Stabilizing neuromuscular contacts reduces motoneuron death caused by paralysis of muscles in neonatal rats.
Transient paralysis of the soleus muscle in neonatal rats leads to permanent muscle weakness, loss of muscle fibres and motoneuron death. Application of leupeptin, an inhibitor of a calcium-activated neutral protease, to the neuromuscular junction is known to enhance the maintenance of neuromuscular contacts during development and axonal sprouting. Here, we show that treatment of soleus muscles with leupeptin as they recover from a period of paralysis rescues motoneurons that would otherwise die. The number of motoneurons to the soleus muscle was established by retrograde labelling with horseradish peroxidase eight to 10 weeks after recovery from paralysis. There were only 38.4 (+/-2.8 S.E.M., n=5) motoneurons innervating the soleus muscle that had been paralysed with alpha-bungarotoxin, compared to 58.2 (+/-3.1 S.E.M., n=5) to the control untreated soleus. Thus, the number of motoneurons to the soleus muscle on the alpha-bungarotoxin-treated side was 66.9% (+/-6.2% S.E.M., n=5) of the control side. In those animals where paralysis of the soleus muscle was followed three days later by treatment with leupeptin, the number of labelled motoneurons on the treated side of the spinal cord was 61.5 (+/-4.6 S.E.M., n=4) and that on the contralateral untreated control side was 59 (+/-3.8 S.E.M., n=4). This improvement in motoneuron survival in the leupeptin-treated animals is also confirmed by counts of the number of motor units in the soleus muscle obtained by recording muscle tension. In animals that had their soleus muscles paralysed at birth, only 21 (+/-0.7 S.E.M., n=5) motor units were present, compared to 30 motor units in control muscles. When the paralysed soleus muscle was subsequently treated with leupeptin, the number of remaining motor units in the muscle was 29.8 (+/- 1.0 S.E.M., n=5). In addition, the force output of the soleus muscles that had undergone a period of neonatal paralysis was calculated for both the NaCl- and leupeptin-treated animals. The results showed that paralysis at birth results in a reduction in weight and force output of the soleus muscle, which is not improved following treatment with leupeptin. This study shows that application of leupeptin to the soleus muscle after alpha-bungarotoxin-induced paralysis rescues motoneurons to the soleus that would otherwise die. This effect is most likely due to stabilization of their neuromuscularjunctions.
Harding
D I
DI
Department of Anatomy and Developmental Biology, The Centre for Neuroscience, University College London, UK.
Greensmith
L
L
Vrbová
G
G
D000818:Animals; D000831:Animals, Newborn; D002038:Bungarotoxins / Q000633:toxicity; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D002452:Cell Count / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018680:Cholinergic Antagonists / Q000633:toxicity; D007537:Isometric Contraction / Q000187:drug effects; D007976:Leupeptins / Q000494:pharmacology / Q000627:therapeutic use*; D009046:Motor Neurons / Q000473:pathology*; D018482:Muscle, Skeletal / Q000294:innervation*; D009469:Neuromuscular Junction / Q000187:drug effects* / Q000648:ultrastructure; D009498:Neurotoxins / Q000633:toxicity; D010243:Paralysis / Q000139:chemically induced / Q000188:drug therapy / Q000503:physiopathology / Q000517:prevention & control*; D011480:Protease Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011950:Receptors, Cholinergic / Q000187:drug effects
D002038:Bungarotoxins; D018680:Cholinergic Antagonists; D007976:Leupeptins; D009498:Neurotoxins; D011480:Protease Inhibitors; D011950:Receptors, Cholinergic; D002154:Calpain; C032854:leupeptin