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Environmental conditions prerequisite for complete limb regeneration in the postmetamorphic adult land-phase salamander, Ambystoma.

Historically, postmetamorphic adult land-phase salamanders have been shown to exhibit minimal to nonexistent limb regeneration. Hence, it has been generally accepted that these forms have lost the intrinsic capacity to regenerate a limb. Due to the experimental protocols used, an alternate explanation is also possible: that this intrinsic capacity cannot be expressed when the salamanders are maintained under adverse laboratory environmental conditions. Therefore, this study addresses two questions: 1) What are the optimal environmental conditions for long-term survival of adult land-phase salamanders; and 2) will complete limb regeneration occur in these salamanders if they are maintained under survival conditions. A mixed population of adult Ambystoma were tested under varying conditions of habitat, temperature, humidity, photoperiod, and food source. Complete limb regeneration was possible in 100% of four species of adult postmetamorphic land-phase Ambystoma salamanders given the proper environmental laboratory conditions of a peat moss and potting soil habitat with a controlled temperature of 25 degrees C +/- 5 degrees C, 70% or greater humidity, a 12/12 light/dark photoperiod, a diet including nightcrawlers released into their respective terraria, and an extended observation time of up to 370 days postamputation (dpa). Regeneration was completed during the following range periods for the adult salamanders: A. annulatum, 324 to 370 dpa; A. maculatum, 255 to 300 dpa; A. texanum, 215 to 250 dpa; and A. tigranum, 155 to 180 dpa.

Ambystoma↗

Cytoarchitecture of the tectum mesencephali in salamanders: a Golgi and HRP study.

The tectum mesencephali of salamanders shows a morphology that has long been considered primitive when compared with that of frogs. The alternative hypothesis is that the salamander brain is secondarily simplified. In order to test these two hypotheses, the cytoarchitecture of the tectum and the projections of tectal neurons were studied in 11 species of salamanders. Application of the Golgi method reveals three major morphological types. Type 1 has a very wide dendritic arborization mostly confined to the deep fiber layers, and somata are always located within the most superficial part of the periventricular gray matter. Type 2 possesses a wide to medium-size dendritic arborization. In subtype 2a the somata are located in the uppermost part of the gray, and dendrites always reach the uppermost layer of retinal afferents; in subtype 2b the somata are found in deeper parts of the gray, and dendrites arborize in the deeper layers of retinal afferents; and in subtype 2c the somata are also located in deeper parts, but the wide dendritic arborization is confined to deep fiber layers. Type 3 shows the narrowest dendritic arbors that always reach the upper two tectal fiber layers. The somata are found at any depth of the gray matter. HRP experiments reveal a correlation between morphological differences and the projections of tectal neurons. Type 1- and type 2c-like cells constitute the uncrossed tecto-bulbo-spinal tract, whereas type 1- and type 2a-like cells and migrated large spindle-shaped cells (Salamandra) constitute the crossed tecto-bulbo-spinal tract. Type 3-like neurons project to thalamic, pretectal, and isthmic termination sites. The HRP experiments also demonstrate the existence of two classes of mesencephalic trigeminal cells. A comparison shows that salamanders and frogs possess very similar functional and morphological types of tectal cells. However, tectal cells of salamanders show a "juvenile" morphology, and the number of migrated cells is about 10 times higher in frogs compared to salamanders. Both phenomena are seen as the result of secondary simplification of brain structures in the context of paedomorphosis.

Animals↗

Social monogamy in a territorial salamander.

Social monogamy, which does not necessarily imply mating or genetic monogamy, is important in the formation of male-female pair associations. We operationally define social monogamy as occurring when two heterosexual adults, exclusive of kin-directed behaviour, direct significantly less aggression and significantly more submission towards each other, and/or spend significantly more time associating with each other relative to other adult heterosexual conspecifics. Long-term pair associations (i.e. those lasting through a lengthy breeding season) that are characteristic of social monogamy are common in some taxa but are virtually unknown in amphibians. Recent studies, however, have suggested that red-backed salamanders, Plethodon cinereus, have complex (for amphibians) social systems. Our laboratory experiments tested the hypothesis that red-backed salamanders found in pairs in the forest display behaviours consistent with social monogamy. During the summer noncourtship season, newly collected male-females pairs showed no preference to associate with their partners more than with a novel conspecific of the opposite sex. However, during the autumn courtship season, paired males and females significantly directed preferential behaviours towards their partners rather than towards a surrogate or a novel paired salamander. Focal animals showed no significant preferences when presented with their partner and a novel single salamander, but they never directed preferential behaviours towards a novel salamander (whether paired or single) or a surrogate. These results are the first to suggest that a salamander species engages in social monogamy. Furthermore, our results suggest that social monogamy may not inhibit paired males and females from displaying alternative strategies: preferring partners when extrapair associations may be disadvantageous (i.e. the extrapair animal is already paired) but not preferring partners when extrapair associations may be advantageous (i.e. the extrapair animal is single). Copyright 2000 The Association for the Study of Animal Behaviour.

