Inactivity-induced motor nerve terminal sprouting in amphibian skeletal muscles chronically blocked by alpha-bungarotoxin.

There is a positive correlation between contractile inactivity and the initiation of motor neuron sprouting. However, the exact mechanism responsible for this neuronal growth remains obscure. In a previous study (M. M. Wines and M.S. Letinsky, 1988, J. Neurosci. 8: 3909-3919) we investigated this phenomenon by inducing chronic contractile inactivity of an amphibian muscle by exposure to formamide and found that motor neuron sprouting occurs in the presence of normal pressynaptic transmitter release and propagated muscle fiber action potentials. The present study investigates motor neuron sprouting in response to inactivity produced when neuromuscular transmission is blocked by chronic exposure to alpha-bungarotoxin (alpha-BTX). The alpha-BTX-induced muscle paralysis was maintained for 1-63 days by repetitive application of the toxin to the cutaneous pectoris muscle of adult Rana pipiens. During the chronic alpha-BTX treatment end-plate potentials were reduced below threshold, which therefore removed both muscle fiber action potentials and contractile activity. Our findings showed only terminal sprouting. Also, higher sprouting frequencies (up to 100% of the observed terminals) were observed after chronic alpha-BTX treatment, compared to the sprouting response induced by formamide treatment. In view of our earlier formamide results, these observations suggest that the inhibition of the postsynaptic acetylcholine response, and consequently inhibition of muscle fiber electrical and contractile activity, produces a stronger stimulus to motor neuron sprouting than the presence of contractile inactivity alone coupled with normal synaptic transmission and muscle electrical activity.

Motor nerve terminal staining combined with catecholamine histofluorescence or immunocytochemistry.

A number of excellent techniques are available to stain and characterize different types of neurons and nerve terminals. However, because these different techniques are frequently not compatible, their usefulness in determining the relationships between specific axons and neuromuscular junctions is often limited. The goal was to develop specific procedures for simultaneous visualization of different types of unmyelinated axons and motor nerve terminals in the same preparation. First we modified the formaldehyde/glutaraldehyde staining solutions of the aqueous aldehyde fluorescence technique (Faglu) to observe catecholamine containing axons in whole mount amphibian skeletal muscle. The compatibility of this modified staining solution with other histological procedures made it possible to stain both motor nerve terminals with tetrazolium salts and, in the same preparation, to observe unmyelinated axons with aldehyde-induced catecholamine histofluorescence. This same general formaldehyde/glutaraldehyde staining procedure was also used with immunocytochemical techniques to visualize fluorescent antibody stained nerves and motor nerve terminals in the same whole mount preparation.

Partial denervation of amphibian skeletal muscle does not induce neuronal growth at the node of Ranvier.

Following partial denervation of a striated muscle sprouting occurs at the nodes of Ranvier and terminal arborizations of the intact motor neurons. We studied the sprouting response of intact frog (Rana pipiens) motor neurons to partial denervation of the cutaneous pectoris muscle (CP). In the 5-19 days following partial denervation of the CP muscle, tetranitroblue tetrazolium-stained motor neurons exhibited sprouting. However, the mixed response of both nodal and terminal sprouting characteristic to mammalian muscle was replaced by a preferential response of only terminal sprouting. Histological examination showed that in many cases these terminal sprouts appeared to reinnervate abandoned junctional sites on adjacent denervated fibers. Given that nodal sprouting failed to occur for the duration of the experiment, these preliminary results suggest that the cues responsible for nodal sprout formation in the amphibian may differ from those in the mammal.

Critical period for the androgenic block of neuromuscular synapse elimination.

