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Intramuscular nerve sprouting induced by CNTF is associated with increases in CGRP content in mouse motor nerve terminals
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Neuroscience Letters 219 (1996) 60 64
NEUROSCIEHC[ LETTERS
Intramuscular nerve sprouting induced by C N T F is associated with increases in CGRP content in mouse motor nerve terminals Olga Tarabal, Jordi Calder6, Josep E. Esquerda* Unitat de Neurobiologia Cellular, Departament de Cibncies Mbdiques Bgtsiques, Facultat de Medicina. Universitat de Lleida, 44, E25198 Lleida. Catalonia, Spain
Received 14 June 1996; revised version received 30 September 1996; accepted 2 October 1996
Abstract
It is known that motor nerve terminal sprouting induced by either nerve injury or muscle paralysis is associated with an increase in calcitonin gene-related peptide (CGRP) content in the soma of motoneurons and in motor endplates. In the present study, CGRP-like immunoreactivity (CGRP-LI) was determined in motor endplates of animals in which nerve terminal sprouting had been induced by exogenous application of ciliary neurotrophic factor (CNTF). After 18 days of CNTF treatment we observed a significant increase in CGRP-LI in motor endplates. The results indicate that CGRP is upregulated when motor nerve outgrowth is induced, even in the absence of muscle paralysis or nerve lesion. Keywords: Calcitonin gene-related peptide; Ciliary neurotrophic factor; Mouse; Nerve sprouting; Neuromuscular junction; Plasticity
Calcitonin gene-related peptide (CGRP) is a neuropeptide distributed in a large number of neurons in the central and peripheral nervous system (see [14] for a review). In craneal and spinal motoneurons CGRP is present in the majority of cell bodies where the peptide is synthesized [11,21,28], transported anterogradely to nerve terminals and released [16,27]. CGRP-immunoreactivity has been observed in motor nerve terminals although in adults it has been detected only in certain muscles [7]. Over the last few years, a number of studies have suggested that C G R P is important in the regulation of certain aspects of nerve-muscle interaction. For example, (1) during development, motoneuronal CGRP content changes in a period coincident with the maturation of neuromuscular synapses [20], (2) after peripheral nerve injury CGRP is upregulated in cell bodies and accumulates in reinnervating nerve terminals [1,4,24] (3) intramuscular nerve sprouting induced by paralyzing toxins (i.e. botulinum toxin or tetrodotoxin) is associated with increased levels of CGRP in both motoneuron somas and nerve terminals [22,25]. Nevertheless, it has been reported that exogenous
* Corresponding author. Tel.: +34 73 702427; fax: +34 73 702426.
administration of CGRP is able to block the sprouting capacity of nerve terminals in paralyzed muscles [26]. Since in muscle cells CGRP is able to locally stimulate the synthesis of acetylcholine receptors [8,9,20] it seems reasonable to suppose that when CGRP accumulates in the tips of growing nerves it may contribute to the induction of new postsynaptic structures on the non-innervated muscle surfaces. In this case, CGRP upregulation might be an essential element in the formation of endplates during motoneuronal regeneration and plasticity. However, in regenerating nerves or after paralysis with BoTx, CGRP may be accumulated by causes not directly related to nerve growth, i.e. as a consequence of disturbances on release and/or transport. CGRP may also be unspecifically upregulated in the general context of cell body reaction to nerve injury or paralysis. On the other hand, muscle paralysis upregulates some muscle derived growth factors such as insulin-like growth factors (IGFs) that, in turn, stimulate motor nerve outgrowth [15]. Exogenous application of growth factors like IGF, neurotrophin-4 (NT-4) and ciliary neurotrophic factor (CNTF) stimulates intramuscular nerve sprouting in normal muscles, mimicking the effects of factors that could be released after muscle paralysis [5,6,10,12].
