A method for the demonstration of adrenergic nerve fibres in peripheral nerves

Summary The adrenergic nerve fibres running from the ganglia to the innervated tissues usually have too low a content of noradrenaline to be clearly visualized with the histochemical fluorescence method of Falck and Hillarp. They can easily be demonstrated, however, as early as 24 hours after axotomy (crushing or constriction of the nerves) due to the rapid accumulation of what is probably noradrenaline taking place proximally to the lesion. The fibres can be visualized even more clearly if axotomy is combined with the administration of l-dopa and with monoamine oxidase inhibition. In this way the presence, distribution and direction of adrenergic fibres can be directly studied in peripheral nerves.For generous supplies of drugs we are indepted to Swedish Ciba, Stockholm (reserpine) and Swedish Pfizer, Stockholm (nialamide). The investigation has been supported by research grants from the United States Public Health Service (NB 02854-04), the Swedish Medical Research Council, and Knut and Alice Wallenbergs Foundation.     

Myogenic defect in muscular dystrophy of the chicken.

Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at $\text{3}\text{1}\text{2}$ days of incubation (stage 19–20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves.     

The fine structure of cephalopod blood vessels III. Vessel innervation

Summary The cephalic aorta of Octopus vulgaris has a fairly complete endothelium lining the lumen, a thick complete basement membrane, a layer of circularly orientated and a layer of longitudinally orientated muscle fibres. Presumptive synaptic endings are of two types. In the circular muscle, axons containing vesicles, contact club-shaped projections of the muscle. The gap between the pre- and postsynaptic membranes is less than 100 Å and in some places apparently forms a tight junction. The second type of ending has been found in the longitudinal muscle; here axons full of vesicles end on the muscle. The ending is enclosed by a mesaxon of muscle and the synaptic gap is approximately 100 Å. In the smaller blood vessels, axons end on myofilament-containing pericytes of blood vessels (equivalent to small arterioles). The endings contain vesicles and have a synaptic gap of 100 Å. Only some of the pericytes seem to be innervated and transmission between one pericyte to another may be mediated by specialized junctions between the cells. The smaller non-myofilament containing vessels (equivalent to capillaries) are not thought to be innervated.We would like to thank Professor J. Z. Young and Dr. E. G. Gray for advice and encouragement, Mrs. Jane Astafiev for drawing Figs. 1 and 12, Mr. S. Waterman for photography, and Miss Cheryl Martin for secretarial assistance.     

Waveform generation of the electric organ discharge inGymnotus carapo

Summary The electric organ (EO) ofGymnotus carapo was studied using different neurohistological techniques including conventional electron microscopy. The electric tissue extends along the fish body from the pectoral girdle to the tip of the tail, constituting a single, undivided organ. However, taking into account the number, arrangement, and innervation of the electrocytes, it is possible to divide the EO into three different portions. The more rostral portion is included within the ventral wall of the abdominal cavity. It consists of singly and doubly innervated electrocytes arranged in two rows at each side of the midline. Innervation of this zone is supplied by the first 5–7 segmental nerves and by the anterior electromotor nerves. Segmental nerves terminate on the rostral faces of doubly innervated electrocytes; axons stemming from the anterior electromotor nerves end on the caudal faces of both doubly and singly innervated electrocytes. There is an intermediate body-tail region in which the electrocytes are arranged in four dorsoventral tubes (tubes 1 to 4) on each side of the midline. In this zone, doubly innervated electrocytes (confined within tube 1) coexist together with singly innervated ones, receiving nerve terminals on their caudal faces (tubes 2, 3, and 4). The innervation characteristics appear modified at more distal portions of the tail where the doubly innervated electrocytes of tube 1 are replaced by singly innervated units. The most distal portion of the EO (approximately its terminal 30%) consists of numerous, homogeneously innervated electrocytes with nerve endings distributed exclusively on their caudal faces. Nerve supply to the intermediate and distal regions derives from the posterior electromotor nerves (PENs) which appear as well-defined anatomical entities beyond the level of metamere XXVII. At the bodytail and more distal regions the innervation pattern of the EO is particularly complex. Thin nerve trunks arise from the PENs and project ventrally toward the electrocyte tubes. Before reaching the electric tissue the electromotor axons branch frequently. Our anatomical studies indicate that the EO is heterogeneous, a feature consistent with most recent electrophysiological and biophysical experiments.Abbreviations AEN anterior electromotor nerve - EMN electromotoneurons - EO electric organ - EOD electric organ discharge - LLN lateral line nerve - PEN posterior electromotor nerve     