Juvenile androgen treatment during developmental synapse elimination changes the pattern of innervation in the adult levator ani (LA), an androgen-sensitive muscle (Jordan, Letinsky, and Arnold, 1989b). Most notably, such adult muscles contain an unusually high number of muscle fibers that are innervated by two or more axons indicating that these fibers are multiply innervated. Juvenile androgen treatment also increases the adult level of preterminal branching, the number of junctional sites per adult fiber, and the size of adult LA muscle fibers and motoneurons in the spinal nucleus of the bulbocavernosus (SNB). The present study was designed to determine when in development androgen treatment is most effective in maintaining multiple innervation in adulthood and whether there are different critical periods for the different effects of juvenile androgen treatment. Male rats were castrated on 7, 21, or 34 days after birth (roughly corresponding to the beginning, middle, and end of synapse elimination in the LA muscle) and treated daily with testosterone propionate for the next 2 weeks. All rats were sacrificed at 9 weeks and their spinal cords and LA muscles were stained and analyzed. Only during the first treatment period (7-20) did androgen treatment result in increased levels of multiple innervation at 9 weeks. During this period, androgen also increased the number of junctional sites per fiber and the size of SNB somata but did not influence the adult level of preterminal branching or the diameter of adult LA muscle fibers. Androgen treatment during the two later periods increased the level of preterminal branching and the size of LA muscle fibers without influencing the level of multiple innervation.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of alpha-melanocyte-stimulating hormone-like immunoreactivity in the innervation of amphibian skeletal muscle.

Amphibian motor nerve terminals are sensitive to a wide variety of peptides, including alpha-melanocyte-stimulating hormone (alpha-MSH). We determined the presence and distribution of alpha-MSH-like immunoreactivity (alpha-MSHLI) in the innervation of the cutaneous pectoris muscle from bullfrog (Rana catesbeiana) tadpoles and postmetamorphic froglets, and adult frogs (R. catesbeiana and R. pipiens). alpha-MSHLI was found in unmyelinated, noncholinergic axons, in motor axons, and in motor nerve terminals. In motor axons, alpha-MSHLI was predominantly associated with neurofilaments. The distribution of this form of alpha-MSHLI changed during development and seasonally in adult frogs. The possible functional roles of this alpha-MSHLI are discussed.

The role of gonadal hormones in neuromuscular synapse elimination in rats. I. Androgen delays the loss of multiple innervation in the levator ani muscle.

The normal period of synapse elimination in the androgen-sensitive levator ani (LA) muscle occurs between 2 and 4 weeks after birth, well after the period of synapse elimination for most other rat muscles. To evaluate whether gonadal androgen might be involved in the delayed development of single innervation in the LA, we compared the time course of synapse elimination in LA muscles that lacked endogenous gonadal androgen or were exposed to exogenous androgen. Tetranitroblue tetrazolium was used to stain neuromuscular connections. Our results suggest that both endogenous and exogenous androgen delay the normal process of synapse elimination. Removing endogenous androgen resulted in lower levels of multiple innervation in the LA, suggesting that androgen may normally influence synapse elimination. Moreover, androgen treatment prevented much of the normal loss of multiple innervation in the LA. Androgen treatment during the normal period of synapse elimination also increased the diameter of LA muscle fibers, enhanced the development of preterminal branching, and increased the number of junctional sites on some LA fibers. Because androgen did not appear to induce the formation of new synapses through sprouting, we conclude that androgen maintained multiple innervation in the LA by preventing the normal ontogenetic process of synapse elimination.

The role of gonadal hormones in neuromuscular synapse elimination in rats. II. Multiple innervation persists in the adult levator ani muscle after juvenile androgen treatment.

