Mudpuppy Necturus Research Articles

Environmental DNA Assessment Reveals Restoration Success for Mudpuppies (Necturus maculosus)

Mudpuppies (Necturus maculosus) are secretive, fully aquatic salamanders with a range that spans much of the eastern United States and Canada including the Great Lakes region. Although this species was once abundant, there have been widespread declines due to habitat loss and modification, pollution, lampricide use, and overcollection. We compared environmental DNA (eDNA) and trapping surveys conducted from 2014 to 2016 to determine Mudpuppy occupancy along the St. Clair-Detroit River System, where this indicator species could be a gauge for success of ongoing restoration. Mudpuppy eDNA was detected at all sites with positive trapping records, as well as one site where individuals have not been trapped previously. Sites with shoreline restoration had the highest occupancy estimates, whereas deep-water restoration did not affect Mudpuppy occupancy. Additionally, eDNA surveys resulted in higher detection probability than setline and minnow trap survey methods, illustrating the benefit of using eDNA to detect secretive species. This study demonstrates the success of restoration efforts in increasing the occupancy of an indicator species and can be used as a template for other restoration initiatives.

Yersinia ruckeri Isolated from Common Mudpuppy Necturus maculosus.

During a routine health inspection of apparently healthy wild-caught common mudpuppies Necturus maculosus, the bacteria Yersinia ruckeri was isolated and the identity confirmed using biochemical and molecular methods. This represents the first isolation of Y. ruckeri from an amphibian. This finding increases the known host range capable of harboring this important fish pathogen and could have serious management implications for aquaculture. Furthermore, addressing wild amphibians in fish hatchery biosecurity plans is discussed.

Cd, Cu, Zn, Se, and Metallothioneins in Two Amphibians, Necturus maculosus (Amphibia, Caudata) and Bufo bufo (Amphibia, Anura)

The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85μg/L) for up to 40days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1μg/g in kidneys and gills (0.64-0.95 and 0.52-0.76; n = 4), whereas the levels stayed below 0.5 in liver (0.14-0.29; n = 4) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn-thioneins in liver and Zn,Cu-thioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd-thionein and in pellet extract of kidneys as Zn,Cu,Cd-thioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56-5.0μg/g >0.03-0.72μg/g; n = 11, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all r > 0.80; α = 0.05, n = 5), and between Cd and Se in kidney (r = 0.76; n = 5) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd-thioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.

ISOLATION AND PRIMARY CULTURE OF NECTURUS MACULOSUS (AMPHIBIA: URODELA) HEPATOCYTES

In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7 x 10(5) viable hepatocytes per gram body weight with an average viability of 86 +/- 5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8 degrees C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.

Morphology of brachial segments in mudpuppy (Necturus maculosus) spinal cord studied with confocal and electron microscopy.

The isolated brachial spinal cord of Necturus maculosus is useful for studies of neural networks underlying forelimb locomotion, but information about its cellular morphology is scarce. We addressed this issue by using confocal and electron microscopy. Remarkably, the central region of gray matter was aneural and consisted exclusively of a tenuous meshwork of glial fibers and large extracellular spaces. Somata of motoneurons (MNs) and interneurons (INs), labeled by retrograde transport of fluorescent tracers from ventral roots and axons in the ventrolateral funiculus, respectively, were confined within a gray neuropil layer abutting the white matter borders, whereas their dendrites projected widely throughout the white matter. About one-third of labeled INs were found contralaterally, with axons crossing ventral to a thick layer of ependyma surrounding the central canal. Lateral MN dendrites proliferated under the pial surface to form a dense, thin (1-2 microm) plexus immediately beneath a thin layer of glial fibrillary acidic protein-positive glia limitans. The latter contained arrays of unusual tubular structures (diameter 200-400 nm, length 3 microm) that resembled mitochondria but lacked double membranes or cristae. Dorsal roots (DRs) produced dense presynaptic arbors within a wedge-shaped afferent termination zone medial to the dorsal root entry, within which dendrites of MNs and INs 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 designing electrophysiological experiments to study the neural circuits involved in locomotor pattern generation.