Journal Article↗

Laser vibrometric studies of sound-induced motion of the body walls and lungs of salamanders and lizards: implications for lung-based hearing.

A laser Doppler vibrometer was used to measure the acoustic responses of different body surfaces of several species of salamanders and lizards. The lateral body wall over the lung displayed sound-induced motion up to 30 dB greater than the lateral head surface from 300-1,000 Hz in salamanders and from 200-2,500 Hz in lizards. The lateral body wall of lungless plethodontid salamanders showed no such enhanced motion to sound. The lateral body wall of lizards was more responsive than their tympanum to sound frequencies below about 1,250-2,000 Hz. The frequency of the peak response of lizard body walls matched the resonant frequency of a Helmholtz resonator with the volume and dimensions of their lungs. In contrast, the frequency of peak response of salamander body walls was well below the resonant frequencies calculated for both Helmholtz resonators and closed tubes with the dimensions and volumes of their lungs. Nonetheless, filling the lungs with saline dramatically reduced the responsiveness of the lateral body walls of both the lunged salamanders and the lizards. As previously demonstrated in anuran amphibians, the lateral body wall and lungs of salamanders and lizards may function in sound reception, especially at relatively low frequencies.

Acoustics↗

Mercury bioaccumulation in northern two-lined salamanders from streams in the northeastern United States.

Mercury (Hg) bioaccumulation in salamanders has received little attention despite widespread Hg contamination of aquatic ecosystems and worldwide amphibian declines. Here we report concentrations of methyl Hg (MeHg) and total Hg in larval northern two-lined salamanders (Eurycea bislineata bislineata) collected from streams in Acadia National Park (ANP), Maine, and Bear Brook Watershed, Maine (BBWM; a paired, gauged watershed treated with bimonthly applications (25 kg/ha/yr) of ammonium sulfate [(NH4)2SO4]) since 1989), and Shenandoah National Park (SNP), Virginia. MeHg comprised 73-97% of total Hg in the larval salamander composite samples from ANP. At BBWM we detected significantly higher total. Hg levels in larvae from the (NH4)2SO4 treatment watershed. At ANP total Hg concentrations in salamander larvae were significantly higher from streams in unburned watersheds in contrast with larval samples collected from streams located in watersheds burned by the 1947 Bar Harbor fire. Additionally, total Hg levels were significantly higher in salamander larvae collected at ANP in contrast with SNP. Our results suggest that watershed-scale attributes including fire history, whole-catchment (NH4)2SO4 additions, wetland extent, and forest cover type influence mercury bioaccumulation in salamanders inhabiting lotic environments. We also discuss the use of this species as an indicator of Hg bioaccumulation in stream ecosystems.

Animals↗

Identification of a non-mammalian leptin-like gene: characterization and expression in the tiger salamander (Ambystoma tigrinum).

Leptin is well established as a multifunctional cytokine in mammals. However, little is known about the evolution of the leptin gene in other vertebrates. A recently published set of ESTs from the tiger salamander (Ambystoma tigrinum) contains a sequence sharing 56% nucleotide sequence identity with the human leptin cDNA. To confirm that the EST is naturally expressed in the salamander, a 409bp cDNA was amplified by RT-PCR of salamander testis and stomach mRNAs. The coding sequence of the cDNA is predicted to encode 169 amino acids, and the mature peptide to consist of 146 residues, as in mammals. Although the overall amino acid identity with mammalian leptins is only 29%, the salamander and mammalian peptides share common structural features. An intron was identified between coding exons providing evidence that the sequence is present in the salamander genome. Phylogenetic analysis showed a rate of molecular divergence consistent with the accepted view of vertebrate evolution. The pattern of tissue expression of the leptin-like cDNA differed between metamorphosed adult individuals of different sizes suggesting possible developmental regulation. Expression was most prominent in the skin and testis, but was also detected in tissues in which leptin mRNA is present in mammals, including the fat body, stomach, and muscle. The characterization of a salamander leptin-like gene provides a basis for understanding how the structure and functions of leptin have altered during the evolution of tetrapod vertebrates.

