Axolotl Larvae Research Articles

The Source of Melanocytes in Ortho- and Heterotopic Tail Regenerates of Axolotls and the Dependence of the Regenerative Response on the Presence of Neural Tissue

We studied the regeneration of orthotopic and heterotopic tails in larval axolotls. First, we analyzed tail regeneration following reciprocal exchange of cuffs of tail integument between dark-colored (wild-type) and yellow-colored (hybrid) larval animals. Second, we studied tail regeneration in larval axolotls following transplantation of cuffs of tail integument from metamorphosed dark-colored conspecifics and from an adult fire salamander. In all cases, the amputation planes involved the transplanted integumental cuffs. In the first experiment, the regenerated tails showed the color of the host animals, not that of the transplanted cuffs. This suggests that the melanocytes of the regenerated tails were derived from the host hypodermis. Following transplantation of metamorphosed skin from axolotls and a fire salamander onto larval axolotls, the metamorphosed epidermis reverted to a larval condition. This indicates that the state of differentiation of the metamorphosed epidermis was not permanent. Rather, in order to maintain the metamorphosed epidermal structure, a continuous exposure of the animals to sufficient levels of thyroid hormones was required. Transplantation of tail buds from yellow-colored onto dark-colored axolotl embryos caused the formation of yellow-colored tails both in the head and the anterior limb region of the hosts. Incomplete resection of these heterotopic tails was followed by tail regeneration, while no tail regeneration occurred following complete resection of the heterotopic tails. Successful tail regeneration depended on the presence of neural tissue along the resection plane.

Microplastics are transferred in a trophic web between zooplankton and the amphibian Axolotl (Ambystoma mexicanum): Effects on their feeding behavior

Microplastics are contaminants that are often ingested, bioaccumulated, and transferred through food chain, affecting aquatic ecosystems. Zooplankton is susceptible to ingesting microplastics, so it is probably a vector that transfers microplastics to higher trophic levels. Cladocerans are a diet of amphibian larvae. Amphibians' survival is under threat worldwide due to their sensitivity to pollution. This work aimed to determine the transfer of zooplankton microplastics to Ambystoma mexicanum larvae (known as axolotl), for which we evaluated two responses in the first five weeks of development: 1) functional response of the larvae and 2) examination of their feces. Cladocerans were exposed to a diet of phytoplankton and microplastics to use them as food for axolotl larvae. Five axolotl larvae were randomly selected each week, each fed with a different concentration of cladocerans, and prey consumption was recorded for each larva. Finally, all the feces of the axolotls expelled after feeding were digested and examined under a light microscope. We found that microplastics were transferred from the cladocerans to A. mexicanum. Microplastics negatively affected the larval feeding behavior since the axolotls fed zooplankton exposed to microplastics consumed less prey than those of the control group. We also observed that microplastics were present in 78% of the feces. Microplastics reduce the feeding of juvenile A. mexicanum, an endemic amphibian of Mexico, in critical danger of extinction.

Evi5 is required for Xenopus limb and tail regeneration.

Amphibians such as salamanders and the African clawed frog Xenopus are great models for regeneration studies because they can fully regenerate their lost organs. While axolotl can regenerate damaged organs throughout its lifetime, Xenopus has a limited regeneration capacity after metamorphosis. The ecotropic viral integrative factor 5 (Evi5) is of great interest because its expression is highly upregulated in the limb blastema of axolotls, but remains unchanged in the fibroblastema of post-metamorphic frogs. Yet, its role in regeneration-competent contexts in Xenopus has not been fully analyzed. Here we show that Evi5 is upregulated in Xenopus tadpoles after limb and tail amputation, as in axolotls. Down-regulation of Evi5 with morpholino antisense oligos (Mo) impairs limb development and limb blastema formation in Xenopus tadpoles. Mechanistically, we show that Evi5 knockdown significantly reduces proliferation of limb blastema cells and causes apoptosis, blocking the formation of regeneration blastema. RNA-sequencing analysis reveals that in addition to reduced PDGFα and TGFβ signaling pathways that are required for regeneration, evi5 Mo downregulates lysine demethylases Kdm6b and Kdm7a. And knockdown of Kdm6b or Kdm7a causes defective limb regeneration. Evi5 knockdown also impedes tail regeneration in Xenopus tadpoles and axolotl larvae, suggesting a conserved function of Evi5 in appendage regeneration. Thus, our results demonstrate that Evi5 plays a critical role in appendage regeneration in amphibians.

A histological study of normal and pathological limb regeneration in the Mexican axolotl Ambystoma mexicanum.