In the previous study (Jordan et al., 1989), we demonstrated that androgen treatment of juvenile male rats inhibits the elimination of synapses in the levator ani (LA) muscle. In the present study, we asked whether synapse elimination would occur once this juvenile androgen treatment ended. Castrated male rats were given androgen during a juvenile treatment period (7-34 d) and were killed 4 or 8 weeks after the end of androgen treatment (at 9 or 13 weeks after birth). The adult pattern of innervation in the LA was assessed (1) anatomically by counting the number of stained motor axons innervating single muscle fibers and (2) electrophysiologically by counting the number of components in intracellularly recorded endplate potentials. Based on the number of stained motor axons, the LA from juvenile androgen-treated castrates had as much multiple innervation 1 and 2 months after the end of androgen treatment (at 9 and 13 weeks) as was present during androgen treatment at 4 weeks. This suggests that no further synapse loss occurred in the LA once androgen treatment ended. Based on electrophysiological measures, adult LA muscles previously exposed to androgen were found to contain significantly more polyneuronal innervation than normal adult LA muscles. Juvenile androgen treatment also increased the size but not the number of motoneurons in the spinal nucleus of the bulbocavernosus which contains LA motoneurons. Thus, the increased level of multiple innervation in the LA is not due to a higher than normal number of motoneurons innervating this muscle. Because multiple innervation persists in the LA in the absence of continued androgen treatment, androgen may have permanently prevented synapse elimination.(ABSTRACT TRUNCATED AT 250 WORDS)

Motor nerve terminal sprouting in formamide-treated inactive amphibian skeletal muscle.

Motor axons can form sprouts from their terminal arborizations in response to partial denervation, and when exposed to pharmacological blocking agents like TTX, botulinum toxins alpha-bungarotoxin, or curare. Each of these experimental procedures has cessation of muscle contractile activity as a common feature. We tested the specific role of muscle fiber inactivity in regulating nerve terminal sprouting by chronically treating adult frog (Rana pipiens) cutaneous pectoris muscles with formamide. Exposure to formamide, unlike the other compounds used to study sprouting, selectively inhibits muscle contractions without blocking pre- or postsynaptic transmission or muscle fiber action potentials. Repeated formamide applications were used to achieve chronic block of muscle contractile activity in vivo for up to 6 weeks. Motor axons in formamide-treated inactive muscle sprouted only from their terminal arborizations, but not from nodes of Ranvier. The onset of this sprouting was protracted compared with that seen in pharmacologically blocked mammalian muscles, and sprouts in formamide-treated muscles were more complex and ornate. The frequency of sprouting terminals was less in these formamide-treated muscles than that seen after alternate methods of contractile block, and this suggests that contractile inactivity alone serves as only a moderate cue for sprouting. The possibility is discussed that the prolific sprouting seen following neurotoxin administration may, in fact, be due to perturbations in synaptic transmission or muscle electrical activity rather than muscle fiber inactivity.

Synapse elimination occurs late in the hormone-sensitive levator ani muscle of the rat.

Using tetranitroblue tetrazolium (TNBT) to stain neuromuscular synapses, we compared the development of the adult pattern of innervation in two fast-twitch muscles in the rat: the androgen-sensitive levator ani (LA) and the extensor digitorum longus (EDL), which is not thought to be androgen sensitive. We found that about 18% of adult LA muscle fibers, but only about 2% of adult EDL fibers, are multiply innervated. Moreover, synapse elimination occurs substantially later in the LA compared with the EDL. At 2 weeks after birth, the EDL is already predominantly singly innervated, whereas the LA is still predominantly multiply innervated. The apparent delay in the normal time course of synapse elimination in the LA corresponds to a similar delay in other aspects of neuromuscular development (the time course of appearance of axonal retraction bulbs, the growth of fibers, and the development of adult motor terminal morphology). Finally, motor terminals change during synapse elimination from morphologies resembling growth cones to the adult form of neuromuscular synapses. Because the period of synapse elimination is significantly different for muscles that differ in their androgen sensitivity, hormonal sensitivity may represent an important property of motoneurons or muscle fibers influencing the normal time course of neuromuscular synapse elimination in rats. Thus, androgen might regulate the normal ontogenetic process of synapse elimination.

Correlated muscle and nerve development in the bullfrog cutaneous pectoris.