Effects of testosterone on a sexually dimorphic frog muscle: Repeated in vivo observations and androgen receptor distribution

In the present study the sexually dimorphic, androgen-sensitive flexor carpi radialis muscle (FCR) in male Xenopus laevis was viewed repeatedly in vivo to assess the influence of testosterone on muscle fiber size over a period of up to 12 weeks. Regions of the muscle innervated by different spinal nerves responded differently to testosterone treatment. Muscle fibers innervated by spinal nerve 2 (SN2) hypertrophied within 7 days in frogs that had been castrated and given testosterone-filled implants. This initial hypertrophy was followed by a return to normal fiber size a week late, after which fiber size slowly increased again. In castrated males with empty implants, muscle fibers innervated by SN2 gradually atrophied. Fibers innervated by spinal nerve 3 (SN3) were not affected by androgen replacement or withdrawal. The sartorius, a control muscle that is neither sexually dimorphic nor particularly androgen sensitive, was also unaffected. The in vivo observations were confirmed by measurements of muscle fiber cross-sectional areas in frozen sections of whole forelimbs. At 8 and 12 weeks after castration, cross-sectional areas of fibers innervated by SN2 were significantly larger in frogs provided with testosterone than in castrates without testosterone. No difference was found in the SN2 region or in the anconeus caput scapulare (triceps), another control muscle. Immunocytochemistry employing an antibody against the androgen receptor (AR) indicated that the receptor is present in myonuclei of all muscles of the forelimb. While no difference in labeling intensity was detected, the number of AR-containing nuclei per muscle fiber cross-section was higher in fibers innervated by SN2 than in those innervated by SN3, and was yet lower in the triceps. This suggests that regulation of androgen sensitivity may occur via muscle fiber. ARs, although an influence of the nerve may also contribute. 1994 John Wiley & Sons, Inc.     

Nerve supply and distribution of cholinesterase activity in the external nose of the mole

Summary Nerve supply and the distribution of cholinesterase activity were studied in the skin of the external nose of seven moles using a simplified Bielschowsky-Gross silver method and Koelle's histochemical technique.The sensory units of the mole's nose or the organs of Eimer are surrounded by blood sinuses which facilitate their movements during mechanical stimulation. All nerve fibres of the plexus deep to the basal cell layer of Eimer's organ ultimately become intra-epidermal endings. Contrary to the findings of earlier investigators, Merkel's discs, Pacinian corpuscles and Ruffini corpuscles have not been observed at the base of Eimer's organ. In the superficial layer of the plexus, the Schwann sheath cells increase in number, undergo modification and give a positive cholinesterase reaction.It is suggested that the organ of Eimer, the specialised nerve plexus deep to it and the surrounding blood sinus together constitute the touch receptor on a similar principle of transmission by leverage as in the tactile hair or the intermediate ridge of the papillary ridge.The role of the intra-epidermal nerve endings of the mole's nose as tactile receptors is disputed. A suggestion is made that tnese nerves may constitute pain and temperature receptors and that several modalities of sensation may be carried to the brain along one and the same medullated axon.We gratefully acknowledge the technical assistance of Miss Jill Hocknell. Our thanks are also due to Mr. C. J. Duncan and the staff of the Photography Department for their aid with the photographic work. We are particularly grateful to Mr. D. Burgess of the Ministry of Agriculture and Fisheries for kindly supplying us with live moles. One of the authors (N.C.) acknowledges an equipment grant from the Royal Society.     