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)13174-8
o . Tarabal et al. / Neuroscience Letters 219 (1996) 6 0 - 6 4
It has been suggested that in adult neuromuscular junctions a balance exists between CGRP and muscle derived trophic factors: tropbic factors released by the muscle upregulate the expression of CGRP in motoneurons, while CGRP downregulates muscle trophic factors leading to endplate stabilization [22]. To further evaluate this hypothesis, we have tested whether the imbalance in this system, elicited by exogenous neutrophic factor overloading, can alter the normal levels of CGRP in neuromuscular junctions. We show here that CNTF injected over the muscle is able to induce both nerve terminal sprouting and accumulation of CGRP in motor nerve terminals. Male CD1 mice of 2 0 - 2 5 g weight were injected subcutaneously over the surface of the right gluteus muscle with 500 ng/day of CNTF (Research Biochemical Int., Natick, MA, USA). CNTF was dissolved in sterile phosphate-buffered saline (PBS) containing bovine serum albumin at 100 /zg/ml. CNTF (20 /zl) solution was injected daily for 18 days using a Hamilton syringe. The same volume and regimen of injections were made with the vehicle over the left gluteus muscle. This muscle was chosen because it is flat and very thin, facilitating the spread of CNTF, which is normally restricted [12]. Another group of animals were bilaterally injected with just the vehicle. Mice were anesthetized with chloral hydrate (450 mg/kg body weight) and killed by intracardiac perfusion of 100 ml of physiological saline solution followed by a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The gluteus muscle was dissected, removed and left overnight in the same fixative. Following cryoprotection with several changes of 20% sucrose w/v in 0.1 M PB, samples were frozen and longitudinal sections (20 /zm thick) were obtained. CGRP-LI was revealed by incubating sections with a rabbit antibody against synthetic rat CGRP (Peninsula Labs., Belmont, CA, USA), diluted 1/500 in PBS, for 12 h at 4°C. After several washes, sections were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti200 E
61
rabbit IgG antibody (Sigma, St. Louis, MO, USA; diluted 1/40 in PBS) mixed with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated o~-bungarotoxin (Bgtx, Molecular Probes, Eugene, OR, USA; 1 #g/ml) for 1 h at room temperature. Sections were mounted in Fluoprep (Biomerieux, Charbonnier les Bains, France). CGRP-LI was evaluated in at least 150 endplates per muscle by means of an epifluorescence microscope with selective filter sets for fluorescein and rhodamine. Motor endplates were considered CGRP positive when the immunoreactivity was clearly distinguishable from the background. Some specimens were also examined using a confocal laser-scanning microscope (Zeiss LSM-310, Oberkochen, Germany) equipped with 543 nm helium/neon and 488 nm argon ion lasers and with appropriate barrier filters. An oil immersion lens ( x 100, NA 1.3) was used with the following setting parameters: 0.39/zm pixel size, 31 pinhole aperture, 32 s scan speed. Images were digitalized at a resolution of eight bits into an array of 512 x 512 pixels, processed and stored in the microscope's own software. The average intensity of CGRP-LI signal was measured within delimited areas of motor nerve terminals. Between 20 and 35 endplates were analyzed per muscle. For morphological examination of motor endings, gluteus muscles were fixed with 10% formalin for at least 15 days. Free-floating frozen sections (40 /zm thick) were processed for silver impregnation according to GrosBielschowschy's method. In each muscle, nerve terminals from 2 0 - 5 0 randomly selected endplates were drawn using a camera lucida and their lengths were measured with a digital table analyzer (MOP Videoplan, Kronton, Germany). Results from CNTF and vehicle-treated muscles were statistically compared by Student's t-test. In line with previous reports by Gurney et al. [12], we found that chronic administration of locally applied CNTF (500 ng/day) induced terminal motor nerve sprouting (Fig. 1A). Measurements of total length of endplate presynaptic branches in silver stained gluteus muscles evidenced that
180
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160
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°o vehicle-injected
o
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Fig. 1. Length (A) and CGRP-LI intensity (B) of motor nerve terminals from gluteus muscle after 18 days of CNTF or saline (control) treatment. Nerve terminals were stained with a silver impregnation. In (A), each point represents the data (mean _+SEM) from 20-50 motor endplates examined per muscle (four animals). In (B), average CGRP-LI intensity was measured within nerve terminals using a confocal laser-scanningmicroscope. Data (mean -+ SEM) were obtained after measuring 20-35 endplates per muscle (four animals). *P < 0.05 versus vehicle-injected muscles, Student's t-test.
62
O. Tarabal et al. / Neuroscience Letters 219 (1996) 60-64
II IIIII
b
Fig. 2. Confocal laser-scanning micrographs of gluteus muscle endplates from control animals (a,c) and after 18 days of CNTF treatment (b,d,e). Endplates were double-labeled with TRITC-~-Bgtx (red) and CGRP with fluorescent (FITC) immunostaining (green). Positive CGRP-LI is present in motor axons (arrowheads) of both control (a) and CNTF treated muscles (b,d). Sprouts emerging from CNTF muscles have a highly intense positive CGRP-LI signal (arrows in (b)). In presynaptic nerve terminals CGRP-LI is distributed as small granules that are more prominent in CNTF treated muscles (encircled in (d) and (e)) than in those treated with saline (encircled in (c)). Scale bars, (a,b,e) 25 /~m; (c,d) 10/~m.