The development of the cutaneous pectoris muscle was studied and compared with the differentiation of its peripheral nerve in bullfrog (Rana catesbeiana) tadpoles and frogs by light and electron microscopic techniques. This muscle preparation was chosen for this study because it possesses a number of advantages for (and has become a model system for) the study of correlated nerve-muscle development. At the earliest stage examined (stage XI) the presumptive muscle did not contain any contractile or morphologically distinguishable myotubes, but was contacted by the well-defined cutaneous pectoris nerve trunk. Myotubes were present at stage XII, the same time that nerve-associated acetylcholine receptor aggregations and nerve-evoked muscle contractions were first observed. The adult number of axons was present in the cutaneous pectoris nerve at stage XII, but no axons were myelinated. Gradually thereafter, the number of muscle fibers increased and the cutaneous pectoris axons became myelinated. By stages XX and XXI, but prior to metamorphic climax (stage XXV), the adult numbers of cutaneous pectoris muscle fibers and myelinated and unmyelinated nerve fibers were present. These numbers did not change significantly between stages XX and XXI, through metamorphosis, and in the adult, even during the period of the most rapid loss of multiple innervation in the first 2 weeks after metamorphosis. These results show that the nerve was present and in contact with the cutaneous pectoris muscle from the earliest stages of development prior to muscle differentiation, at a time when the muscle was a disorganized mass of undifferentiated cells. Such early contact suggests that the nerve may have a significant influence on muscle maturation.

Neuromuscular junction development in the cutaneous pectoris muscle of Rana catesbeiana.

Synaptic specializations were studied in the developing cutaneous pectoris muscle of Rana catesbeiana tadpoles and froglets to correlate nerve terminal morphology (by light and electron microscopy), accumulation of acetylcholine receptors, and the ability of the muscle to contract following nerve stimulation. This correlated approach was used to determine the developmental timing and possible causal relationship of events in nerve and muscle maturation at the neuromuscular junction. Initially, the cutaneous pectoris nerve trunk was present in the undifferentiated presumptive cutaneous pectoris mesenchyme, prior to muscle maturation. At stage XII when the muscle was first able to contract weakly in response to nerve stimulation, the motor nerve terminal endings were simple bulbous enlargements associated with diffuse subneural aggregations of acetylcholine receptors (indicated by diffuse speckles of rhodamine alpha-bungarotoxin fluorescence). Before stage XII no rhodamine alpha-bungarotoxin fluorescence was present anywhere in the muscle. The first stage in the organization of acetylcholine receptors at the neuromuscular junction was the accumulation of diffuse speckles of fluorescence beneath the terminal enlargements. This was followed by the clustering of receptors into small polygonal areas at each synaptic site, and finally the organization of receptors into parallel linear rows. Presumably this final stage was associated with formation of junctional folds. By stage XV the synapses were multiply innervated and had developed acetylcholinesterase activity. The general nerve terminal morphology and pattern of accumulation of acetylcholine receptors at cutaneous pectoris neuromuscular junctions were similar to those of the adult throughout metamorphic climax except that they still contained more than one motor axon. After metamorphic climax, elimination of multiple innervation occurred.

Structure of motor nerve terminals in chickens with hereditary muscular dystrophy.

The structure and size of 1-week to 1-year-old normal (line 412) and dystrophic (line 413) chicken motor nerve terminals were studied using combined pre- and postsynaptic histologic endplate staining. The main result is that adult dystrophic terminals have abnormal structure and are significantly smaller than normal. These differences occurred progressively during development. At 1 week ex ovo, dystrophic motor nerve terminals were similar to normals in size and appearance. By 8 weeks, differences between normal and dystrophic terminal size and structural organization began to emerge. Qualitatively, beginning at 8 weeks and becoming more frequent by 1 year of age (the endpoint of this study), dystrophic motor endplates differed from normal in having: generally smaller synaptic boutons, often separated by extremely thin branching interconnectives; increasing incidence of multiple innervation; and frequent occurrences of apparent partial or total denervation, terminal sprouting, and reinnervation.

Staining normal and experimental motor nerve terminals with tetrazolium salts.