Cytochemistry of 5-hydroxytryptamine at the electron microscope level

Summary The nerves of rat pineal gland are known to contain norepinephrine and 5-hydroxytryptamine. With the glutaraldehyde-dichromate reaction for the cytochemical localization of unsubstituted catechol- and indoleamines, dense reactive granules could be demonstrated in such endings. A similar reaction was observed in the adrenergic nerves supplying the vas deferens and storing exclusively norepinephrine. Formaldehyde fixation, prior to the glutaraldehyde-dichromate treatment, interferes with the reaction given by catecholamines not affecting the indolic reactive sites. After this combined procedure pineal nerves still exhibited the dense reactive granules, while these were not found in the nerves of the vas deferens. Following bilateral cervical sympathectomy reactive granules disappeared from the perivascular processes of the pineal gland. No reaction could be observed in the cytoplasm of parenchymal cells neither in their perivascular processes.These cytochemical results suggest that both catecholamines and 5-hydroxytryptamine are contained within the granulated vesicles of pineal nerves.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina and U.S.Air Force (AF-AFOSR 963-66).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. We want to express our gratitude to Prof. E. De Robertis for his constant help and encouragement; and to Miss Nélida Fernández Oranges, Mr. Raúl Castelli and Mr. Alberto Sáenz for their skillful technical assistance.     

The podial pit — a new structure in the echinoidDiadema antillarum Philippi

Summary A glandular pit with an orally directed opening has been revealed at the base of the aboral podia, in the precise region known to be the most photosensitive area of the skin. The pit is associated with a thickening of the superficial nerve felt to form the podial organ. The fine structure is unremarkable aside from the large number of fine nerves housed in spaces between the cells of the lining epithelium. The underlying nerve felt is composed of densely packed varicose axons with little evidence of synapses and no indication of sub-cellular photosensory specialization. It is tentatively suggested that the remarkably superficial location of the fine nerve fibres may be correlated with the relatively high photosensitivity of this region.One of us (N. Millott) is greatly indebted to the Science Research Council for grants providing most of the equipment used. We are also grateful to the Zoological Society of London, especially to Dr.H. G. Vevers. Our thanks are also due to Messrs.R. L. Jones andZ. Podhorodecki for assistance.     

II. Ultrastructure of the type B sense organ of the specific lateral line system of Gymnarchus niloticus

Summary The type B cutaneous receptor represents one of the three kinds of specific organs of the lateral line system of Gymnarchus niloticus (Szabo and Barets, 1963). Electron microscopic observations reveal that the elementary unit (Fig. 18) of the type B organ consists of a well organized assembly of different epithelial elements grouped around each sensory cell. Several such units compose a type B organ innervated by a single myelinated nerve fiber.The cytoplasm of the sensory cell is characterised by a deep invagination lined by long and densely packed microvilli covered by a jelly-like substance. This jelly substance of allongated stylet form is situated in an intraepidermal cavity, overlaid by vacuolised epithelial cells oriented perpendicularly toward the external epidermal surface.Certain morphological characteristics of the organ B allows the conclusion that this organ is one of the possible electroreceptors of the Gymnarchus niloticus.This work was supported by grant No. 659535 of the Direction de Recherches et Moyens d'Essais (D.R.M.E.) to Dr. Szabo.This work was carried out on the electronmicroscope of the Service de Microscopie Electronique du Laboratoire de Médecine Experimentale (RCA) et du Laboratoire d'Histologie Normale et Pathologique du Système Nerveux (Siemens).With the technical assistance of Miss L. Seguin and Mr. C. Pennarun.     

Fine structure of the Paraventricular organ of Xenopus laevis tadpoles

Summary The ultrastructure of the Paraventricular organ in the hypothalamus of Xenopus laevis tadpoles is described. It appeares that the Paraventricular organ of this anuran species is homologous with the Organon vasculosum hypothalami or the Paraventricular organ of other vertebrates.The Paraventricular organ of Xenopus laevis is characterized by an ependymal lining with only few cilia and by two types of nerve cells. Both types of nerve cells have ventricular processes, protruding into the lumen of the third ventricle and forming a network. The protrusions bear cilia of the 8+1 pattern. It has been possible to distinguish both types of nerve cells on account of their dense-core vesicles. A secretory function of both cell types is suggested.In a region close to the Paraventricular organ, another granulated type of nerve cell has been observed. A relationship between these cells and the preoptic nucleus is discussed.The author thanks Prof. Dr. P. G. W. J. van Oordt for his helpful comments and criticism, Mr. H. van Kooten for photographic assistance and Mr. F. Dijk for technical assistance.     

