Motor neurotrophic factor in denervated adult skeletal muscle

J.R. Slack; S. Pockett

Motor neurotrophic factor in denervated adult skeletal muscle

Susan Pockett

1982, Brain Research

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This study explores the presence of a motor neurotrophic factor in denervated adult skeletal muscle that may facilitate the survival of embryonic motor neurons. The research compares the effects of extracts from denervated muscle versus innervated muscle on neuron survival in vitro, using horseradish peroxidase labeling to identify motor neurons. Results indicate that denervated muscle extracts significantly improve neuron survival compared to those from innervated muscle, suggesting the existence of a soluble factor associated with denervated muscle that promotes motor neuron viability.

Brain Research, 247 (1982) 138-140 Elsevier Biomedical Press 138 Motor neurotrophic factor in denervated adult skeletal muscle J. R. SLACK and S. POCKETT Department of Physiology, University of Auckland School of Medicine, Auckland (New Zealand) (Accepted May 1lth, 1982) Key words: neurotrophic factor - - denervated skeletal muscle - - nerve terminal sprouting The presence in a muscle of denervated muscle fibers causes intact nerve terminals to sprout 4. A number of studies have provided indirect evidence that this sprouting is caused by a humoral factor released from inactive muscle fibers 4. However, the effective diffusional range of the sprouting signal within a muscle is only of the order of one muscle fiber diameter or less 3& Thus a viable alternative possibility for the sprouting mechanism is that surface changes on the denervated muscle fibers somehow permit sprouting of intact nerve terminals on adjacent innervated fibers a. If a soluble motor nerve sprout factor does exist, it would be expected by analogy with N G F also to facilitate survival of embryonic motor neurons (as N G F both facilitates survival of embryonic cells 10 and causes sprouting of adult 2 adrenergic neurons). We report here preliminary evidence that denervated adult rat muscle (where sprouting occurs) does contain a factor that permits survival of embryonic motor neurons. Innervated adult muscle (where sprouting does not occur) does not contain such a factor. We used a bioassay for motor neurotrophic factor, similar to that reported by Bennett et a13. A brief description of the method is given here. Under sterile condition 5/zl of a 30 ~ solution of horseradish peroxidase (HRP, Sigma type VI) was injected into the right thigh muscles of 6-7 day chick embryos in ovo. A 50/zm diameter glass microsyringe connected to a 25 #1 Hamilton syringe was used. The right lumbo-sacral hemi cords were removed from embryos which survived for 5 h after injection. Care was taken to remove dorsal root ganglia and meninges. Hemi cords from between 1 and 4 embryos were 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press pooled and finely chopped in Ca-Mgofree Hanks buffered saline solution (CMF-HBSS). Spinal cord cells including motor neurons retrogradely labeled with H R P were then dissociated by incubation for 30 min at 37 °C in 5 ml of CMF-HBSS containing 0.5 ~ trypsin (Flow Laboratories, Irvine Scotland). Following trypsinization the tissue was centrifuged for 1 min at 400 g. The tissue pellet was suspended in an equal volume of Eagles Basal Medium (BME, Flow Laboratories) containing foetal calf serum (FCS, Flow Laboratories, 8 vol. BME + 1 vol. FCS). Tissue was resuspended in 3 changes of fresh medium. It was then triturated 20 times in 0.6 ml per hemicord. A Pasteur pipette with flamed tip was used for trituration. The cell suspension was carefully added to growth medium in 35 mm plastic tissue culture dishes (Nuncleon), previously coated with rat tail collagen 6. Contents of each dish were as follows: 0.25 ml cell suspension (in 8 vol. BME q- 1 vol. FCS), 1.10 ml medium (8 vol. BME q- 1 vol. FCS containing 2 mM glutamine and 1000 U per ml penicillin), and 0.15 ml supplement. The supplement consisted of either HBSS, 50 ~ chick embryo extract (CEE) 7, or soluble extracts from skeletal muscle, prepared as described below. A single labeled hemicord thus provided sufficient cells for two dishes. Cells were incubated in a saturated atmosphere of 5 ~ CO2 in air at 37 °C. To reveal dissociated motor neurons the contents of each dish were carefully fixed for 30 min in 2.5 glutaraldehyde in 0.2 M cacodylate buffer. After careful washing in 0.2 M cacodylate buffer, cells were reacted for H R P by the amino-ethylcarbamide method 1. Brick red HRP-positive cells were counted 139 immediately after mounting in glycerol. At least 100 fields in each dish, chosen in the same systematic order for all dishes, were scored for HRP-containing cells. Overall 400 × bright field magnification was used. Only large ( > 10 #m) labeled cells were counted. We confirm the findings of Bennett et al. 1 that after two days in culture, dissociated HRP-prelabeled cells survive under appropriate conditions and retain sufficient H R P to enable them to be visualized. The morphology of our cells was similar to those of Bennett et al. 1. The reaction product cannot be generated by endogenous peroxidases (except red blood cells) for no product was observed in the absence of injected HRP. The HRP-positive cells seen in culture cannot be dorsal root-ganglion cells as these were removed prior to cell dissociation. HRPpositive cells must therefore be motor neurons, the axons of which had projected to the site of H R P injection. Under our conditions, 913 4- 22 (mean 4S.E.M.) HRP-positive cells were present per dish 3 h after plating-out, irrespective of whether HBSS or CEE were