O. Tarabal et al. / Neuroscience Letters 219 (1996) 6 0 - 6 4
after 18 days of CNTF treatment significant nerve terminal outgrowth was induced. To avoid possible bias in measured data resulting from limited muscle penetration of CNTF, only motor endplates located in the most superficial layers of muscle were examined. Endplate morphology showed both terminal and nodal sprouts, the latter emerging from the node of Ranvier closest to the motor endplates. Although CNTF may have systemic effects stimulating motor nerve sprouting when injected in large doses [17], the doses used here did not induce detectable sprouting in the motor endplates from contralateral muscles. CNTF treatment is also associated with an increase of CGRP-LI in motor endplates, as evidenced by the presence of a higher proportion of endplates showing positive CGRP-LI when compared with contralateral and normal (vehicle treated) muscles. In control muscles only around 10% of the motor endplates showed positive CGRP-LI, whereas in CNTF injected muscles this value increased 5.5-fold. In randomly selected endplates, the morphology of nerve terminals and the intensity of fluorescence representing the CGRP-content were evaluated in high resolution images obtained with a confocal microscope (Fig. 1B and 2). CGRP-LI was located inside motor axons and their presynaptic terminal branches. In the latter, positive CGRP-LI homogeneously filled the whole presynaptic compartment, being accompanied by fine bright punctated granules, which presumably correspond to the large synaptic vesicles in which CGRP is accumulated [19]. The content of CGRP per motor endplate was notably higher after CNTF treatment representing a 4-fold increase in the intensity of fluorescence measured inside nerve terminals; in which there were also more prominent CGRP-LI positive granules (Fig. 1B and 2b,d,e). In some neuromuscular synapses from CNTF treated muscles, terminal sprouts with positive CGRP-LI were seen escaping from the endplate postsynaptic region delimited by fluorescence Bgtx binding. (Fig. 2b). All these results suggest that motoneuronal CGRP changes in parallel with motor endplate remodeling. Previous reports have demonstrated that motoneural CGRP is upregulated in a variety of situations that share as a common feature a reactive response of motor axons to either nerve injury or muscle paralysis [4,22,24,25]. In these cases, the response leads to a functional restoration of the neuromuscular system implying endplate regeneration and remodeling. In the present paper we demonstrate that CGRP is also increased when motor nerve sprouting is induced by exogenous growth factor administration in the absence of muscle paralysis, which suggests a more direct correlation between CGRP and motor terminal outgrowth. The low levels of CGRP present in normal muscles might be related to the subtle plastic changes that take place in adult motor nerve terminals. In fact, neuromuscular synapse remodeling has been shown to occur in normal muscles [13,18]. These changes optimally adjust the size
63
and extension of nerve terminals to the functional requirements for efficient synaptic transmission, in accordance with muscle contractile activity, fiber properties and volume [3]. Reorganization of motor nerve terminal architecture should be coupled with corresponding changes in the assembly of postsynaptic structures. Since it seems that nerve terminal outgrowth precedes the differentiation of its postsynaptic counterpart (i.e. accumulation of acetylcholinesterase and acetylcholine receptors [2,29], signals derived from growing axons need to be present to induce the corresponding changes at the muscle surface. According to our experimental data and current knowledge about CGRP, this peptide fits with the expected properties of the above mentioned axonal-derived signal. In turn, muscle-derived signals should regulate, in an activity-dependent way, the stability of presynaptic nerve terminals [5,6,10,15]. As has been suggested by Sala et al. [22], CGRP levels may reflect the resulting balance of exchanging signals between motor nerve terminals and muscle, and additionally regulate the plastic status of a given motor endplate. Because CNTF is not normally present in the muscle [23], its effects on CGRP regulation must be accomplished in a physiological situation by other muscle-derived trophic factors such as IGF and NT-4 [5,6,10,15]. More studies