Adrenergic innervation of the male reproductive ducts in some mammals

Summary The distribution of adrenergic nerves to the male reproductive ducts were studied by applying the highly specific method of Falck and Hillarp for the cellular demonstration of the adrenergic transmitter. In rats, guinea-pigs, and rabbits, the ducts of the testis and the caput epididymidis were found to lack adrenergic varicose terminals, as they also lack cholinesterase positive structures. Adrenergic terminals extend in the vas deferens and epididymis from the prostatic end as far as the upper mid-corpus level of the ductus epididymidis, occurring in the muscle layers and, except in the rat, in the lamina propria. In the cat, ductuli efferentes and caput epididymidis also receive an adrenergic innervation.For skilful technical assistance we are grateful to Mrs. Ulla Flyger and Miss Berith Hansson. The investigation has been supported by a research grant (B 66-257) from the Swedish Medical Research Council.On sabbatical leave from the Biology Department, University of Oregon, Eugene, Oregon, with the aid of a Senior Research Fellowship from the National Council of Child Health and Human Development, National Institutes of Health, U.S. Department of Health, Welfare and Education. The use of facilities of the Department of Physiology I, Karolinska Institutet, with the kind permission of Professor U. S. von Euler and Research Docent R. Eliasson during the course of this study also is greatly appreciated.     

The pattern of innervation of a polyneural muscle: Axolotl iliotibialis

Summary The pattern of innervation on individual iliotibialis muscle fibres from axolotl (Ambystoma mexicanum) has been investigated histologically and elctrophysiologically. These polyneural fibres were found to be innervated on average at five end plate sites. The sites were distributed irregularly along each fibre. Average end plate length was found to be approxiamtely 70 m. Most end plates were separated by less than 1000 m; 26% by less than 150 m; the average separation was 516 m. Advantage was taken of the dual innervation of the muscle to investigate the separation between synaptic terminals from different axons. Some individual fibres were found to be innervated by axons from two different spinal nerves. End plate sites on dually innervated fibres were located by ACh iontophoresis. 30% of such sites were found to be innervated by more than one axon terminal. The average separation of such sites was found to be 950 m. Four different axons were found to innervate some individual muscle fibres. It is suggested that the unusual ability of axolotl muscle fibres to accept synaptic terminals from different axons at closely adjacent sites may be a major factor underlying selective reinnervation in this animal.Vacation student assisted by The Dale Fund of the Physiological Society     

Anomalous resistance changes following application of the neurotransmitterl-Glutamate in insect muscle

Summary Bath application ofl-glutamate, to larval dipteran muscle, at concentrations between 10^–6 and 10^–4 M will cause a depolarisation of the muscle membrane potential associated with an increase in muscle input resistance. At concentrations above 10^–4 M there is usually a transient decrease in input resistance preceding the resistance increase. l-aspartate at concentrations above 10^–5 M causes membrane depolarisations which are always associated with an increase in muscle input resistance.The pharmacology of the receptors regulating membrane depolarisations associated with increases in input resistance has been compared with the receptors gating synaptically activated cation channels and found to show significant differences in sensitivity to various ligands.     

Nonacceptance of innervation by innervated neonatal rat muscle

Denervated adult muscle accepts innervation and has high levels of extrajunctional acetylcholine (ACh) receptor, compared to innervated adult muscle. If the high receptor density or any externally oriented part of the receptor molecule permitted or triggered functional synaptogenesis, then innervated neonatal muscle, with its known high extrajunctional sensitivity, should also accept extra synapses from implanted motor nerves. This prediction was tested by implanting the common peroneal nerve into innervated lateral gastrocnemius muscle in 25 neonatal rats and studying the innervation achieved 1–8 weeks later. With one exception, zero or negligible twitch tensions were obtained when the implanted nerve was stimulated. Intracellular recording in two cases showed no evidence of subthresholdevoked potentials in surface muscle fibers. In contrast, when the original motor nerve was cut at the time of common peroneal nerve implantation, reinnervation occurred as soon as 4 days later, and substantial indirect twitches (most observed qualitatively) were invariably found 6–7 days after operation. Four to eight weeks after nerve implantation into denervated muscle, substantial twitch tensions were obtained upon stimulation of the implanted nerve. α-Bungarotoxin binding to extrajunctional ACh receptors per unit surface area was similar in innervated neonatal and denervated adult muscle. Therefore, nonacceptance of additional functional innervation in neonatal muscle implies that a high average density of extrajunctional ACh receptor is not sufficient to permit or trigger functional neuromuscular junction formation.     