Ambystoma↗

Ontogenetic evidence for the Paleozoic ancestry of salamanders.

The phylogenetic positions of frogs, salamanders, and caecilians have been difficult to establish. Data matrices based primarily on Paleozoic taxa support a monophyletic origin of all Lissamphibia but have resulted in widely divergent hypotheses of the nature of their common ancestor. Analysis that concentrates on the character states of the stem taxa of the extant orders, in contrast, suggests a polyphyletic origin from divergent Paleozoic clades. Comparison of patterns of larval development in Paleozoic and modern amphibians provides a means to test previous phylogenies based primarily on adult characteristics. This proves to be highly informative in the case of the origin of salamanders. Putative ancestors of salamanders are recognized from the Permo-Carboniferous boundary of Germany on the basis of ontogenetic changes observed in fossil remains of larval growth series. The entire developmental sequence from hatching to metamorphosis is revealed in an assemblage of over 600 specimens from a single locality, all belonging to the genus Apateon. Apateon forms the most speciose genus of the neotenic temnospondyl family Branchiosauridae. The sequence of ossification of individual bones and the changing configuration of the skull closely parallel those observed in the development of primitive living salamanders. These fossils provide a model of how derived features of the salamander skull may have evolved in the context of feeding specializations that appeared in early larval stages of members of the Branchiosauridae. Larvae of Apateon share many unique derived characters with salamanders of the families Hynobiidae, Salamandridae, and Ambystomatidae, which have not been recognized in any other group of Paleozoic amphibians.

Animals↗

Conserved vertebrate chromosome segments in the large salamander genome.

Urodele amphibians (salamanders) are important models for embryological, physiological, and natural history research and are also a biomedically important group because they are the only vertebrates capable of regenerating entire organ systems. To enhance the utility of salamanders for biomedical research and for understanding genome evolution, genetic linkage analysis was used to identify chromosome segments that are homologous between ambystomatid salamanders and distantly related vertebrate model organisms. A total of 347 loci (AFLPs, RAPDs, and protein-coding loci) were mapped using an interspecific meiotic mapping panel (Ambystoma mexicanum and A. tigrinum tigrinum; family Ambystomatidae). Genome size in Ambystoma was estimated to be 7291 cM, the largest linkage map estimate reported for any organism. However, the relatively large size of the salamander genome did not hinder efforts to map and identify conserved syntenies from a small sample of 24 protein-coding loci. Chromosomal segments that are conserved between fishes and mammals are also conserved in these salamanders. Thus, comparative gene mapping appears to be an efficient strategy for identifying orthologous loci between ambystomatid salamanders and genomically well-characterized vertebrate model organisms.

Animals↗

Motor neurons and motor columns of the anterior spinal cord of salamanders: posthatching development and phylogenetic distribution.

The posthatching development of rostral (1st-4th) spinal motor neurons was studied in ten species of salamanders, using horseradish peroxidase and cobaltic lysine tracing techniques. Development of spinal motor neurons differs among species in association with differences in life history and general developmental patterns (i.e., between species with aquatic larvae versus those with direct development, with or without ontogenetic repatterning). In the plesiomorphic state, represented by species with aquatic larvae, five types of motor neurons are present: (1) large, multipolar neurons, believed to be primary motor neurons; (2) medial, pear-shaped neurons; (3) larger, spindle-shaped neurons, which increase in number during posthatching development; (4) cone-shaped neurons, and (5) bilaterally arborizing neurons (found only at the rostral pole of the first spinal nucleus). Direct-developing desmognathine salamanders have the plesiomorphic set of motor neurons, but appear to lack Mauthner neurons. Direct-developing plethodontine salamanders have cone-shaped, pear-shaped, and spindle-shaped neurons, but lack primary motor neurons and Mauthner neurons. Direct-developing bolitoglossine salamanders, which exhibit both pedomorphosis and ontogenetic repatterning, have only medial, pear-shaped neurons, and lack primary motor neurons, spindle-shaped neurons, cone-shaped and bilaterally arborizing neurons. At all developmental stages in all species studied, pear-shaped neurons are always found in medial positions and spindle-shaped neurons are always found in lateral positions. Spindle-shaped neurons are found more laterally as development proceeds. The medial and lateral motor columns of salamanders and amniotes differ in their connections with peripheral targets (i.e., axial muscles vs. limbs). This implies a lack of homology of neuron types in salamanders and amniotes, which has been obscured by the current terminology.

