A simple method for local delivery of various substances to the rat neuromuscular system.

We report here a simple method for the local delivery of various substances to the neuromuscular system in developing and adult rats. This method permits continuous treatment of tissues with a compound over a period of days. Alternative drug delivery systems are unsuitable in neonates. Osmotic pumps are too large and repetitive injections damage the tissues in neonatal rats. Our delivery system provides an adaptable means by which we can directly apply substances in various concentrations in implants of differing sizes. Substances are incorporated into flexible, non-toxic silicone rubber. Strips are cut from the rubber for implantation alongside the muscle or nerve in the anaesthetised animal. The size of the strip is tailored to the length of the muscle or nerve requiring the treatment. Release of the substance from the implant occurs over a period of days and if a longer period of treatment is required, the initial strip can be replaced with a second and even a third implant. We have tested the effects of the substances applied in this manner both physiologically, by examination of muscle function, and morphologically, by muscle histology and retrograde labelling of motoneurones. We have successfully used this method for the application of various groups of substances, including neurotoxins, channel blockers (K+, Ca2+ and Cl-), calcium-chelating agents, protease inhibitors and ionic salts.

The survival of embryonic cardiomyocytes transplanted into damaged host rat myocardium.

In this study, attempts were made to replace damaged myocardium of adult rats with embryonic grafts. To this purpose pieces of embryonic ventricular myocardium were prelabelled with 4',6-diamidino-2-phenylindole and placed into a damaged area of the host myocardium. The hearts containing the grafts were then examined between 2 days and 5-7 months later. Initially the 4'-6-diamindino-2-phenylindole labelled cells were localized only at the site of grafting, but by 2-5 weeks they migrated along the ventricular surface of the heart. Nevertheless the greatest density of grafted cells was always found in the damaged area. At all time points studied, the myogenic phenotype of the 4'-6-diamindino-2-phenylindole-labelled cells was maintained, as the cells contained myosin heavy chains. In addition, immunolabelling with antibodies against cardiac gap junction proteins revealed that initially gap junctions were scattered within the transplanted tissue but with time they became more organised, firstly by alignment into rows along the developing myofibres and then into structures that resembled intercalated discs. Thus the grafted embryonic cardiac myocytes survived in an adult host myocardium and expressed characteristics typical of heart cells.

A simple method for cardiac surgery in rats.

In our laboratory we have developed a relatively simple method for cardiac surgery in rats. The operation is carried out through a small incision in the chest wall using inexpensive equipment. This method allows for the delivery of tissue fragments and cells from a donor rat to an intact or damaged area of ventricular myocardium of a host rat, with easy subsequent localisation of the transplanted/grafted tissue. The rats recover well after the surgery and survive for long periods of time. The technique could also be used for the direct injection of chemicals or molecular probes into the heart. In our experiments we have found that embryonic rat cardiomyocytes that have been transplanted into adult host rat ventricular myocardium using this method survive and develop characteristics typical of heart muscle, thus indicating that using this technique the host heart offers a favourable environment for the transplanted embryonic heart cells.

Stabilizing neuromuscular contacts increases motoneuron survival after neonatal nerve injury in rats.

Following sciatic nerve crush at birth the rat soleus muscle is rendered permanently weak. This reduction in muscle force is caused by the loss of a proportion of its motoneurons. Furthermore, motoneurons that survive and reach the muscle fail to reoccupy a sufficient number of denervated muscle fibres to compensate for the loss of neurons. Both the loss of motoneurons and poor reinnervation may be due to the inability of the regenerating axons to establish and maintain neuromuscular contacts. Application of leupeptin, an inhibitor of a calcium-activated neutral protease and some serine proteases, is known to help in the maintenance of neuromuscular contacts during development and axonal sprouting. Here we examined whether protecting new neuromuscular contacts formed between regenerating axons and denervated muscle fibres after nerve injury, would influence the survival of motoneurons and improve muscle recovery. This study shows that in muscles treated with leupeptin the reduction in weight and force output after nerve crush at birth was significantly less than in those that were untreated. Moreover, the number of motor units in the leupeptin-treated muscles was significantly higher than in untreated muscles. Thus, treating regenerating nerve terminals with leupeptin during early stages of reinnervation rescues motoneurons and improves muscle recovery.

Survival of embryonic cardiac myocytes transplanted into host rat soleus muscle.

