Neurotransmitter candidates in the retina of the mudpuppy,Necturus maculosus

Summary Neurons accumulating (³H)-glycine and (³H) GABA were demonstrated with the use of autoradiography. Both were accumulated by different types of amacrine cells, similar those of goldfish. (³H)-GABA was also accumulated by horizontal cells, again similar to the goldfish. These results and physiological studies from other laboratories suggest that GABA and glycine are neurotransmitter candidates in amacrine cells of the mudpuppy.Immunoreactive neuropeptide Y (NPY), glucagon, vasoactive intestinal peptide (VIP), somatostatin, substance P, and neurotensin were found in different types of stratified amacrine cells. Weakly immunoreactive enkephalin and bombesin processes were also seen in the inner plexiform layer. Gastrin-immunoreactive neurons were not detectable.Endogenous 5-hydroxytryptamine was visualized immunohistochemically in a population of diffuse amacrine cells and some cells in the ganglion cell layer. This suggests that 5-hydroxytryptamine may be a neurotransmitter in the retina of the mudpuppy.     

Internal convective and conductive heat loss in the mudpuppy,Necturus maculosus

• 1.1. Intestinal temperatures of 10 mudpuppies (Necturus maculosus) subjected to an instantaneous decrease of 6.0°C in water temperature were monitored while animals were alive, alive with gills tied and dead.
• 2.2. The rates of cooling: alive > alive gills tied > dead, were consistent in all animals.
• 3.3. Comparisons of data for live and dead mudpuppies indicated that conduction accounted for a mean of 68.5 and therefore convection 31.5% of total heat loss in these experiments.
• 4.4. The external gills were the site of approximately half (54.4%) of total internal convective heat transfer with the remainder occurring through the body surface.

     

The effects of ATP and cAMP on the thermal tolerance of the mudpuppy,Necturus maculosus

• 1.1.|Data from previous studies suggest that cellular thermal tolerance depends in part on the availability of high-energy metabolites. To determine if a similar phenomenon is involved in the regulation of thermal tolerance in whole organisms, we treated the mudpuppy, Necturus maculosus, with drugs presumed to elevate or depress hypothalamic levels of either adenosine triphosphate (ATP) or cyclic adenosine monophosphate (cAMP).
• 2.2.|Injections, into the third ventricle, of ATP, cAMP, and theophylline (which elevates endogenous levels of cAMP) elicited dose-related increases in the critical thermal maximum (CTM).
• 3.3.|A dose-related decrease in the CTM resulted when animals were treated with 2-deoxy-D-glucose, a nonmetabolizable glucose analogue.

     

Amiloride does not block taste transduction in the mudpuppy, Necturus maculosus

Activity of the glossopharyngeal nerve was recorded with bipolarsilver wire electrodes while taste stimuli were applied to thelingual surface in anesthetized mudpuppies. Taste stimuli wereinjected into a continuous stream of distilled water which wasrunning over the tongue, KCl, CaCl₂ and LiCl₂ at 0.4 M elicitedbrisk responses, as did HCl at 0.2 M and quinine at 6 x 10^–4M. Sucrose, glucose and saccharin did not elicit responses.Twenty amino acids were surveyed for their ability to evokea response at 0.04 M: 1-arginine, 1-valine, 1-phenylalanine,1-tryptophan, 1-tyrosine, 1-glutamic acid, 1-lysine and histidinealways evoked responses, whereas other amino acids either didnot evoke responses or only occasionally evoked responses. Thesupernatants from solutions of minced worms and minnows andPurina Trout Chow were effective taste stimuli. Pre-adaptingthe tongue to Ringer's solution by running a continuous streamof Ringer's solution over it eliminated responses to quinineand decreased responses to NaCl. Pre-adapting the tongue to10^–4 to 10^–3 M amiloride, a potent sodium channelblocker, did not alter the responses to NaCl, LiCl, or othertaste stimuli.     

Scanning electron microscope observations of saccular ultrastructure in the mudpuppy (Necturus maculosus)

Summary Scanning electron microscopy revealed two types of hair cells in the sacculus of an amphibian, the mudpuppy (Necturus maculosus). Both types were surrounded by microvilli-covered sustentacular cells. The peripheral hair cells have shorter, thinner stereocilia and longer kinocilia than the hair cells in the central macula. The hair cells generally were found to be oriented with their stereocilia gradient directed toward the periphery of the macula. A nearly semicircular stria separated those directed forward and outward from those directed rearward and outward. Two basic types of otoconia were found in the otolith, and X-ray analysis revealed the entire otolith to be composed of aragonite.We would like to thank Dean E. Hillman for suggestions regarding fixation and interpretation, R. Eric Lombard for assistance with amphibian morphology and for helpful discussions during the course of this work, H. R. Wenk for performing X-ray analysis of otolith and Sister Loretta Shimondle for technical assistance. Research sponsored by the National Science Foundation Grant GK-3845 and the United States Public Health Service Grant GM-17523.     