Animals↗

Mechanics of lung ventilation in a post-metamorphic salamander, Ambystoma Tigrinum.

The mechanics of lung ventilation in frogs and aquatic salamanders has been well characterized, whereas lung ventilation in terrestrial-phase (post-metamorphic) salamanders has received little attention. We used electromyography (EMG), X-ray videography, standard videography and buccal and body cavity pressure measurements to characterize the ventilation mechanics of adult (post-metamorphic) tiger salamanders (Ambystoma tigrinum). Three results emerged: (i) under terrestrial conditions or when floating at the surface of the water, adult A. tigrinum breathed through their nares using a two-stroke buccal pump; (ii) in addition to this narial two-stroke pump, adult tiger salamanders also gulped air in through their mouths using a modified two-stroke buccal pump when in an aquatic environment; and (iii) exhalation in adult tiger salamanders is active during aquatic gulping breaths, whereas exhalation appears to be passive during terrestrial breathing at rest. Active exhalation in aquatic breaths is indicated by an increase in body cavity pressure during exhalation and associated EMG activity in the lateral hypaxial musculature, particularly the M. transversus abdominis. In terrestrial breathing, no EMG activity in the lateral hypaxial muscles is generally present, and body cavity pressure decreases during exhalation. In aquatic breaths, tidal volume is larger than in terrestrial breaths, and breathing frequency is much lower (approximately 1 breath 10 min(-)(1 )versus 4-6 breaths min(-)(1)). The use of hypaxial muscles to power active exhalation in the aquatic environment may result from the need for more complete exhalation and larger tidal volumes when breathing infrequently. This hypothesis is supported by previous findings that terrestrial frogs ventilate their lungs with small tidal volumes and exhale passively, whereas aquatic frogs and salamanders use large tidal volumes and and exhale actively.

Ambystoma↗

Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum.

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.

Ambystoma↗

Pathology, isolation, and preliminary molecular characterization of a novel iridovirus from tiger salamanders in Saskatchewan.

All iridovirus was confirmed to be the cause of an epizootic in larval and adult tiger salamanders (Ambystoma tigrinum diaboli) from four separate ponds in southern Saskatchewan (Canada) during the summer of 1997. This organism also is suspected, based on electron microscopic findings, to be the cause of mortality of larval tiger salamanders in a pond over 200 km to the north during the same year. Salamanders developed a generalized viremia which resulted in various lesions including: necrotizing, vesicular and ulcerative dermatitis; gastrointestinal ulceration; and necrosis of hepatic, splenic, renal, lymphoid, and hematopoietic tissues. In cells associated with these lesions, large lightly basophilic cytoplasmic inclusions and vacuolated nuclei with marginated chromatin were consistently found. Virus was isolated from tissue homogenates of infected salamanders following inoculation of epithelioma papilloma cyprini (EPC) cells. The virus, provisionally designated Regina ranavirus (RRV), was initially identified as an iridovirus by electron microscopy. Subsequent molecular characterization, including partial sequence analysis of the major capsid protein (MCP) gene, confirmed this assignment and established that RRV was a ranavirus distinct from frog virus 3 (FV3) and other members of the genus Ranavirus. Intraperitoneal inoculation of 5 x 10(6.23) TCID50 of the field isolate caused mortality in inoculated salamanders at 13 days post infection. Field, clinical, and molecular studies jointly suggest that the etiological agent of recent salamander mortalities is a highly infectious novel ranavirus.

Ambystoma↗

Molecular cloning of a rhodopsin gene from salamander rods.