Small fragments of embryonic hearts were transplanted either alongside or into a skeletal muscle (soleus) of an adult host. The implanted tissue grew, and survived for at least 6 months after transplantation. The graft was well vascularized and established a network of blood vessels that communicated with the host's circulation. This communication appeared to be established by the proliferation of blood vessels from the graft into the host tissues. The grafted tissue was rhythmically active and the rate of these contractions was similar to that of adult rat hearts. The frequency of the spontaneous contractions could be modified by acetylcholine. Exposure to acetylcholine lead to a reversible slowing of the rate of beating. The presence of gap junctions in the transplanted tissue was revealed by visualizing connexin 43 with a specific antibody. During early periods after grafting the gap junctions were scattered within the graft but over time they became aligned into rows, to prepare for the formation of intercalated discs. Thus embryonic heart grafted into, or alongside skeletal muscle is able to acquire a considerable degree of differentiation.

Neuromuscular contacts of expanded motor units in rat soleus muscles are rescued by leupeptin.

In the soleus muscle of the rat following section of the L5 ventral ramus (partial denervation) the remaining motor axons increase their territory by sprouting. Nerve sprouts are first seen two to three days after the operation, their number peaks at 10-14 days and subsequently remains at this level. The time course of the initial sprouting in partially denervated muscles is not altered by paralysing the muscles with alpha-bungarotoxin, and the initial extent of the sprouting is, if anything, greater in the paralysed muscles. However, unlike in controls, this level of sprouting is not maintained and neuromuscular contacts are lost when muscles recover from the paralysis. The loss of these contacts can be prevented by treatment of these partially denervated paralysed muscles with leupeptin, an inhibitor of calcium-activated neutral protease. Interestingly, more contacts are rescued when leupeptin is applied 10 days after alpha-bungarotoxin treatment, when sprouting has reached high levels, than at three days, when sprouting has just begun. The neuromuscular connections rescued by leupeptin are functional. Maximum tetanic tension produced by untreated soleus muscles two to five months after partial denervation is 66 +/- 9% of contralateral control muscles, but only 39 +/- 8% when the muscles were paralysed with alpha-bungarotoxin for 12-14 days after partial denervation. However, when partially denervated paralysed muscles were treated with leupeptin three and 10 days after alpha-bungarotoxin treatment their tension output is 74 +/- 3% and 81 +/- 8%, respectively. After partial denervation alone, motor units are twice their normal size. Short-term paralysis with alpha-bungarotoxin prevents this increase in motor unit territory. However, the application of leupeptin to the paralysed muscles rescues neuromuscular contacts, allowing motor unit size to remain expanded, at around 2-2.5-fold. Thus, following recovery from temporary paralysis with alpha-bungarotoxin, there is a sudden withdrawal of neuromuscular contacts and these can be rescued by treatment with leupeptin.

Inactivity induces muscle hypertrophy and redistribution of myosin isozymes in chicken anterior latissimus dorsi muscle.

Denervation of the anterior latissimus dorsi (ALD) muscle causes transient muscle fibre hypertrophy and leads to an increase of the SM1 myosin isoform. We tested whether the changes that take place after denervation can be attributed to loss of muscle activity which follows denervation. Neuromuscular activity was prevented by blocking the acetylcholine receptors with alpha-bungarotoxin and thereby paralysing the muscle. Following this treatment, we found increased muscle weight and pronounced hypertrophy of muscle fibres. Also, the proportion of SM1 isomyosin was decreased. Due to the multiple innervation of ALD muscle fibres it is possible to paralyze only part of the muscle. When only a region of the muscle was paralysed a local hypertrophy of fibres was detected, and the change from SM1 to SM2 was most pronounced in the area where activity was blocked. Removal of muscle activity resulted in changes similar to those that occurred after denervation.

Response of developing rat fast muscles to partial denervation.

The changes of motor unit size following partial denervation of the extensor digitorum longus muscles in rat neonates (at five to six days) and later in development (at 18-20 days) were studied. Extensor digitorum longus muscle is innervated mainly by axons from L4 ventral ramus and to a lesser extent by axons from L5 ventral ramus. In neonates the motor units in extensor digitorum longus are large, and they become restricted to their adult size during the first two weeks of life. Six to 10 weeks after removing the major input to extensor digitorum longus, i.e. L4 ventral ramus at five to six days, the motor unit sizes of axons in the remaining L5 ventral ramus decrease from their expanded neonatal territory to their adult smaller size. In spite of partial denervation the motor units remain small throughout the animal's life and the denervated muscle fibres do not become "occupied" by sprouts from the remaining axons of L5 ventral ramus motor nerves. Partial denervation of extensor digitorum longus muscles at 18-20 days by section of the L4 ventral ramus leads to the expected two- to three-fold increase in the size of motor units of L5 ventral ramus. These results are taken to show that fast motor units of neonatal rats are unable to maintain their enlarged peripheral field, while later in development their axons can sprout and occupy an expanded peripheral field.