Nitroblue tetrazolium (NBT) has been used to stain motor nerve terminals and unmyelinated axons in vertebrate skeletal muscle, but undesirable background connective tissue coloration resulted. This procedure was improved by separation of the tetrazolium salt's binding from its subsequent reduction. By uncoupling the binding and reduction steps it was possible (1) to improve nerve terminal staining by using tetranitroblue tetrazolium (TNBT), (2) to counterstain and postfix in osmium tetroxide and (3) to enhance the overall tissue preservation. The separate binding and reduction procedure is compatible with postsynaptic acetylcholinesterase staining. Experimentally manipulated and diseased preparations can be successfully stained, and the requirements for optimal staining in each case are described.

A preparation for studying dystrophic avian muscle and neuromuscular junctions.

The extensor of the second digit of the chicken wing (EDII) is a small, fast-twitch muscle susceptible to chicken muscular dystrophy and well suited for correlated studies of the morphology and physiology of identified nerve and muscle fibers. In cross section, the dystrophic EDII shows morphological abnormalities common to other well-studied dystrophic chicken muscles. However, in contrast to other dystrophic chicken muscles, living endplates can be viewed in the EDII, facilitating electrophysiological studies. A survey of electrophysiological properties of the EDII muscle in birds 5-16 weeks ex ovo revealed that compared to normal preparations, the dystrophic preparations had: (1) lower resting potentials, (2) lower miniature endplate potential frequency, (3) abortive nerve-evoked action potentials recorded extrajunctionally, and (4) multiple muscle fiber action potentials to a single depolarizing current pulse.

Histological staining of pre- and postsynaptic components of amphibian neuromuscular junctions.

We have developed a combined staining technique whereby pre- and postsynaptic structures of the neuromuscular junction can be simultaneously visualized in the light microscope. The general approach was first to stain presynaptic nerve terminals using nitroblue tetrazolium (NBT), which when reduced to its diformazan state coloured the entire nerve terminal arborization blue. When the NBT stain was combined with the Karnovsky acetylcholinesterase (AChE) procedure the blue-coloured nerve terminal processes were vividly outlined by the brown copper ferrocyanide-AChE reaction product. Experiments were performed to ensure that the NBT AChE method stained nerve reliably and that even extremely small neural processes were stained and visualized in the light microscope. This method stained cholinergic neuromuscular junctions and unmyelinated axons in a variety of preparations. Aldehyde pre-and poststaining fixation markedly affected the quality of NBT nerve stain. In addition, glutaraldehyde had a direct role in the staining process. The quality of the nerve terminal staining was affected by the pH and the constituents of the staining solution. The powerful experimental advantage of the NBT-AChE stain for neuromuscular junctions resulted from the sharp colour contrast which made possible accurate determinations of the relationship between the presynaptic nerve terminal arborization and the postsynaptic junctional AChE activity.

Structure of developing frog neuromuscular junctions.

Developing neuromuscular junctions in the cutaneous pectoris muscle from tadpoles and and postmetamorphic frogs were studied in the light microscope. Presynaptic nerve terminals and postsynaptic acetylcholinesterase (AChE) activity simultaneously demonstrated at these developing junctions using the NBT-AChE method. The earliest nerve contacts studied were small enlargements at the ends of unmyelinated axons. As development progressed, single nerve contacts, often with growth cones, grew in length, generally parallel to the long axis of the myotube. Further endplate maturation was characterized by individual terminal processes developing secondary and tertiary branches, ultimately leading to highly complex terminal arborizations. The initial synaptic contact at developing neuromuscular junctions was made by a single axon, but with further development these same synaptic sites became multiply innervated. The occurrence of multiple innervation was a transient phenomena; the multiple synaptic inputs were eliminated during further maturation. The time course of synapse elimination was protracted, with some multiple innervation even persisting in relatively large adult frogs. The first nerve contacts were generally devoid of observable endplate AChE activity. Early appearance of AChE activity was sometimes graded and in some cases portions of the nerve terminal processes were associated with AChE while other regions of the same terminal arborization were not.