Assessment of rates of deformity in wild frog populations using in situ cages: a case study of Leopard Frogs (Rana pipiens) in Ontario, Canada

High rates of deformity in wild amphibian populations from north-eastern North America have been increasingly reported since 1995. In the St Lawrence River basin (Canada) elevated frequencies of limb and eye deformities in mudpuppies (Necturus maculosus) and leopard frogs (Rana pipiens) were recorded in the early 1990s. A caging study was conducted during 1998 to verify the rates recorded in leopard frogs and pursue the potential causes of deformities seen in juveniles and adults. Week-old leopard frog tadpoles were collected from a reference wetland and maintained through to metamorphosis in cages in previously identified high risk wetlands. Deformity frequencies were measured and compared with frequencies measured in wild populations of leopard frogs inhabiting the same wetlands. The results of caging studies and sampling of wild populations were also compared with corresponding data collected from a reference wetland. No deformities were observed in caged or wild reference animals. Very low deformity frequencies (up to 2.2%) were observed in frogs caged in high risk wetlands, but greater frequencies (3.4-10%) were observed in wild young-of-the-year frogs captured at the same sites. The types of deformities were similar among groups; they included fused, missing or extra digits and disproportionate hindlimb length or eye pupil size. In addition, mortality rates were elevated in two cages in high risk wetlands. In general, the caging procedure was effective in establishing the potential for production of deformities in the waters of a given wetland, but tended to underestimate the rates calculated for samples of wild populations. The ramifications of the first-year findings for similar assessments of amphibian deformity rates and establishment of cause-effect linkages are discussed.

Thyroid hormone receptor genes of neotenic amphibians.

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to alpha and beta genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3',5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRalpha in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRbeta expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRalpha and TRbeta may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis.

Potassium conductance activated during regulatory volume decrease by mudpuppy red blood cells.

The cellular basis of regulatory volume decrease (RVD) by mudpuppy (Necturus maculosus) red blood cells (RBCs) was examined. Volume regulation was inhibited by replacing extracellular Na+ with K+. In contrast, addition of gramicidin (5 microM) to the extracellular medium enhanced RVD. The K(+)-channel blocker quinine (1 mM) also inhibited RVD, and this inhibition was reversed by gramicidin (5 microM). In addition, a 0 Ca(2+)-EGTA Ringer blocked RVD, whereas the Ca2+ ionophore A23187 ( microM) enhanced recovery of cell volume. The stretch-activated ion channel antagonist gadolinium (10 microM) inhibited RVD, and this effect was reversed by A23187 (2 microM). Furthermore, the calmodulin inhibitors pimozide (10 microM) and N-(6-aminohexyl)-5-chloro-1-napthalene-sulfonamide (0.1 mM) blocked RVD, and this inhibition was reversed with gramicidin (5 microM). Consistent with these findings, a K(+)-selective membrane conductance was activated by exposing RBCs to a 0.5x Ringer solution (observed with the whole cell patch clamp technique). This conductance was inhibited by quinine (1 mM), gadolinium (10 microM), and pimozide (10 microM). These results indicate that cell swelling activates a K+ conductance by a Ca(2+)-calmodulin-dependent mechanism and that this channel mediates K+ loss during RVD.

Phylogeny of the Polystomatidae (Platyhelminthes, Monogenea), with particular reference to Polystoma integerrimum

Polystome phylogeny is examined, with emphasis on the dimorphism of the frog parasite Polystoma integerrimum, which exists in a fully differentiated and a neotenic form, and the evolutionary development of exclusively neotenic genera. Protopolystoma, which infects the aquatic toad Xenopus, has essentially the same morphology as the neotenic (branchial) adult of P. integerrimum and is interpreted as a neotenic genus; however, it inhabits the bladder of its host, the infection site of the normal adult of the dimorphic species. The sphyranurid Sphyranura, ectoparasitic on the external gills of the mud puppy Necturus, resembles the two-sucker larva of P. integerrimum in possessing a single pair of haptorial suckers in place of the 6 suckers of adult polystomes, and is probably a neotenic parasite associated with a neotenic host. The neotenic animals in general are parasites of aquatic hosts, and the uterus is lost or reduced in these genera; by contrast, uterine function is greatly enhanced among polystomes infecting amphibians best adapted to terrestrial life.

Action potentials and chemosensitive conductances in the dendrites of olfactory neurons suggest new features for odor transduction.