are needed to address the effects of these factors when administered in vivo on CGRP regulation. In any case the response obtained after CNTF administration imitates that induced by musclederived growth factors. We would like to thank Dr. Joan Ribera for constructive comments, Anna Naco for excellent technical assistance, and Malcolm Hayes for reading the manuscript. This work was supported by a grant from Ministerio de Educaci6n y Ciencia (DGICYT, P B 9 3 0642) and a grant from Ajuntament de Lleida. [1] Arvidsson, U., Johnson, H., Piehl, F., Cullheim, S., HOkfelt, T., Risling, M., Terenius, L. and Ulfhake, B., Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons, Exp. Brain Res., 79 (1990) 212-216. [2] Angaut-Petit, D., Molg6, J., Cornelia, J.X., Faille, L. and Tabti, N., Terminal sprouting in mouse neuromuscular junctions poisoned with botulinum type A toxin: morphological and electrophysiological features, Neuroscience, 37 (1990) 799-808. [3] Balice-Gordon, R.J., Breedlove, S.M., Bernstein, S. and Litchman, J.W., Neuromuscular junctions shrink and expand as muscle fiber size is manipulated: in vivo observations in the androgen-sensitive bulbocavernosus muscle of mice, J. Neurosci., 10 (1990) 26602671. [4] Calder6, J., Casanovas, A., Sorribas, A. and Esquerda, J.E., Calcitonin gene-related peptide in rat spinal cord motoneurons: subcellular distribution and changes induced by axotomy, Neuroscience, 48 (1992) 449-461. [5] Caroni, P. and Grandes, P., Nerve sprouting in innervate adult skeletal muscle induced by exposure to elevated levels of insuline growth factors, J. Cell Biol., 110 (1990) 1307 1317. [6] Caroni, P., Schenider, C., Kiefer, M.C. and Zapf, J., Role of muscle
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[7]
[8]
[9]
[10]
[11]
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[13]
[14] [15]
[16]
[17]
[18]
O. Tarabal et al. / Neuroscience Letters 219 (1996) 6 0 - 6 4
insuline-growth factors in nerve sprouting: suppression of terminal sprouting in paralyzed muscle by IGF-binding protein 4, J. Cell Biol., 125 (1994) 893-902. Csillik, B., Tajti, L., Kovacs, T., Kukla, E., Rakic, P. and Knyiharscsillik, E., Distribution of calcitonin gene-related peptide in vertebrate neuromuscular junction-relationship to the acetilcholine receptor, J. Histochem. Cytochem., 41 (1993) 1547-1555. Fontaine, B., Klarsfeld, A. and Changeux, J.P., Calcitonin generelated peptide and muscle activity regulate acetylcholine receptor c~-subunit mRNA levels by distinct intracellular pathways, J. Cell Biol., 105 (1987) 1337-1342. Fontaine, B., Klarsfeld, A., H6kfelt, T. and Changeux, J.P., Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetilcholine receptors in primary cultures of chick embryo myotubes, Neurosci. Lett., 71 (1986) 5965. Funakoshi, H., Belluardo, N., Arenas, E., Yamamoto, Y., Casabona, A., Persson, H. and Ib~fiez, C.F., Muscle derived neurotrophin-4 as an activity-dependent trophic signal for adult motor neurons, Science, 268 (1995) 1495-1499. Gibson, S.J., Polak, J.M., Giaid, A., Hamid, Q.A., Kar, S., Jones, P.M. and Denny, P., Calcitonin gene-related peptide messenger RNA is expressed in sensory neurons of the dorsal root ganglia and also in spinal motoneurones in man and rat, Neurosci. Lett., 91 (1988) 283-288. Gurney, M.E., Yamamoto, H. and Kwon, Y., Induction of motor neuron sprouting in vivo by ciliary neurotrophic factor and basic fibroblast growth factor, J. Neurosci., 12 (1992) 3241-3247. Herrera, A.A., Banner, L.R. and Nagaya, N., Repeated in vivo observation of frog neuromuscular junctions: remodelling involves concurrent growth and retraction, J. Neurocytol., 19 (1990) 85-99. Ishida-Yamamoto, A. and Tohyama, M., Calcitonin gene-related peptide in the nervous tissue, Prog. Neurobiol., 33 (1989) 355-386. Ishii, D.N., Relationship of insuline growth factor II gene expression in muscle to synaptogenesis, Proc. Natl. Acad. Sci. USA, 86 (1989) 2898-2902. Kashihara, Y., Sakaguchi, M. and Kuno, M., Axonal transport and distribution of endogenous calcitonin gene-related peptide in rat peripheral nerve, J. Neurosci., 9 (1989) 3796-3802. Kwon, Y.W. and Gurney, M.E., Systemic injections of ciliary neurotrophic factor induce sprouting by adult motor neurons, NeuroReport, 5 (1994) 789-792. Litchman, J.W., Magrassi, L. and Purves, D., Visualization of neuromuscular junctions over periods of several months in living mice, J. Neurosci., 7 (1987) 1215-1222.