used to supplement the growth medium. After incubation for two days, supplemented with HBSS, however, only 20 ~ of this number were present (188 4- 10). In cultures supplemented with CEE the number of HRP-positive cells per dish present after two days (819 4- 122) was not significantly different (P > 0.25) from that found 3 h after plating-out. This suggests that the amount of motor neurotrophic factor present in CEE was sufficient to promote complete survival of HRP-labeled motor neurons over two days in culture. This is in accord with the findings of Bennett et al. 1 who also found that myoblast conditioned medium (but not embryonic kidney or embryonic smooth muscle conditioned media) supported survival of most HRP-positive cells for up to two days. Soluble extract from embryonic rat skeletal muscle similarly promoted survivaP. We set out to find whether extracts from denerrated adult muscle would promote survival of HRPlabeled neurons in these culture conditions. Since evidence from partial denervation studies s suggests that 6 days after denervation more sprout factor is released from the endplate than from the extrajunctional regions of denervated muscle fibers, we made 1200¢- :5 looo¸ (1. 4800- o 600¢- 0 400- 200" 0 A B C D Fig. 1. Numbers of HRP-positive neurons per dish surviving for two days in culture medium supplemented in the following way: bar A, medium supplemented with chick embryo extract; bar B, medium supplemented with Hanks Buffered Saline; bar C, media supplemented with soluble extract from endplate containing segments of denervated muscle; bar D, media supplemented with soluble extract from non-endplate segments of innervated muscle. Result from 12 dishes containing cells from 6 embryos isolated in 4 experiments. Error bars represent :t= S.E.M. Bars A and B are significantly different (P < 0.005) as are bars C and D. extracts from the endplate region of denervated muscle fibers. Control extracts were made from the extrajunctional regions of innervated muscle fibers (since innervated endplates may release small quantities of neurotrophic factor). Six- to eight-week-old white mice (Bantin and Kingman, Hull) were used. Endplate zones from soleus muscles denervated 6 days previously by resecting the sciatic nerve in the thigh or non-endplate segments of normal innervated soleus muscles were excised under sterile conditions. Segments from 6 denervated and from 6 innervated muscles were pooled in one ml of HBSS (approximately 60 mg muscle wet weight per ml). All extracts were homogenized for 45 s in an Ultraturrax homogenizer set at 75 ~ maximum speed under sterile conditions. Extracts were then centrifuged at 2,950 g for 15 min. The clear supernatants were pipetted-off and stored frozen. Fig. 1 shows that extracts from endplate-containing segments of adult denervated muscle do contain atrophic factor which supports survival of embryonic chick motor neurons in vitro. Non-endplate containing segments of innervated muscle contain little or none of such a factor. The difference in content of motor neurotro- 140 phic factor is unlikely to be due to variation o f the quantity o f muscle tissue used for making each extract since extracts o f endplate containing segments invariably contained 50 ~ less muscle tissue per ml than non-endplate extracts. I n conclusion, we have shown that the non-endplate regions o f skeletal muscle contain little if any m o t o r neurotrophic factor. Denervated, endplate containing segments, however, do contain this factor. By analogy with the influence o f N G F , which produces sprouting o f sensitive neurons in vivo, this m o t o r neurotrophic factor f r o m denervated muscle 1 Bennett, M. R., Lai, K. and Nurcombe, V., Identification of embryonic motoneurons in vitro: their survival is dependent on skeletal muscle, Brain Research, 190 (1980) 537-542. 2 Bjerre, B., Bjorklund, A., Moberly, W. and Rosengren, E., Short- and long-term effects of nerve growth factor on the sympathetic nervous system in the adult rat, Brain Research, 94 (1975) 263-277. 3 Brown, M. C., Holland, R. L., Hopkins, W. G. and Keynes, R. I., An assessment of the spread of the signal for terminal sprouting within and between muscles, Brain Research, 210 (1980) 145-151. 4 Brown, M. C., Holland, R. L. and Hopkins, W. G., Motor nerve sprouting, Ann. Rev. Neurosci., 4 (1981) 17-42. 5 Hill, M. A., Stratford, J. D., Nurcome, V. and Bennett, m a y be the humoral sprout factor, the existence o f which is suggested by partial denervation studies. J.R.S. gratefully acknowledges facilities and assistance offered by the Muscular D y s t r o p h y Research Laboratories at Newcastle-Upon-Tyne General Hospital, U.K., where part o f this w o r k was undertaken. This w o r k was supported by a project grant f r o m the New Zealand Medical Research Council. S.P. is the Isaacs Fellow o f the A u c k l a n d Medical Research Foundation. M. R., Partial purification of a factor from skeletal muscle that maintains embryonic motor neurons, Proc. Aust. Physiol. Pharmacol. Soc., 12 (1981) 160P. 6 Masurovsky, E. B. and Peterson, E. R., Photo-reconstituted collagen gel for tissue culture substrates, Exp. Cell Res., 76 (1973) 447-448. 7 Paul, J., Cell and Tissue Culture, Churchill Livingstone, Edinburgh, 1975, 83 pp. 8 Pockett, S. and Slack, J. R., Source of the stimulus for nerve terminal sprouting in partially denervated muscle, Neuroscience, in press. 9 Slack, J. R. and Pockett, S., Terminal sprouting of motoneurones is a local response to a local stimulus, Brain Research, 217 (1981) 368-374. 10 Thoenen, H. and Barde, Y.-A., Physiology of nerve growth factor, PhysioL Rev., 60 (1980) 1284-1335.

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