PURPOSE: Salamander photoreceptor cells have been used widely as models in vision research. However, the salamander opsin genes had not been cloned. The purpose of this study was to clone a salamander rhodopsin and to determine its primary structure and cell type-specific expression. METHODS: Using salamander retina RNA as a template and Xenopus rhodopsin-specific oligonucleotides as primers, reverse transcription and polymerase chain reaction (RT-PCR) were used to amplify and clone a rhodopsin cDNA fragment. This fragment was used as a probe to isolate a full-length cDNA of the rhodopsin from a cDNA library of salamander retina. The dideoxynucleotide chain termination method was used to determine the nucleotide sequence. Single rod and cone cells were isolated by micromanipulation, and the absorbance spectra of the rod outer segments were measured with a photon-counting microspectrophotometer. Individual rod and cone cells were lysed for RT-PCR and Southern blot analysis to detect cell-specific expression of this gene. RESULTS: A 1.2 kb rhodopsin cDNA containing the full-length coding region of rhodopsin has been cloned and sequenced from the larval tiger salamander, Ambystoma tigrinum. This cDNA encodes 354 amino acids that, by hydropathy profile, could form seven transmembrane domains characteristic of other rhodopsins. Sequence identity was found with other amphibian rhodopsins at the nucleic acid (82% to 83%) and the amino acid (88% to 89%) levels. Key amino acids critical for structure and function of rhodopsin have been retained. The mRNA of this rhodopsin was identified in red rod cells (lambda max 506 nm). No expression of the gene was detected in cone cells. CONCLUSIONS: The cloned rhodopsin is a newly isolated member of the G protein-coupled receptor superfamily. This protein is expressed in rods but not in cones.

Ambystoma↗

Endoparasites of plethodontid salamanders from Paradise Brook, New Hampshire.

Totals of 52 dusky salamanders Desmognathus fuscus, 51 two-lined salamanders Eurycea bislineata, 54 red-backed salamanders Plethodon cinereus, and 3 spring salamanders Gyrinophilus porphyriticus (Plethodontidae) collected in June and August 1995 from Paradise Brook, a tributary to Hubbard Brook, New Hampshire, were examined for parasites. Parasites found were Brachycoelium storeriae, Brachycoelium sp., Bothriocephalus rarus, Falcaustra sp., Omeia sp., Batracholandros magnavulvaris, and Cepedietta michiganensis. Eighty-six percent of the red-backed salamanders, a terrestrial species, harbored 1 or more parasites. Among the aquatic and semiaquatic species, 27% of the dusky and 45% of the two-lined salamanders were infected with 1 or more parasites.

Animals↗

Functional morphology of the feeding mechanism in aquatic ambystomatid salamanders.

This study addresses four questions in vertebrate functional morphology through a study of aquatic prey capture in ambystomatid salamanders: How does the feeding mechanism of aquatic salamanders function as a biomechanical system? How similar are the biomechanics of suction feeding in aquatic salamanders and ray-finned fishes? What quantitative relationship does information extracted from electromyograms of striated muscles bear to kinematic patterns and animal performance? and What are the major structural and functional patterns in the evolution of the lower vertebrate skull? During prey capture, larval ambystomatid salamanders display a kinematic pattern similar to that of other lower vertebrates, with peak gape occurring prior to both peak hyoid depression and peak cranial elevation. The depressor mandibulae, rectus cervicis, epaxialis, hypaxialis, and branchiohyoideus muscles are all active for 40-60 msec during the strike and overlap considerably in activity. The two divisions of the adductor mandibulae are active in a continuous burst for 110-130 msec, and the intermandibularis posterior and coracomandibularis are active in a double burst pattern. The antagonistic depressor mandibulae and adductor mandibulae internus become active within 0.2 msec of each other, but the two muscles show very different spike and amplitude patterns during their respective activity periods. Coefficients of variation for kinematic and most electromyographic recordings reach a minimum within a 10 msec time period, just after the mouth starts to open. Pressure within the buccal cavity during the strike reaches a minimum of -25 mmHg, and minimum pressure occurs synchronously with maximum gill bar adduction. The gill bars (bearing gill rakers that interlock with rakers of adjacent arches) clearly function as a resistance within the oral cavity and restrict posterior water influx during mouth opening, creating a unidirectional flow during feeding. Durations of electromyographic activity alone are poor predictors of kinematic patterns. Analyses of spike amplitude explain an additional fraction of the variance in jaw kinematics, whereas the product of spike number and amplitude is the best statistical predictor of kinematic response variables. Larval ambystomatid salamanders retain the two primitive biomechanical systems for opening and closing the mouth present in nontetrapod vertebrates: elevation of the head by the epaxialis and depression of the mandible by the hyoid apparatus.