[19] Matteoli, M., Haimann, C., Torri-tarelli, F., Polak, J.M., Ceccarelli, B. and De Camilli, P., Differential effect of c~-latrotoxin on exocytosis from small vesicles and from large dense core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction, Proc. Natl. Acad. Sci. USA, 85 (1988) 7366-7370. [20] New, H.V. and Mudge, A.W., Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis, Nature, 323 (1986) 809-811. [21] Rosenfeld, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W. and Evans, R.M., Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specifc RNA processing, Nature, 304 (1983) 129-135. [22] Sala, C., Andreose, J.S., Fumagalli, G. and Lomo, T., Calcitonin gene-related peptide: possible role in formation and maintenance of neuromuscular junctions, J. Neurosci., 15 (1995) 520-528. [23] St6ckli, K.A., Lottspeich, F., Sendtner, M., Masiakowski, P., Carroll, P., G6tz, R., Lindholm, D. and Thoenen, H., Molecular cloning, expression and regional distribution of rat ciliary neurotropfiic factor, Nature, 342 (1989) 920-923. [24] Streit, W.J., Dumoulin, F.L., Raivich, G. and Kreutzberg, G.W., Calcitonin gene-related peptide increases in rat facial motoneurons after peripheral nerve transection, Neurosci. Lett., 101 (1989) 143148. [25] Tarabal, O., Calder6, J., Ribera, J., Sorribas, A., L6pez, R., Molg6, J. and Esquerda, J.E., Regulation of motoneuronal calcitonin generelated peptide (CGRP) during axonat growth and neuromuscular synaptic plasticity, Eur. J. Neurosci., 8 (1996) 829-836. [26] Tsujimoto, T. and Kuno, M., Calcitonin gene-related peptide prevents disuse-induced sprouting of rat motor nerve terminals, J. Neurosci., 8 (1988) 3951 3957. [27] Uchida, S., Yamamoto, H., Lio, S., Matsumoto, N., Wang, X.B., Yonehara, N., Imai, Y., Inoki, R. and Yoshida, H., Released of calcitonin gene-related peptide-like immunoreactivity substance from neuromuscular junction by nerve excitation and its action on striated muscle, J. Neurochem., 54 (1990) 1000-1003. [28] Villar, M.J., Huchet, M., H6kfelt, T., Changeux, J.-P., Fahrenkrug, J. and Brown, J.C., Existence and coexistence of calcitonin generelated peptide, vasoactive intestinal polypeptide- and somatostatin-like immunoreactivities in spinal cord motoneurons of developing and post-hatch chicks, Neurosci. Lett., 86 (1988) 114-118. [29] Yee, W.C. and Pestronk, A., Mechanisms of postsynaptic plasticity: remodelling of the functional acetylcholine receptor cluster induced by motor nerve terminal outgrowth, J. Neurosci., 7 (1987) 2019-2024.
Neuroscience Letters 219 (1996) 60 64
NEUROSCIEHC[ LETTERS
Intramuscular nerve sprouting induced by C N T F is associated with increases in CGRP content in mouse motor nerve terminals Olga Tarabal, Jordi Calder6, Josep E. Esquerda* Unitat de Neurobiologia Cellular, Departament de Cibncies Mbdiques Bgtsiques, Facultat de Medicina. Universitat de Lleida, 44, E25198 Lleida. Catalonia, Spain
Received 14 June 1996; revised version received 30 September 1996; accepted 2 October 1996
Abstract
It is known that motor nerve terminal sprouting induced by either nerve injury or muscle paralysis is associated with an increase in calcitonin gene-related peptide (CGRP) content in the soma of motoneurons and in motor endplates. In the present study, CGRP-like immunoreactivity (CGRP-LI) was determined in motor endplates of animals in which nerve terminal sprouting had been induced by exogenous application of ciliary neurotrophic factor (CNTF). After 18 days of CNTF treatment we observed a significant increase in CGRP-LI in motor endplates. The results indicate that CGRP is upregulated when motor nerve outgrowth is induced, even in the absence of muscle paralysis or nerve lesion. Keywords: Calcitonin gene-related peptide; Ciliary neurotrophic factor; Mouse; Nerve sprouting; Neuromuscular junction; Plasticity
Calcitonin gene-related peptide (CGRP) is a neuropeptide distributed in a large number of neurons in the central and peripheral nervous system (see [14] for a review). In craneal and spinal motoneurons CGRP is present in the majority of cell bodies where the peptide is synthesized [11,21,28], transported anterogradely to nerve terminals and released [16,27]. CGRP-immunoreactivity has been observed in motor nerve terminals although in adults it has been detected only in certain muscles [7]. Over the last few years, a number of studies have suggested that C G R P is important in the regulation of certain aspects of nerve-muscle interaction. For example, (1) during development, motoneuronal CGRP content changes in a period coincident with the maturation of neuromuscular synapses [20], (2) after peripheral nerve injury CGRP is upregulated in cell bodies and accumulates in reinnervating nerve terminals [1,4,24] (3) intramuscular nerve sprouting induced by paralyzing toxins (i.e. botulinum toxin or tetrodotoxin) is associated with increased levels of CGRP in both motoneuron somas and nerve terminals [22,25]. Nevertheless, it has been reported that exogenous
* Corresponding author. Tel.: +34 73 702427; fax: +34 73 702426.