Axonal protrusions in the small multiple endings in the extraocular muscles of the rat

Summary A special type of myoneural junction has been observed in the extraocular muscles of the rat with electron microscopy. These axon terminals are derived from unmyelinated nerves and contain synaptic vesicles and mitochondria. The terminals are invested by teloglia cells and separated by a synaptic cleft of about 500 Å from a slow-type muscle fibre. From the nerve ending a pseudopod-like evagination projects into the muscle cell. The membranes of this evagination and the muscle cells are only separated by a narrow cleft of about 100 Å, which is devoid of the basement membrane-like material typical of ordinary myoneural junctions. The evagination contains fewer axonal vesicles than other regions of the terminal axoplasm and the postsynaptic part of the muscle plasma membrane in this special region does not exhibit the postsynaptic thickening characteristic of ordinary myoneural junctions.The author thanks ProfessorAntti Telkkä, M.D., Head of the Electron Microscope Laboratory, University of Helsinki, for permission to use the facilities of the laboratory.     

Unique intertesticular tissue complex in larvae of Heliothis virescens (F.) (Lepidoptera : Noctuidae)

A unique heart-testis complex has been found in the last instar of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera : Noctuidae). This complex consists of a previously undescribed muscle system, herein designated the cardiotesticular muscle, that attaches the heart to the testes, and a ubiquitous connective tissue sheath that overlays this muscle and surrounding fat body and pericardial cells. These interconnected organs form a sieve-like septum enclosing a sinus that contains clusters of hemocytes and lipid droplets. Lipid droplets appear to be released from the surface of the testes sheath. The cardiotesticular muscle is innervated by neuroendocrine nerves that may be the route by which the brain peptide, testis ecdysiotropin, reaches the testes. The cardiotesticular muscle and products sequestered in the cardiotesticular sinus may be involved in the fusion of the paired testes and maturation of the male reproductive system, because they are present only during this stage of development.     

Innervation pattern and electric organ discharge waveform in Gymnotus carapo (Teleostei; Gymnotiformes)

The electrogenic organ (EO) of Gymnotus carapo has two main portions: a posterior region consisting of four bilaterally arranged electrocyte rows; and an anterior portion composed of only two. The lateral row (LR) of the anterior portion contains doubly innervated electrocytes with axon terminals from different nerves on their rostral and caudal faces. The LR is continuous with the most dorsal row of the caudal region. This row also contains doubly innervated electrocytes. The medial row (MR) electrocytes of the anterior region and ventral rows of the caudal region are exclusively caudally innervated. All caudal faces of the anterior or abdominal region are supplied by two nerves which originate from spinal roots VIII to XXI. Roots I to VII give origin to pure rostral nerves whose electromotor axons terminate on the rostral surfaces of the first seven LR electrocytes. A given doubly innervated electrocyte is supplied on its caudal face by a nerve originating several segments (usually seven) posterior to the spinal root supplying its rostral face. Transections of the spinal cord at the level of root VIII isolate the activity of the rostral surfaces of the first electrocytes. The EO discharge (EOD) then appears as a head negative deflection which arises from abdominally located electrocytes. Its monophasic character reveals that the activity remains restricted to the rostral electrocyte surfaces. Damage of the abdominal portion of the EO abolishes the first negative deflection of the normal pulse. Transections of the spinal cord at the level of root XXI isolate the activity of the whole abdominal portion of the EO. Since both doubly and singly innervated electrocytes remain active, the EOD appears biphasic. Comparative studies have shown that the EOD of Hypopomus sp. lacks any early negative wave and correspondingly all its electrocytes are exclusively caudally innervated.     