Odors affect the excitability of an olfactory neuron by altering membrane conductances at the ciliated end of a single, long dendrite. One mechanism to increase the sensitivity of olfactory neurons to odorants would be for their dendrites to support action potentials. We show for the first time that isolated olfactory dendrites from the mudpuppy Necturus maculosus contain a high density of voltage-activated Na+ channels and produce Na-dependent action potentials in response to depolarizing current pulses. Furthermore, all required steps in the transduction process beginning with odor detection and culminating with action potential initiation occur in the ciliated dendrite. We have previously shown that odors can modulate Cl- and K+ conductances in intact olfactory neurons, producing both excitation and inhibition. Here we show that both conductances are also present in the isolated, ciliated dendrite near the site of odor binding, that they are modulated by odors, and that they affect neuronal excitability. Voltage-activated Cl- currents blocked by 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid and niflumic acid were found at greater than five times higher average density in the ciliated dendrite than in the soma, whereas voltage-activated K+ currents inhibited by intracellular Cs+ were distributed on average more uniformly throughout the cell. When ciliated, chemosensitive dendrites were stimulated with the odorant taurine, the responses were similar to those seen in intact cells: Cl- currents were increased in some dendrites, whereas in others Cl- or K+ currents were decreased, and responses washed out during whole-cell recording. The Cl- equilibrium potential for intact neurons bathed in physiological saline was found to be -45 mV using an on-cell voltage-ramp protocol and delayed application of channel blockers. We postulate that transduction of some odors is caused by second messenger-mediated modulation of the resting membrane conductance (as opposed to a specialized generator conductance) in the cilia or apical region of the dendrite, and show how this could alter the firing frequency of olfactory neurons.

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.

Physiological and morphological correlations of horizontal cells in the mudpuppy retina.

1. Horizontal cells (HCs) of the mudpuppy (Necturus maculosus) retina were physiologically characterized with the use of intracellular recordings in a superfused, dark-adapted, retina-eyecup preparation. 2. Physiological analysis included an evaluation of rod versus cone input and a determination of the receptive field size with the use of a displaced slit of light. 3. The morphology of HCs was established through intracellular staining with horseradish peroxidase (HRP) and Lucifer yellow mixed in a single electrode. 4. Three types of horizontal cells were identified, each associated with a distinct morphology. Physiological subtypes included luminosity (L) and chromaticity (C) cells. Morphological diversities included single axon-bearing, multiple axon-bearing and, nonaxon-bearing cells. All C-type HCs lacked axons. 5. Approximately 90% of HCs encountered in this study were L-type cells, which received sign-conserving inputs from both rods and cones. These cell types contained one or more long axons that often stretched greater than 500 microns. This group was morphologically diverse, particularly with respect to variations in the number of axons, but we were unable to correlate this diversity with any unique set of physiological properties. 6. Several C-type HCs were identified (n = 8). These cells depolarized to a low-intensity, short-wavelength (SW) stimulus, whereas they hyperpolarized to high-intensity, long-wavelength stimuli. Morphologically, these cells were axonless (n = 4), with relatively small dendritic fields. 7. A third group of HCs were classified as "short wavelength preferring" HCs (n = 7). These cells responded better to a SW stimulus at all intensity levels. They were thus dissimilar to the common L-type HCs, which showed an apparent rod to cone transition as the stimulus intensity increased, suggestive of a shift from rod to cone preference. Morphologically, these cells were axonless (n = 2), but had broader dendritic fields than the C-type HCs. 8. Our observations indicate that the horizontal cell population of the mudpuppy retina is considerably more complex than previously supposed. The existence of both axon-bearing and axonless HCs, which could be correlated with L- and C-type physiology, implies that HCs may support more than one function in outer retina processing.

Ultrastructure of taste cells and synapses in the mudpuppy Necturus maculosus.