Temporary loss of activity prevents the increase of motor unit size in partially denervated rat soleus muscles.

1. The effects of temporary short-term paralysis on changes of motor unit size and amount of sprouting after partial denervation of the rat soleus were studied. 2. Two to ten months after section of the L5 ventral ramus combined with subsequent treatment with alpha-bungarotoxin (BTX) the tension developed by the operated muscle was 39 +/- 8% (S.E.M., n = 8) of control unoperated soleus muscles. This is much less than the tension produced by partially denervated, NaCl-treated or untreated muscles which was 66 +/- 9% (S.E.M., n = 5) and 66 +/- 12% (S.E.M., n = 5) respectively. The smaller tension developed by the BTX-treated muscles was due to the relatively small size of their motor units. 3. The mean increase of tension output of individual motor units after partial denervation and treatment with NaCl when compared with controls was 194 +/- 15% (S.E.M., n = 4) while in the paralysed muscle this value was only 118 +/- 15% (S.E.M., n = 8). This reduced expansion of motor unit size in the BTX-treated soleus was not caused by a decrease of the size of the muscle fibres. Thus a brief temporary paralysis prevents the expansion of motor unit territory that normally occurs in partially denervated muscles. 4. Examination of the innervation pattern and incidence of sprouting revealed that in partially denervated NaCl-treated muscles 24 +/- 4% (S.E.M., n = 4) of endplates was contacted by either terminal or collateral sprouts, whereas in the BTX-treated muscles only 5 +/- 2% (S.E.M., n = 3) of endplates had been contacted by sprouts. Treatment with BTX alone, without partial denervation, did not affect the tension output of the muscles, but caused 8 +/- 5% (S.E.M., n = 3) of the endplates to become innervated by collateral sprouts, as compared to only 3 +/- 2% (S.E.M., n = 3) in controls.

The effect of muscle activity on motor unit size in partially denervated rat soleus muscles.

It has been shown previously that after section of L5 ventral ramus at 5 days the intact axons of L4 ventral ramus retain their large neonatal peripheral field in the rat soleus muscles. Soleus muscles of 5-day-old rats were partially denervated by section of their major neural input, L5 ventral ramus, and in addition paralysed with alpha-bungarotoxin for 3-5 days. The motor unit size was examined 2 months later. The tension developed by individual motor units from muscles that were partially denervated and in addition temporarily paralysed was much less than that after partial denervation alone. This reduced tension output was not due to muscle atrophy but to a smaller number of muscle fibres supplied by individual axons. Thus, unlike after partial denervation only, motoneurons were unable to maintain their large neonatal territory when the muscle was temporarily paralysed and they were unable to reoccupy this territory after the muscles recovered from the paralysis. The possibility that arrested muscle maturation due to paralysis has a permanent effect on motor unit size is discussed.

The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins.

We describe here the levator auris longus muscle of the mouse as a convenient neuromuscular preparation for the in vitro study of presynaptic effects of drugs and toxins applied in vivo in young or adult mice. The good visibility of its motor axons and terminals using Nomarski optics allows accurate electrophysiological studies of presynaptic signals. In addition, the levator auris longus muscle is sufficiently thin to be stained as a whole mount preparation. Preliminary results indicate that some correlation can be established between changes in time course of the presynaptic signal and the morphology of motor endings after poisoning the levator auris longus muscle with botulinum type A toxin.

Effect of low calcium and protease inhibitors on synapse elimination during postnatal development in the rat soleus muscle.

The mechanisms controlling the reorganisation of synaptic inputs to developing skeletal muscle fibres was studied using electrophysiological and histological methods. In the developing rat soleus muscle there is a rapid reduction of polyneuronal innervation between 9 and 12 days. Reducing the local concentration of calcium by applying chelating agents such as EGTA or BAPTA in vivo to 9-day-old rat soleus muscles over a period of 3 days slowed the rate of elimination of polyneuronal innervation. It was established that the reduction of calcium induced by EGTA or BAPTA was not sufficient to produce a detectable reduction in neuromuscular activity. The possibility that a calcium-dependent enzyme such as CANP may play a role in synapse reorganisation was therefore tested. Local application of inhibitors of calcium-activated neutral protease (CANP), leupeptin or E-64, to 9-day-old rat soleus muscles over 3 days had similar effects to those of EGTA or BAPTA, i.e. the elimination of polyneuronal innervation that usually takes place was much slower. Since the inhibition of thiol proteases had similar effects on synapse elimination as a reduction of calcium concentration, it is concluded that CANP is important in the reorganisation of the developing neuromuscular junction.