Competitive interaction between foreign nerves innervating frog skeletal muscle.

1. Competition between two foreign nerves innervating frog skeletal muscle has been studied by using pairs of somatic motor nerves (s.m.n.s) or one s.m.n. and the preganglionic splanchnic nerve (s.p.n.) implanted into a denervated sartorius muscle that has been transplanted to the lymph sac of the back. 2. A single s.m.n. implanted into the muscle succeeded in innervating essentially every fibre within 2--3 months; tetanic stimulation of the nerve elicited 9--100% of the maximal direct tetanus tension. Most of the e.p.p.s were suprathreshold, since a single indirect stimulus evoked a twitch 60--100% as large as that to a direct stimulus. 3. If two s.m.n.s were implanted simultaneously, tetanic stimulation of either elicited 80--100% of the maximal tension to direct stimulation. If one nerve was implanted 2--3 months before the other, the second, although usually less effective than the first, normally innervated 50--100% of the fibres, with approximately the same time course of innervation as a single s.m.n. 4. Mutual synaptic repression was seen on examination of twitch tensions. With either simultaneous or staggered innervation, stimulation of each s.m.n. resulted in a twitch of 30--50% of the total direct twitch tension, with little overlap between the fields driven by the two nerves. Intracellular recordings showed that the distribution of subthreshold and spike-producing e.p.p.s reflected the existence of separate twitch fields. Even if one s.m.n. was implanted several months before the other and had time to establish suprathreshold junctions on most muscle fibres, an s.m.n. implanted later was able to reduce sharply the effectiveness of many junctions from the earlier nerve while itself innervating most muscle fibres. 5. The subthreshold e.p.p.s had low quantal content, typically ten or fewer quanta/e.p.p. The min e.p.p. frequency was very low, while min e.p.p. amplitude appeared to be normal. 6. In the vast majority of muscle fibres, junctions from the two nerves were not within recording distance of each other. Hence, we infer that the competitive interaction was mediated somehow via the muscle fibre. 7. The preganglionic splanchnic nerve, which also successfully reinnervated frog skeletal muscle, competed with a foreign s.m.n. in ways which differ qualitatively from the competition by a second s.m.n. In the presence of a s.m.n., synapses of the s.p.n. were almost universally subthreshold. However, if the s.p.n. was implanted 2--3 months before the s.m.n., the s.m.n. was prevented for several months from innervating fibres driven by the s.p.n. This delay in s.m.n. reinnervation was greater than if the first nerve implanted was also an s.m.n. 8. After 6--8 months of dual innervation by s.m.n. and s.p.n., the s.m.n. became almost totally dominant. However, if the s.m.n. was then sectioned, the s.p.n. became as effective, within approximately 1 week, as it would have been in the absence of the s.m.n.

Precision of reinnervation of original postsynaptic sites in frog muscle after a nerve crush.

Regenerating neuromuscular junctions in the cutaneous pectoris muscle of the frog were examined by light and electron microscopy up to three months after crushing the motor nerve. The aim was to determine the precision of reinnervation of the original synaptic sites. More than 95% of the original postsynaptic membrane is recovered by nerve terminals and little, if any, synaptic contact is made on other portions of the muscle fibre surface. Even after prolonged denervation when the Schwann cells have retracted from 70-80% of the postsynaptic membrane, regenerating terminals return to and cover a large fraction of it. Although synapses are confined to the original synaptic sites, the pattern of innervation of muscle fibres is altered in several ways: (a) regenerating axon terminals can fail to branch leaving small stretches of postsynaptic membrane uncovered; (b) two terminal branches can lie side by side over a stretch of postsynaptic membrane normally occupied by one terminal; and (c) after growing along a stretch of postsynaptic membrane on one muscle fibre, terminals can leave it to end either in extracellular space or on the postsynaptic membrane of another fibre. Altogether the results demonstrate a strong and specific affinity between the original synaptic sites and regenerating nerve terminals.