Taste buds in the mudpuppy Necturus maculosus were examined with electron microscopy. Three cell types (dark, light, and basal) were identified and reconstructed from serial thick sections. Dark and light cells extend from the basal lamina to the surface of the tongue. The apical process of the dark cells was usually quite lamellar when viewed in cross section, in contrast to light cells, whose apical process appeared more cylindrical. Basal cells are situated at the base of the bud and do not extend processes to the surface of the tongue. The cytoplasm of basal cells contains numerous clear and dense-cored vesicles. Small, spinelike processes (2-3 microns in length) project outward from the basal cells into the cytoplasm of the surrounding tast receptor cells. Morphologically, basal cells in mudpuppy taste buds resemble Merkel cells. Unmyelinated afferent nerve fibers enter the taste bud at the base and course through the lower portion of the bud. Synapses were found between taste receptor cells and nerve fibers, between basal cells and nerve fibers, and between basal cells and taste receptor cells. Over 65% of the synapses observed in the mudpuppy taste bud involved the basal cell. These findings suggest that basal cells play some role in chemosensory signal processing or integration of the taste response.

Electrogenic Na/HCO3 cotransport across basolateral membrane of isolated perfused Necturus proximal tubule.

This study was undertaken to determine whether the proximal tubule of the mud puppy Necturus maculosus possesses a basolateral Na/HCO3 cotransporter. We examined the effects on basolateral membrane potential (Vbl) and intracellular pH (pHi) of 1) lowering basolateral [HCO3-] at constant PCO2, and 2) replacing Na+ with N-methyl-D-glucamine. Vbl and pHi were measured with Ling-Gerard and liquid-membrane pH microelectrodes, respectively, in isolated tubules perfused in vitro. We found that decreasing basolateral [HCO3-] from 10 mM (pH 7.5) to 2 mM (pH 6.8) resulted in an immediate depolarization of 14.9 mV, and a pHi decrease of 0.35. SITS (4-acetamido-4'-isothiocyanostibene-2,2'-disulfonic acid, 0.5 mM) inhibited the HCO3-induced depolarization by 87% and inhibited the initial rate of the pHi decrease by 79%. Replacement of basolateral Na+ with N-methyl-D-glucamine resulted in an immediate depolarization of 11.3 mV, and a pHi decrease of 0.36. SITS inhibited the zero Na-induced depolarization by 86% and the initial rate of the pHi decrease by 81%. Nominal removal of basolateral HCO3- (replaced with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) inhibited the zero Na-induced depolarization by 64%, whereas nominal removal of Na+ inhibited the 2 mM HCO3-induced depolarization by 67%. Replacement of all basolateral Cl- with glucuronate did not inhibit the changes in Vbl induced by changing [HCO3-] or [Na+]. Observations similar to those described above have been made previously on Ambystoma proximal tubules, and attributed to an electrogenic Na/HCO3 cotransport mechanism that carries HCO3-, Na+, and net negative charge in the same direction. We conclude that Necturus proximal tubules possess a similar, if not identical, electrogenic Na/HCO3 cotransport mechanism.

Regenerative impulses in taste cells.

Taste cells and nongustatory epithelial cells in the isolated lingual mucosa from the mud puppy Necturus maculosus were impaled with microelectrodes. The taste cells, but not surrounding epithelial cells, were electrically excitable when directly stimulated with current passed through the recording electrode. Action potentials produced by taste cells had both a sodium and a calcium component.

Effect of Temperature on Sprint Performance in the Frog Xenopus laevis and the Salamander Necturus maculosus

Sprint performance is strongly temperature dependent in the mudpuppy Necturus maculosus and the African clawed frog Xenopus laevis. In experiments designed to control the effects of physical training and temperature acclimation on sprint performance, the time to swim 1.5 m was lowest at 15 C in Necturus, and at 27 C in Xenopus, and increased as temperature was either raised or lowered from these optima. These temperatures are similar to those selected by these species in laboratory thermal gradients.

Transient adaptation and sensitization in the retina of Necturus.

Responses to repetitive stimulation were monitored at several retinal levels in the eyecup of the mudpuppy Necturus maculosus. When alternating sequences of low-intensity small and large spots were presented, two effects were found, which could be localized to the proximal retina: (a) response decrement (RD), in which, after the first small spot response, subsequent small spot responses are decreased in amplitude and (b) transient response enhancement (TRE), in which the first small spot response after a large spot sequence is larger than preceding or subsequent small spot responses. RD and TRE are absent or weak in sustained on or off responses (horizontal and bipolar cells, and ON and OFF ganglion cell post-stimulus time histograms (PSTH) but are particularly well developed in the on/off responses of the proximal retina (proximal negative response, M-wave, PSTHs of ON/OFF ganglion cells, and intracellular responses from on/off neurons and Müller cells). RD and TRE appear to arise from a stimulus-evoked slow depolarization in on/off neurons that interacts with the amplitude of succeeding responses. We conclude that RD and TRE are a form of neural adaptation that is largely specific to the on/off channels of the proximal retina.

Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones

1. Synaptic transmission was studied in visually identified parasympathetic ganglion cells that modulate the heart beat of the mudpuppy Necturus maculosus).2. The brief pulse of acetylcholine (ACh) released from terminals of the vagus nerve after each impulse can produce two distinct post-synaptic responses in individual principal cells of the ganglion: (i) within a milli-second of release, ACh generates a rapid and strong excitatory post-synaptic potential (e.p.s.p.) that normally initiates a post-synaptic impulse; (ii) this excitation is usually followed by a slow hyperpolarizing inhibitory post-synaptic potential (i.p.s.p.) that lasts for several seconds. The magnitude and time course of the i.p.s.p. depends on the frequency and number of vagal stimuli. When the hydrolysis of ACh is inhibited by prostigmine, a train of nerve stimuli may be followed by an i.p.s.p. lasting half a minute or longer.3. The rapid e.p.s.p. and slow i.p.s.p. result from the direct action of ACh on two different types of chemoreceptors in the post-synaptic membrane of the principal cell. The e.p.s.p. can be preferentially blocked by the nicotinic antagonist dihydro-beta-erythroidine (5 x 10(-7)M), while the i.p.s.p. is selectively blocked by the muscarinic antagonist atropine (5 x 10(-9)M).4. Potentials resembling nerve-evoked e.p.s.p.s and i.p.s.p.s can be produced by iontophoretic release of ACh from micropipettes onto the post-synaptic membrane. Application of the muscarinic agonist bethanechol generates exclusively inhibitory responses.5. The reversal potential for the i.p.s.p. is about -105 mV, which is approximately the equilibrium potential for potassium (E(K)). When the external K(+) concentration is altered, the reversal potential for inhibition is shifted to the new value of E(K) as expected from the Nernst equation. Changes in the external Na(+) and Cl(-) concentrations have no appreciable effect on the reversal potential. Thus, the i.p.s.p. is the result of a conductance increase for K(+).6. The conductance change producing the i.p.s.p. is voltage sensitive. When the membrane potential is shifted from -40 to -60 mV, the i.p.s.p becomes larger and longer. Beyond -60 mV the inhibitory response decreases in proportion to the driving force on K(+) without any further change in time course.7. The inhibitory response produced by an iontophoretically applied pulse of bethanechol has a delayed onset of about 150 msec at 24 degrees C. The early portion of this response, including the delay, is proportional to t(3), where t is time. The proportionality factor (the apparent rate constant) decreases elevenfold when the temperature is lowered by 10 degrees C. This suggests that a multi-step process is involved in the activation of the conductance increase that leads to the inhibitory response. Inhibitory responses with similar kinetics were produced in heart muscles of the mudpuppy upon application of ACh.

The permeability of the skin of a neotenous urodele amphibian, the mudpuppy Necturus maculosus.

1. The permeability of the isolated skin of a neotenous urodele amphibian, the mudpuppy Necturus maculosus, to Na, Cl, urea and water was measured. 2. Unidirectional transcutaneous flux measurements and the action of ouabain and amiloride, showed that there was normally no active Cl or Na transport, nor a Cl/Cl exchange diffusion process. 3. Amphotericin B initiated a transcutaneous potential difference and short-circuit current, which could be inhibited by ouabain. 4. The short-circuit current was nearly equivalent to the net Na Transport and this was also inhibited by ouabain. 5. A transcutaneous active Na transport mechanism thus appears to be incipient in the mudpuppy but is limited by a low permeability of the outer barrier of the cells. 6. Vasotocin increased the skin's diffusion permeability for water but had no effect on the influx of Na or urea. 7. The function of Necturus skin is in several respects unique compared to that of other amphibians.

Effects of prolactin on hypophysectomized mud puppies Necturus maculosus.

Effects of prolactin on hypophysectomized mud puppies Necturus maculosus.