administration of CGRP is able to block the sprouting capacity of nerve terminals in paralyzed muscles [26]. Since in muscle cells CGRP is able to locally stimulate the synthesis of acetylcholine receptors [8,9,20] it seems reasonable to suppose that when CGRP accumulates in the tips of growing nerves it may contribute to the induction of new postsynaptic structures on the non-innervated muscle surfaces. In this case, CGRP upregulation might be an essential element in the formation of endplates during motoneuronal regeneration and plasticity. However, in regenerating nerves or after paralysis with BoTx, CGRP may be accumulated by causes not directly related to nerve growth, i.e. as a consequence of disturbances on release and/or transport. CGRP may also be unspecifically upregulated in the general context of cell body reaction to nerve injury or paralysis. On the other hand, muscle paralysis upregulates some muscle derived growth factors such as insulin-like growth factors (IGFs) that, in turn, stimulate motor nerve outgrowth [15]. Exogenous application of growth factors like IGF, neurotrophin-4 (NT-4) and ciliary neurotrophic factor (CNTF) stimulates intramuscular nerve sprouting in normal muscles, mimicking the effects of factors that could be released after muscle paralysis [5,6,10,12].
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)13174-8
o . Tarabal et al. / Neuroscience Letters 219 (1996) 6 0 - 6 4
It has been suggested that in adult neuromuscular junctions a balance exists between CGRP and muscle derived trophic factors: tropbic factors released by the muscle upregulate the expression of CGRP in motoneurons, while CGRP downregulates muscle trophic factors leading to endplate stabilization [22]. To further evaluate this hypothesis, we have tested whether the imbalance in this system, elicited by exogenous neutrophic factor overloading, can alter the normal levels of CGRP in neuromuscular junctions. We show here that CNTF injected over the muscle is able to induce both nerve terminal sprouting and accumulation of CGRP in motor nerve terminals. Male CD1 mice of 2 0 - 2 5 g weight were injected subcutaneously over the surface of the right gluteus muscle with 500 ng/day of CNTF (Research Biochemical Int., Natick, MA, USA). CNTF was dissolved in sterile phosphate-buffered saline (PBS) containing bovine serum albumin at 100 /zg/ml. CNTF (20 /zl) solution was injected daily for 18 days using a Hamilton syringe. The same volume and regimen of injections were made with the vehicle over the left gluteus muscle. This muscle was chosen because it is flat and very thin, facilitating the spread of CNTF, which is normally restricted [12]. Another group of animals were bilaterally injected with just the vehicle. Mice were anesthetized with chloral hydrate (450 mg/kg body weight) and killed by intracardiac perfusion of 100 ml of physiological saline solution followed by a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The gluteus muscle was dissected, removed and left overnight in the same fixative. Following cryoprotection with several changes of 20% sucrose w/v in 0.1 M PB, samples were frozen and longitudinal sections (20 /zm thick) were obtained. CGRP-LI was revealed by incubating sections with a rabbit antibody against synthetic rat CGRP (Peninsula Labs., Belmont, CA, USA), diluted 1/500 in PBS, for 12 h at 4°C. After several washes, sections were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti200 E
61
