Schwann cells at
the synapse (terminal-Schwann cells) mediate this growthpromoting signal. This is based on the fact that these cells
can respond rapidly to changes in transmitter release (Reist
and Smith, 1992
; Georgiou et al., 1994
) and that they appear to be causally involved in most if not all forms of
nerve sprouting at the neuromuscular junction (Reynolds
and Woolf, 1992
; Son and Thompson, 1995
). Out of a total
of 22 transgenic lines, we never found any evidence of transgene expression in peripheral nerve or terminal-Schwann cells. To search for signs of terminal-Schwann cell activation in the transgenic mice, we analyzed endogenous
GAP-43, GFAP, and low-affinity NGF receptor immunoreactivity on corresponding muscle sections. These experiments did not reveal obvious differences to nontransgenic
mice (data not shown), suggesting that transgene expression may not have induced a reactive state in terminalSchwann cells. It therefore seems most likely that, if they
indeed mediate these growth reactions, terminal-Schwann
cells in transgenic and nontransgenic mice respond in the
same manner to reduced transmitter release, and that the
presence of GAP-43 or CAP-23 affects the subsequent
sprouting reaction. One puzzling observation was that within the same muscle, neuromuscular junctions with vigorous sprouting coexisted side-by-side with junctions that
showed no detectable sprouting. This was obvious in all
transgenic lines and in paralyzed muscle from nontransgenic mice. Transgene presence greatly enhanced both the
extent of sprouting at single endplates and the proportion
of endplates with sprouting. Although we currently have
no explanation for this phenomenon, one possibility is that for sprouting to occur a threshold must be reached. This in
turn may either be a property intrinsic to the nerve or may
involve both the nerve and the terminal-Schwann cell.
-bungarotoxin-labeled synaptic structures
displayed a pronounced increase in branching index and
overall synaptic area. In contrast, we detected no extrajunctional AChR clusters in these muscles, suggesting that
ultraterminal sprouts did not induce synaptic structures.
Significantly, in nontransgenic mice, treatment with Bot-A
failed to induce synaptic features resembling those in
GAP-43- or CAP-23-overexpressing mice, regardless of
the extent and duration of muscle paralysis. These results
suggest that, within the synaptic region, presynaptic branching and growth due to the presence of GAP-43 or
CAP-23 induced corresponding growth of synaptic structures in muscle. This phenomenon is probably related to
nerve terminal outgrowth, the terminal branch elongation
reaction detected in nontransgenic mice in response to
Bot-A-induced paralysis (Pestronk and Drachman, 1978b
).
; Stewart et al., 1996
). As suggested
recently, this may reflect homeostasis of synaptic transmission (Stewart et al., 1996
), possibly involving corresponding changes in active zone density (Budnik et al., 1990
;
Tsujimoto et al., 1990
; Woytowicz et al., 1994
; Stewart et al.,
1996
). These questions will be adressed by an analysis of
ultrastructural parameters at the neuromuscular junction
of the transgenic mice.
). In spite of substantial transgene expression in dentate gyrus granule cells,
none of the CAP-23-overexpressing lines displayed comparable massive sprouting reactions. These findings further argue for the existence of significant functional differences between GAP-43 and CAP-23. However, because it
is technically impossible to resolve the terminal arbors of
single neurons in these experiments, the informative value
of these data is limited, and spontaneous nerve sprouting
in the adult central nervous system will have to be analyzed at a much higher resolution.
Received for publication 23 August 1996 and in revised form 13 November 1996.
Address all correspondence to Pico Caroni, Friedrich Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland. Tel.: 41-51-6973727. Fax: 4161-6973976. E-mail: caroni{at}fmi.chWe are grateful to S. Arber, S. Kaech, and T. Laux (Friedrich Miescher Institute) for critically reading the manuscript. We thank H.-R. Brenner (Department of Physiology, University of Basel; electrophysiology on nerve-muscle explants) and F. Botteri (Friedrich Miescher Institute; generation of transgenic mice) for help with some of the experiments.
AChR, acetylcholine receptor; Bot-A, Botulinum toxin-A; CAP, cortical cytoskeleton-associated protein; GAP, growth-associated protein; MARCKS, myristoylated alanine-rich C kinase substrate; PKC, protein kinase C.