Fine structure of the abdominal epidermis of the adult mudpuppy,Necturus maculosus (Rafinesque)

Summary The ventral epidermis of adult Necturus maculosus has been studied using electron and light microscopy. Many larval characteristics of amphibian epidermal structure are retained in adult Necturus. The epidermis is a stratified epithelium consisting of four cell layers and five cell types. Major differences compared with other adult amphibians are: (1) the absence of a well defined moulting cycle together with an apparently diminished synthetic and mitotic activity in the stratum germinativum; (2) an outermost cell layer (stratum mucosum) that is unkeratinized and appears to synthesize a mucous layer; and (3) numerous large club-shaped Leydig cells which span the epidermis between the cells of the stratum germinativum and stratum mucosum. The apical region of the stratum granulosum and stratum mucosum cells shows evidence of extensive synthesis. The stratum mucosum appears to be involved in the secretion of vesicular contents onto the outermost surface of the epithelium. The external surfaces of the stratum mucosum cells possess numerous microridges which are supported by an intricate network of cytofilaments in the apical region of these cells. The significance of these features is discussed in relation to the physiology and ecology of this species.     

Modulation of Cl-, K+, and nonselective cation conductances by taurine in olfactory receptor neurons of the mudpuppy Necturus maculosus

Odors are transduced by processes that modulate the membrane conductance of olfactory receptor neurons. Olfactory neurons from the aquatic salamander, Necturus maculosus, were acutely isolated without enzymes and studied with a resistive whole-cell method to minimize loss of soluble intracellular constituents. 55 of 224 neurons responded to the test compound taurine at concentrations between 10 nM and 100 microM. Four different conductance changes were elicited by taurine: an increased Cl- conductance (33%), an increased nonselective cation conductance (15%), a decreased Cl- conductance (15%), and a decreased K+ conductance (15%); in addition, responses too small to be characterized were elicited in some neurons. In most cases, taurine appeared to modulate only a single conductance in any particular cell. Modulation of each conductance was dose dependent, and each response ran down quickly in the normal whole-cell mode, presumably due to washout of a diffusible component in the transduction pathway. Modulation of taurine-sensitive conductances caused either inhibitory or excitatory responses. A similar diversity of responses in vivo would produce a complex pattern of electrical activity that could encode the identity and characteristics of an odor.     

Gas exchange and habitat selection in the aquatic salamanders Necturus maculosus and Cryptobranchus alleganiensis

Summary The standard metabolic rate (SMR) and critical O₂ tension (P_c) of water-breathing mudpuppies and hellbenders were determined at 20° C using open-system respirometry. Both species are metabolic O₂ regulators, although the P_c of hellbenders (90 mmHg) is much higher than that of mudpuppies (40 mmHg). The SMR of the two species in water saturated with air was similar (19.5 and 20.0 l O₂/g·h for Cryptobranchus and Necturus, respectively) and not different from that of salamanders in general. Both species were able to survive for at least 5–11 days in severely hypoxic water (9–10 mmHg) by breathing air, indicating that the lungs are functional accessory respiratory structures.We conclude that hellbenders are restricted to relatively cool and flowing waters because of their limited gas exchange capabilities, particularly with regard to their limited aerobic scope for activity and slow recovery from exercise. Necturus maculosus is much more tolerant of hypoxia, but it is not known if they can inhabit areas were hypoxia is combined with hypercarbia.     

Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.     

Anatomical organization of motoneurons and interneurons in the mudpuppy (Necturus maculosus) brachial spinal cord: the neural substrate for central pattern generation

The isolated brachial spinal cord of the mudpuppy is useful for studies of neural networks underlying forelimb locomotion, but information about its anatomy is scarce. We addressed this issue by combining retrograde labeling with fluorescent tracers and confocal microscopy. Remarkably, the central region of gray matter was aneural and contained only a tenuous meshwork of glial fibers and large extracellular spaces. Somata of motoneurons (MNs) and interneurons (INs), labeled retrogradely from ventral roots or axons in the ventro-lateral funiculus, respectively, were confined within a gray neuropil layer abutting the white matter borders, while their dendrites projected widely throughout the white matter. A considerable fraction of labeled INs was found contralaterally with axons crossing beneath a thick layer of ependyma surrounding the central canal. Dorsal roots (DRs) produced dense presynaptic arbors within a restricted dorsal region containing afferent terminations, within which dorsally directed MN and IN dendrites mingled with dense collections of synaptic boutons. Our data suggest that a major fraction of synaptic interactions takes place within the white matter. This study provides a detailed foundation for electrophysiological experiments aimed at elucidating the neural circuits involved in locomotor pattern generation.     