rabbit IgG antibody (Sigma, St. Louis, MO, USA; diluted 1/40 in PBS) mixed with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated o~-bungarotoxin (Bgtx, Molecular Probes, Eugene, OR, USA; 1 #g/ml) for 1 h at room temperature. Sections were mounted in Fluoprep (Biomerieux, Charbonnier les Bains, France). CGRP-LI was evaluated in at least 150 endplates per muscle by means of an epifluorescence microscope with selective filter sets for fluorescein and rhodamine. Motor endplates were considered CGRP positive when the immunoreactivity was clearly distinguishable from the background. Some specimens were also examined using a confocal laser-scanning microscope (Zeiss LSM-310, Oberkochen, Germany) equipped with 543 nm helium/neon and 488 nm argon ion lasers and with appropriate barrier filters. An oil immersion lens ( x 100, NA 1.3) was used with the following setting parameters: 0.39/zm pixel size, 31 pinhole aperture, 32 s scan speed. Images were digitalized at a resolution of eight bits into an array of 512 x 512 pixels, processed and stored in the microscope's own software. The average intensity of CGRP-LI signal was measured within delimited areas of motor nerve terminals. Between 20 and 35 endplates were analyzed per muscle. For morphological examination of motor endings, gluteus muscles were fixed with 10% formalin for at least 15 days. Free-floating frozen sections (40 /zm thick) were processed for silver impregnation according to GrosBielschowschy's method. In each muscle, nerve terminals from 2 0 - 5 0 randomly selected endplates were drawn using a camera lucida and their lengths were measured with a digital table analyzer (MOP Videoplan, Kronton, Germany). Results from CNTF and vehicle-treated muscles were statistically compared by Student's t-test. In line with previous reports by Gurney et al. [12], we found that chronic administration of locally applied CNTF (500 ng/day) induced terminal motor nerve sprouting (Fig. 1A). Measurements of total length of endplate presynaptic branches in silver stained gluteus muscles evidenced that
180
-~ lOO .E
160
~
80
120
~
60
100
E
.E
O9
140
Q)
80
"5
==
_~ 40
60 40
"E •- 20
v
..1
20 0
m
CNTFqnjected animals
°o vehicle-injected
o
animals
Fig. 1. Length (A) and CGRP-LI intensity (B) of motor nerve terminals from gluteus muscle after 18 days of CNTF or saline (control) treatment. Nerve terminals were stained with a silver impregnation. In (A), each point represents the data (mean _+SEM) from 20-50 motor endplates examined per muscle (four animals). In (B), average CGRP-LI intensity was measured within nerve terminals using a confocal laser-scanningmicroscope. Data (mean -+ SEM) were obtained after measuring 20-35 endplates per muscle (four animals). *P < 0.05 versus vehicle-injected muscles, Student's t-test.
62
O. Tarabal et al. / Neuroscience Letters 219 (1996) 60-64
II IIIII
b
Fig. 2. Confocal laser-scanning micrographs of gluteus muscle endplates from control animals (a,c) and after 18 days of CNTF treatment (b,d,e). Endplates were double-labeled with TRITC-~-Bgtx (red) and CGRP with fluorescent (FITC) immunostaining (green). Positive CGRP-LI is present in motor axons (arrowheads) of both control (a) and CNTF treated muscles (b,d). Sprouts emerging from CNTF muscles have a highly intense positive CGRP-LI signal (arrows in (b)). In presynaptic nerve terminals CGRP-LI is distributed as small granules that are more prominent in CNTF treated muscles (encircled in (d) and (e)) than in those treated with saline (encircled in (c)). Scale bars, (a,b,e) 25 /~m; (c,d) 10/~m.
