Albert A. Herrera

Glial Cells Maintain Synaptic Structure and Function and Promote Development of the Neuromuscular Junction In Vivo

To investigate the in vivo role of glial cells in synaptic function, maintenance, and development, we have developed an approach to selectively ablate perisynaptic Schwann cells (PSCs), the glial cells at the neuromuscular junction (NMJ), en masse from live frog muscles. In adults, following acute PSC ablation, synaptic structure and function were not altered. However, 1 week after PSC ablation, presynaptic function decreased by approximately half, while postsynaptic function was unchanged. Retraction of nerve terminals increased over 10-fold at PSC-ablated NMJs. Furthermore, nerve-evoked muscle twitch tension was reduced. In tadpoles, repeated in vivo observations revealed that PSC processes lead nerve terminal growth. In the absence of PSCs, growth and addition of synapses was dramatically reduced, and existing synapses underwent widespread retraction. Our findings provide in vivo evidence that glial cells maintain presynaptic structure and function at adult synapses and are vital for the growth and stability of developing synapses.

Repeated,in vivo observation of frog neuromuscular junctions: remodelling involves concurrent growth and retraction

SummaryThe fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide was used as a vital stain to study remodelling of motor nerve terminals in sartorius muscles of living frogs (Rana pipiens). Identified terminals were observed twicein vivo at intervals of 87–192 days. After the second observation, muscles were fixed and stained with the nitroblue tetrazolium method for nerve terminals and with cholinesterase stain. Observations were made of 243 junctions in 26 frogs.Most nerve terminals grew during the observation interval, with an average increase in total terminal length of 29%. This growth involved substantial remodelling. Within single junctions, the change in size was the net result of differing degrees of growth or shrinkage in individual nerve terminal branches. At least one new terminal branch appeared in 25% of the junctions. Terminal retraction was also common, with branch shortening seen in 60% of junctions and the complete disappearance of a branch in 12%. In one case the original axonal input retracted completely and the junction was partially reinnervated by a terminal sprout from a junction on an adjacent fibre. Some discrepancies between histological andin vivo observations of remodelling were noted. These observations confirm that frog neuromuscular junctions are highly dynamic synapses, subject to profound structural remodelling throughout adult life.

The role of perisynaptic Schwann cells in development of neuromuscular junctions in the frog (xenopus laevis)

Fluorescence microscopy was used to study the behavior of perisynaptic Schwann cells (PSCs) in relation to motor nerve terminals and postsynaptic clusters of acetylcholine receptors, during the development of the neuromuscular junction (NMJ) in the frog Xenopus laevis. Pectoral (supracoracoideus) muscles were labeled with monoclonal antibody 2A12 for Schwann cells, the dye FM4-64 for nerve terminals (NTs), alpha-bungarotoxin for acetylcholine receptors (AChRs), and Hoechst 33258 for cellular nuclei, in animals from tadpole stage 57 to fully grown adults. When muscle fibers first appeared in stage 57, NMJs consisted of tightly apposed NTs and AChRs and were only partially covered with PSCs or their processes. Within a few stages, PSCs fully occupied and overgrew the NMJs, extending fine sprouts between a few micrometers and hundreds of micrometers beyond the borders of the junction. Sprouts of PSCs were most abundant during the time when secondary myogenesis, synaptogenesis, and synaptic growth occurred at their highest rates. PSCs were recruited to NMJs during synaptic growth, at rates between 1.3 PSCs/100 microm junctional length early on and 0.4 PSCs/100 microm later. Shortly after metamorphosis, PSC sprouts disappeared and NMJs acquired the adult appearance, in which PSCs, NTs, and AChRs were mostly congruent. The results suggest that, although PSCs may not be required for initial nerve-muscle contacts, PSCs sprouts lead synaptic growth and play a role in the extension and maturation of developing NMJs.

A comparison of active zone structure in frog neuromuscular junctions from two fast muscles with different synaptic efficacy

SummaryTo search for ultrastructural correlates of differences in synaptic safety factor and neurotransmitter release, neuromuscular junctions from the cutaneous pectoris and cutaneous dorsi muscles of the grass frogRana pipiens were freeze fractured. Synaptic efficacy in these muscles was determined by the extent to which isometric twitch tension could be blocked by lowering [Ca2+] in the bathing solution. We found that junctions in the cutaneous pectoris were significantly more effective than those of the cutaneous dorsi. Morphometric analysis of 16 junctions from each type of muscle showed significant differences in some aspects of active zone structure. Cutaneous pectoris terminals had longer active zone segments and active zones spaced more closely together. This resulted in 20% more active zone length per unit terminal length in the cutaneous pectoris. Cutaneous dorsi terminals had active zones that were more often segmented into two or more sections at a single junctional fold. Mean active zone length per junctional fold and the number of active zone particles per micrometre of active zone length were not significantly different. As a result of the somewhat larger terminal width in the cutaneous dorsi, the percentage of terminal width occupied by active zone was greater in the cutaneous pectoris. As an attempt to indirectly estimate active zone spacing with the light microscope, we applied rhodamine-conjugated alpha bungarotoxin to neuromuscular junctions from the cutaneous pectoris and cutaneous dorsi. No significant difference in the spacing of fluorescently labelled acetylcholine receptor bands was found between the two types of junctions. Our results indicated that the greater active zone length per unit terminal length in the cutaneous pectoris was associated with its increased synaptic efficacy. In addition the continuity and particle organization of active zones may have contributed to the observed differences in synaptic safety factor at frog neuromuscular junctions.