Distribution of monoamine oxidase activity in tissues of the urodeles Ambystoma tigrinum (tiger salamander) and Necturus maculosus (mudpuppy)

1. Monoamine oxidase (MAO) activity was determined fluorometrically in tissues of adult mudpuppies, and pre- (young) and post- (adult) metamorphic tiger salamanders. 2. From responses to specific inhibitors it was determined that 95% activity was MAO type A in all tissues. 3. In young salamanders MAO activity was greater in brain and intestine of males than of females, and was considerably higher in kidney of both sexes and in intestine of males compared to adults. 4. MAO activity was distributed differently in the mudpuppy compared to the salamander. Intestine and liver contained high activity and brain had relatively little MAO activity compared to salamander. 5. The apparent Michaelis constant of MAO activity in the different groups and tissues was generally similar, suggesting a similarity of the MAO molecule.     

Histamine and histamine receptors: behavioral thermoregulation in the salamander Necturus maculosus

Low doses (0.01, 0.1 mg/kg, i.p.) of histamine (HA) caused selection of significantly lower temperatures, and higher doses (0.5, 1.0 mg/kg) increased temperatures by mudpuppies in linear thermal gradients. Injection of the HA precursor, L-histidine (500 mg/kg) produced an increase in the temperatures selected. Results from injections of HA H1-receptor agonist (2-pyridylethylamine) and antagonist (pyrilamine), and H2-receptor agonist (dimaprit) and antagonist (cimetidine) had significant effects on thermoregulation; H1-receptors may mediate behavioral hyperthermia and H2-receptors behavioral hypothermia. Responses to these histaminic compounds are significantly influenced by the time of day at which the responses are measured and by season and acclimation temperature. The equivalent behavioral responses in both endotherms and ectotherms to agents which produce physiological hyperthermia and hypothermia are probably behavioral hypothermia ("cold seeking") and behavioral hyperthermia ("heat seeking"), respectively.     

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.     

Electron microscopic and autoradiographic studies on vitellogenesis in Necturus maculosus

Electron microscope studies on Necturus maculosus oocytes ranging in size from 1.1–1.5 mm in diameter indicate the primary proteinaceous yolk to arise within structures referred to in other amphibian oocytes as yolk precursor sacs or bodies. The origin of these yolk precursor sacs appears to result from the activity of the Golgi complexes which form multivesicular and granular-vesicular bodies, the limiting membrane of which is at times incomplete. During differentiation, the yolk precursor sacs contain small vesicles similar in size to Golgi vesicles, larger vesicles similar to vesicular elements of the agranular endoplasmic reticulum and, on occasion, a portion of a mitochondrion. The interior of these sacs becomes granular, perhaps by a dissolution of the components just described, and soon becomes organized into a crystalline configuration. In oocytes 2.0–2.5 mm in diameter, an extensive micropinocytotic activity begins, continues throughout vitellogenesis, and constitutes the primary mechanism for the formation of secondary yolk protein. Numerous coated and smooth-surfaced vesicles, as well as electron-dense and electronlucent ones, fuse in the cortical ooplasm to form progressively larger yolk platelets.     

Thyroid hormone receptor expression in the obligatory paedomorphic salamander Necturus maculosus

Amphibian metamorphosis is under the strict control of thyroid hormones (TH). These hormones induce metamorphosis by controlling gene expression through binding to thyroid hormone receptors (TRs). Necturus maculosus is considered to be an obligatory paedomorphic Amphibian since metamorphosis never occurs spontaneously and cannot be induced by pharmacological means. Since metamorphosis depends on the acquisition of response of tadpole tissues to thyroid hormone, we aimed to determine TR gene expression patterns in Necturus maculosus as well as the expression of two TH-related genes: Cytosolic Thyroid Hormone-Binding Protein (CTHBP)-M2-pyruvate kinase, a gene encoding a cytosolic TH binding protein and stromelysin 3, a direct TH target gene in Xenopus laevis. Tissue samples were obtained from specimens of Necturus maculosus. We performed in situ hybridization using non-cross-hybridizing RNA probes obtained from the cloned Necturus TRalpha and TRbeta genes. We found clear expression of Necturus TRalpha gene in several tissues including the central nervous system, epithelial cells of digestive and urinary organs, as well as myocardium and skeletal muscle. TRbeta was also expressed in the brain. In other tissues, hybridization signals were too low to draw reliable conclusions about their precise distribution. In addition, we observed that the expression of CTHBP and ST3 is largely distinct from that of TRs. The fact that we observed a clear expression of TRalpha and TRbeta which are evolutionary conserved, suggests that Necturus tissues express TRs. Our results thus indicate that, in contrast to previously held hypotheses, Necturus tissues are TH responsive.