O. Tarabal et al. / Neuroscience Letters 219 (1996) 6 0 - 6 4
after 18 days of CNTF treatment significant nerve terminal outgrowth was induced. To avoid possible bias in measured data resulting from limited muscle penetration of CNTF, only motor endplates located in the most superficial layers of muscle were examined. Endplate morphology showed both terminal and nodal sprouts, the latter emerging from the node of Ranvier closest to the motor endplates. Although CNTF may have systemic effects stimulating motor nerve sprouting when injected in large doses [17], the doses used here did not induce detectable sprouting in the motor endplates from contralateral muscles. CNTF treatment is also associated with an increase of CGRP-LI in motor endplates, as evidenced by the presence of a higher proportion of endplates showing positive CGRP-LI when compared with contralateral and normal (vehicle treated) muscles. In control muscles only around 10% of the motor endplates showed positive CGRP-LI, whereas in CNTF injected muscles this value increased 5.5-fold. In randomly selected endplates, the morphology of nerve terminals and the intensity of fluorescence representing the CGRP-content were evaluated in high resolution images obtained with a confocal microscope (Fig. 1B and 2). CGRP-LI was located inside motor axons and their presynaptic terminal branches. In the latter, positive CGRP-LI homogeneously filled the whole presynaptic compartment, being accompanied by fine bright punctated granules, which presumably correspond to the large synaptic vesicles in which CGRP is accumulated [19]. The content of CGRP per motor endplate was notably higher after CNTF treatment representing a 4-fold increase in the intensity of fluorescence measured inside nerve terminals; in which there were also more prominent CGRP-LI positive granules (Fig. 1B and 2b,d,e). In some neuromuscular synapses from CNTF treated muscles, terminal sprouts with positive CGRP-LI were seen escaping from the endplate postsynaptic region delimited by fluorescence Bgtx binding. (Fig. 2b). All these results suggest that motoneuronal CGRP changes in parallel with motor endplate remodeling. Previous reports have demonstrated that motoneural CGRP is upregulated in a variety of situations that share as a common feature a reactive response of motor axons to either nerve injury or muscle paralysis [4,22,24,25]. In these cases, the response leads to a functional restoration of the neuromuscular system implying endplate regeneration and remodeling. In the present paper we demonstrate that CGRP is also increased when motor nerve sprouting is induced by exogenous growth factor administration in the absence of muscle paralysis, which suggests a more direct correlation between CGRP and motor terminal outgrowth. The low levels of CGRP present in normal muscles might be related to the subtle plastic changes that take place in adult motor nerve terminals. In fact, neuromuscular synapse remodeling has been shown to occur in normal muscles [13,18]. These changes optimally adjust the size
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and extension of nerve terminals to the functional requirements for efficient synaptic transmission, in accordance with muscle contractile activity, fiber properties and volume [3]. Reorganization of motor nerve terminal architecture should be coupled with corresponding changes in the assembly of postsynaptic structures. Since it seems that nerve terminal outgrowth precedes the differentiation of its postsynaptic counterpart (i.e. accumulation of acetylcholinesterase and acetylcholine receptors [2,29], signals derived from growing axons need to be present to induce the corresponding changes at the muscle surface. According to our experimental data and current knowledge about CGRP, this peptide fits with the expected properties of the above mentioned axonal-derived signal. In turn, muscle-derived signals should regulate, in an activity-dependent way, the stability of presynaptic nerve terminals [5,6,10,15]. As has been suggested by Sala et al. [22], CGRP levels may reflect the resulting balance of exchanging signals between motor nerve terminals and muscle, and additionally regulate the plastic status of a given motor endplate. Because CNTF is not normally present in the muscle [23], its effects on CGRP regulation must be accomplished in a physiological situation by other muscle-derived trophic factors such as IGF and NT-4 [5,6,10,15]. More studies are needed to address the effects of these factors when administered in vivo on CGRP regulation. In any case the response obtained after CNTF administration imitates that induced by musclederived growth factors. We would like to thank Dr. Joan Ribera for constructive comments, Anna Naco for excellent technical assistance, and Malcolm Hayes for reading the manuscript. This work was supported by a grant from Ministerio de Educaci6n y Ciencia (DGICYT, P B 9 3 0642) and a grant from Ajuntament de Lleida. [1] Arvidsson, U., Johnson, H., Piehl, F., Cullheim, S., HOkfelt, T., Risling, M., Terenius, L. and Ulfhake, B., Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons, Exp. Brain Res., 79 (1990) 212-216. [2] Angaut-Petit, D., Molg6, J., Cornelia, J.X., Faille, L. and Tabti, N., Terminal sprouting in mouse neuromuscular junctions poisoned with botulinum type A toxin: morphological and electrophysiological features, Neuroscience, 37 (1990) 799-808. [3] Balice-Gordon, R.J., Breedlove, S.M., Bernstein, S. and Litchman, J.W., Neuromuscular junctions shrink and expand as muscle fiber size is manipulated: in vivo observations in the androgen-sensitive bulbocavernosus muscle of mice, J. Neurosci., 10 (1990) 26602671. [4] Calder6, J., Casanovas, A., Sorribas, A. and Esquerda, J.E., Calcitonin gene-related peptide in rat spinal cord motoneurons: subcellular distribution and changes induced by axotomy, Neuroscience, 48 (1992) 449-461. [5] Caroni, P. and Grandes, P., Nerve sprouting in innervate adult skeletal muscle induced by exposure to elevated levels of insuline growth factors, J. Cell Biol., 110 (1990) 1307 1317. [6] Caroni, P., Schenider, C., Kiefer, M.C. and Zapf, J., Role of muscle
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