Fine structure of smooth muscle and neuromuscular junctions in the foot of Helix aspersa

Summary The smooth muscle cells in the foot of Helix aspersa are arranged in bundles which interweave to form a complex mesh. In the peripheral cytoplasm of the muscle cells there is a system of interconnected obliquely and longitudinally orientated tubules. The full extent of this system has not been determined; its possible function in relation to Ca⁺⁺ storage and excitation-contraction coupling is discussed. Longitudinal tubules are present among the myofilaments and in association with mitochondria. Distributed throughout the myofilaments are elliptically shaped dense bodies, the fine structure of which resembles an accumulation of thin filaments. Located on the plasma membrane of the muscle cells are dense areas; the fine structure and relationships of these cellular elements resemble desmosomes. They may serve as attachment points for thin, cytoplasmic filaments (not necessarily myofilaments). The muscle cells are innervated by axons which diverge from a coarse, neural plexus (the sole plexus). The axons initially come into close contact with the muscle cells and then pass over their surfaces for up to 35 before being gradually enveloped by flange-like protrusions of the muscle cells. These axons contain either, (i) agranular vesicles (600 Å in diameter), (ii) agranular and very dense granular vesicles (1000 Å in diameter) or (iii) agranular and less dense, granular vesicles (1000 Å in diameter). The possible role of these inclusions as sites of excitatory and inhibitory transmitters is discussed.I wish to thank Professor G. Burnstock for making laboratory facilities available. This work has been supported by the Australian Research Grants Committee.     

Spontaneous and neurally evoked contractions of visceral muscles in the oviduct of Locusta migratoria

The oviducts of Locusta migratoria are innervated by a pair of nerves which arise from, the seventh abdominal ganglion. A distinctive network of striated muscle fibres occurs in the oviducts. The lateral oviducts and common oviduct consist of an inner circular layer of muscle and an outer longitudinal layer of muscle. At the junction of the lateral and common oviduct an additional thin longitudinal layer is found adjacent to the basement epithelium. The oviducts contracted spontaneously when isolated from the central nervous system. These myogenic contractions took the form of peristaltic contractions in the lateral oviduct, and intermittent phasic-like contractions of the posterior regions of the lateral oviduct and the common oviduct. These phasic-like contractions were associated with individual complex potentials recorded extracellularly from the muscle fibres. In locusts that had been interrupted in the process of egg laying, there were large-amplitude action potentials, firing in a bursting pattern, in the oviducal nerves. These large action potentials were absent in locusts that had not been egg-laying. These action potentials were associated with both bioelectric potentials and mechanical events in the posterior region of the lateral oviduct and the common oviduct. Electrical stimulation of the oviducal nerve mimicked the effects of spontaneous action potentials, resulting in the appearance of monophasic potentials and contractions. The contractions were graded and dependent upon both frequency and duration of stimulation. It is concluded that the oviducts of Locusta are both myogenic and neurogenic. The role of these contractions in oviposition is discussed.     

Regeneration and supernumerary limb formation under sparsely innervated conditions

Normally, urodele limb regeneration is nerve-dependent. Reduction in nerve-dependency has been reported for regenerating, transplanted newt limbs (Singer and Mutterperl, '63). Aneurogenic limbs can regenerate without nerves (Yntema, '59). Induction of supernumerary limbs may be obtained from aneurogenic limbs of larval Ambystoma after transplantation orthotopically to innervated larvae and with normal nerve ingrowth to the limb transplant prevented by repeated section of brachial nerves. Of the 13 (of 43) grafts with supernumeraries, nerve counts showed 11 with 0–5; 1 with 5–10; and 1 with 20⁺ fibers. Orthotopically grafted aneurogenic limbs allowed to become innervated showed 14 supernumeraries in 49 grafts. This supernumerary limb induction is thus not nerve-dependent. Normally, innervated larval Ambystoma limbs grafted orthotopically and heteroplastically regenerated in 17 of 37 cases after repeated section of brachial nerves. Of the 17 regenerates nerve counts showed 4 with 0–5; 5 with 5–10; 7 with 10–19; and 1 with 20⁺ fibers. Larval limbs heteroplastically transplanted may require very few or no nerves for regeneration.