Ambystoma↗

Behavioral and physiological responses of the terrestrial life stages of the Jefferson salamander, Ambystoma jeffersonianum, to low soil pH.

Terrestrial metamorphs of the salamander, Ambystoma jeffersonianum, were exposed to four levels of pH on artificial substrates in the laboratory. Short-term exposure (7 d) to pH levels between 3.5 and 5.0 had no effect on total body mass, Na+, K+, Ca2+, and Mg2+ concentrations. However, at 14 d in both laboratory experiments and in field microcosms, whole body water and Na+ concentrations were significantly lower at the lower pH level. Terrestrial metamorphs and adults of the Jefferson salamander preferred artificial substrates that were higher in pH. Ponds not utilized for breeding all had adjacent soil pH below 3.7. Pond water and soil pH were directly and highly correlated. Soil pH may indirectly affect breeding success of Jefferson salamanders by influencing pond pH or directly by physiologically stressing the terrestrial life stages. Laboratory data implicate direct effects on the physiology of terrestrial young of the year salamanders by induction of loss of body water and Na+. Apparently, soil pH is a critical factor not only in the selection of vernal ponds by Jefferson salamander adults for breeding, but in determining the likelihood of reproductive success as judged by recruitment into the adult population.

Ambystoma↗

Molecular characterization of major histocompatibility complex class II alleles in wild tiger salamanders (Ambystoma tigrinum).

Major histocompatibility complex (MHC) class II genes are usually among the most polymorphic in vertebrate genomes because of their critical role (antigen presentation) in immune response. Prior to this study, the MHC was poorly characterized in tiger salamanders (Ambystoma tigrinum), but the congeneric axolotl (Ambystoma mexicanum) is thought to have an unusual MHC. Most notably, axolotl class II genes lack allelic variation and possess a splice variant without a full peptide binding region (PBR). The axolotl is considered immunodeficient, but it is unclear how or to what extent MHC genetics and immunodeficiency are interrelated. To study the evolution of MHC genes in urodele amphibians, we describe for the first time an expressed polymorphic class II gene in wild tiger salamanders. We sequenced the PBR of a class II gene from wild A. tigrinum (n=33) and identified nine distinct alleles. Observed heterozygosity was 73%, and there were a total of 46 polymorphic sites, most of which correspond to amino acid positions that bind peptides. Patterns of nucleotide substitutions exhibit the signature of diversifying selection, but no recombination was detected. Not surprisingly, trans-species evolution of tiger salamander and axolotl class II alleles was apparent. We have no direct data on the immunodeficiency of tiger salamanders, but the levels of polymorphism in our study population should suffice to bind a variety of foreign peptides (unlike axolotls). Our tiger salamander data suggest that the monomorphism and immunodeficiencies associated with axolotl class II genes is a relic of their unique historical demography, not their phylogenetic legacy.

Alleles↗

Bulbospinal and intraspinal connections in normal and regenerated salamander spinal cord.

The salamander is the only limbed adult vertebrate which can regenerate portions of cervical, thoracic, or lumbar spinal cord. While the salamander has been a popular model for regeneration of the spinal cord, it is still not known what portions of the nervous system participate in the regeneration process. In the experiments reported here we examine the bulbospinal and intraspinal projections to the lumbar spinal cord in normal and regenerated salamanders (Notophthalmus viridescens). HRP application to the lumbar enlargement of normal salamanders labeled cells in the ventral thalamus, the rostral tegmentum in the proposed homolog of the red nucleus, the reticular neurons of the rhombencephalon, and the midline regions of the rhombencephalon which are possibly equivalent to raphe nuclei of other vertebrates. In the brachial spinal cord HRP-labeled cells were located in dorsal, intermediate, and ventral regions of the spinal gray matter and tended to be located at the periphery of the gray matter. To examine the spinal circuitry of regenerated salamanders, animals received complete spinal transections at the junction of the thoracic and lumbar spinal cord, abolishing all spontaneous coordinated hindlimb and tail movements. Animals exhibited walking and swimming within 60 days at which time a pledget of HRP was inserted into a gap in the spinal cord made by a transection 10.0 mm (six animals) or 5.0 mm (one animal) caudal to the first lesion. On average, the number of HRP labeled brain stem neurons in regenerated animals was 40% of that found in normal animals. The number of labeled cells in the brachial spinal cord was within the range of normal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Amputation, Surgical↗