The use and effects of vital fluorescent dyes: observation of motor nerve terminals and satellite cells in living frog muscles

SummarySeveral different fluorescent mitochondrial dyes were tested as vital stains for motor nerve terminals and other cells in frog skeletal muscles. It was found that 3,3′diethyloxadicarbocyanine iodide and 4-(4-diethylaminostyryl)-N-methylpyridinium iodide were most useful. Both dyes labelled motor nerve terminals with high reliability. Electrophysiological and morphological control experiments showed that these dyes could be used to repeatedly observe neuromuscular junctions in living animals without affecting synaptic growth or remodelling. The importance of appropriate controls was emphasized by the finding that illumination, if excessively intense or prolonged, can cause physiological and structural damage to nerve terminals. Additional observations indicated that these dyes may be useful for determining the mitochondrial content, and therefore oxidative capacity, of living muscle fibres. It was also found that the fluorescent dyes labelled cells identified as muscle satellite cells, and that these myoblast precursors could be visualized in fixed whole mounts with a nitroblue tetrazolium stain. Both methods were used to study reactive cells that were closely associated with muscle fibres in lesioned muscles. Mitochondrial dyes also labelled the microvasculature, associated axons and other cells.

Motor axon sprouting in frog sartorius muscles is not altered by contralateral axotomy

SummarySartorius muscles of the frogRana pipiens were used to study the incidence of motor nerve sprouting in normal unoperated muscles, in experimental muscles contralateral to axotomy of the sartorius nerve, and in sham-operated control muscles. Muscles were stained with either a combination of nitroblue tetrazolium nerve terminal stain and cholinesterase stain or with a combination of silver nerve terminal stain and cholinesterase stain. Each endplate that could be clearly seen was classified into one or more of the following categories: normal endplates without sprouts, three types of terminal sprouts, preterminal sprouts, nodal sprouts, sprouts of unknown origin and destination, and doubly innervated gutters. A quantitative study of 318 endplates from nine unoperated muscles, 779 endplates from 45 experimental muscles, and 694 endplates from 41 control muscles showed that all muscles had a high incidence of motor nerve sprouting and other forms of remodelling (20–28% of all endplates). There were, however, no significant differences between experimental, control, and unoperated muscles when results obtained with the same stains were compared. Results obtained with the two different stains were only slightly different. We conclude that sprouting is a very common but highly variable feature of normal frog neuromuscular junctions, and in the sartorius, contralateral axotomy does not alter this ongoing remodelling.

Precision of Reinnervation and Synaptic Remodeling Observed in Neuromuscular Junctions of Living Frogs

Repeated in vivo observations were used to study regenerated nerve terminals in neuromuscular junctions of the adult frog Rana pipiens. Sartorius junctions in living animals were stained with the fluorescent vital dye RH414 and viewed with video fluorescence microscopy. Each junction was observed in the intact muscle and then again 7, 10, and 13 weeks after nerve crush. At 13 weeks, junctions were determined to be mono- or polyneuronally innervated using intracellular recording. Between 7 and 13 weeks, most identified junctions were reinnervated less precisely and completely than described previously. Although some of the original synaptic gutters were reoccupied by regenerated terminal branches, other gutters were only partially occupied, and many appeared abandoned. Junctions showing precise recapitulation of original terminal arborizations comprised a small number of the total examined, as did those where reinnervation was very imprecise. Striking differences in the precision of reinnervation were found within the muscle such that distal terminals regenerated more precisely and completely than did proximal terminals. Terminals in reinnervated muscles were more dynamic than terminals in unoperated muscles over equivalent times. In singly innervated junctions, terminal growth was favored over regression. In doubly innervated junctions, regressive events were more common. Imprecise reinnervation is explained in terms of multisite innervation of muscle fibers and the activity dependence of synaptic stability. We hypothesize that when axons reinnervate the second or third junctions on a fiber, they do so less precisely, because the activity restored by reinnervation of the first junction renders later sites less attractive or less stable.

Hormonal regulation of motor systems: how androgens control amplexus (clasping) in male frogs

In this article we review the literature concerning the hormonal, neural, and muscular basis of amplexus (clasping) in male frogs and toads. We also present our recent results on the effects of testosterone on the clasping musculature of Xenopus laevis. Amplexus, the prolonged embrace of females by males during mating, has been characterized as a spinal reflex that is subject to modification by higher neural centers. Many of the cells involved in this behavior have high levels of androgen receptors and cellular structure and function can be regulated by androgens. We find that testosterone modifies the flexor carpi radialis muscle (FCR) such that it has an enhanced ability to maintain low levels of tension while retaining the ability to contract rapidly. These properties are well matched to the functional demands of clasping. Histology reveals that there are regional differences in testosterone sensitivity within the FCR. Tension recordings show that the more androgen-sensitive region of the muscle is composed of fibers that contract more slowly than other regions. These results reveal some of the ways in which the FCR is seasonally remodelled for the specialized task of clasping.