Salamanders show unparalleled capacities of tissue regeneration amongst tetrapods (four-legged vertebrates), being able to repair and renew lost or damage body parts, such as tails, jaws, and limbs in a seemingly perfect fashion. Despite countless studies on axolotl (Ambystoma mexicanum) regeneration, only a few studies have thus far compared gross morphological and histological features of the original and regenerated limb skeleton. Therein, most studies have focused on nerves or muscles, while even fewer have provided detailed information about bones and cartilage. This study compares skeletal tissue structures of original and regenerated limbs with respect to tissue level histology. Histological serial sections of 55 axolotl larvae were generated, including 29 limbs that were severed by conspecifics, and 26 that were subject to targeted amputations. Amputations were executed in several larval stages (48, 52, and 53) and at different limb positions (humeral midshaft, above the mesopod). In addition, 3D reconstructions were prepared based on X-ray microtomography scans. The results demonstrate that regenerated forelimbs show a diversity of limb and digit abnormalities as a result of imperfect regeneration. Furthermore, abnormalities were more severe and more frequent in regenerated forelimbs caused by natural bites as compared with regenerated forelimbs after amputation. The results indicate that abnormalities occur frequently after regeneration in larval axolotls contradicting the notion of regeneration generally resulting in perfect limbs.

Normal development in Ambystoma mexicanum: A complementary staging table for the skull based on Alizarin red S staining.

As the role of Ambystoma mexicanum, or the Mexican axolotl, expands in research applications beyond its traditional use in studies of limb regeneration, a staging table that is more anatomically extensive is required. Here, we describe axolotl skull development as it relates to previously established developmental stages that were based on limb development. We find that most key developmental events in the skull correspond to these previously established stages, creating easily recognizable stages of axolotl throughout skull morphogenesis. With this complementary staging table in hand, researchers can stage axolotl larvae when limb data are missing or incomplete, or when cranial data alone is available.

Using dual labeling of thymidine analogs to investigate cell cycle dynamics in the regenerating salamander limb

For almost two centuries, scientists have observed that most forms of regeneration fail to regrow in the absence of nerves, but at the cellular level, how the nerves regulate this phenomenon is still unknown. Understanding how cell cycle kinetics change when the nerve supply is cut would help us understand what role nerves play during regeneration. We used dual labeling of cells using thymidine analogs, 5‐bromo‐2′‐deoxyuridine (BrdU) and 5‐ethynyl‐2‐deoxyuridine (EdU), to quantify the changes in the cell cycle in the denervated regenerating limb of axolotl salamanders. Eight larval axolotls were denervated in their left limbs 12 days after amputation and injected with BrdU and EdU at 24 hour intervals. 72 hours after denervation, the limbs were collected and stained for DNA synthesis (S), mitosis (M), and cell death markers. The duration of S phase of the cell cycle shortened in the actively proliferating cells of denervated regenerating limb almost by half compared to those of the innervated regenerating limb. The total cell cycle lengths differed by about 10%, which suggests that the growth phases (G1 and G2) may have increased in length in the denervated limb. When the nerve supply was cut, less cells returned to cell cycle. Our results suggest that the nerves may regulate the duration of S phase. Future analysis of upregulated and downregulated proteins such as transcription factors during cell proliferation in denervated limb blastema may suggest what pathways are causing the observed changes in the cell cycle.Support or Funding InformationThis project was supported by Summer Scholars Independent Research Fellowship from the Office of Undergraduate Research and Fellowships and NSF grants 1558017 and 1656429.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pattern of Lateralized Behaviors in a Caudate Amphibian, <i>Ambystoma mexicanum</i>

Laterality of brain and behavior at the individual and/or group level is a characteristic of all vertebrate classes. The general pattern of left-right asymmetry in vertebrates is so that left-eye/right-hemisphere system is used to detect socially important images, like conspecifics, and also recognize danger, while the right-eye/left-hemisphere system is used to discriminate prey and food items from inedible objects. However, the pattern of motor and visual lateralization in caudate amphibians is poorly investigated to date. Here we present the first evidence of individual and group level motor and visual lateralization in sexually mature and immature larvae of the Mexican axolotl, Ambystoma mexicanum , thus presenting a comprehensive characteristic of the lateralized behavioral pattern in a salamander species. First, we performed four successive tests to assess asymmetry in rapid C-start and measure its characteristics in responses to startle stimuli in sexually immature larvae. We experimentally showed that axolotls exhibited a leftward population bias in each of four C-start tests, of which the bias was significant in the second test and in the pooled data for all four tests. Second, in mature axolotls we have found right-sided individual level asymmetry and, at the group level, a tendency to react more actively to food items (live chironomid larvae) appeared in their right visual hemifield. Third, individual axolotl larvae been placed in the square aquarium with two opposite mat walls and two opposite mirror walls preferred to stay near to mirror walls, rather than near to mat walls. At the group level, this preference increased over three successive tests and reached significance in the third test and in the pooled data. At both individual and group levels axolotls showed statistically significant lateralization, preferring to inspect the mirror walls with their left eyes in all three tests. Overall, the expressed pattern of lateralization in the Mexican axolotl corresponds to the general pattern of lateralization established for other vertebrates including anuran amphibians.

Embryonic yolk removal affects neither morphology nor escape performance of larval axolotls

Maternal effects, the influences of maternal phenotype on the phenotypes of her offspring, mediate early ontogenetic traits through maternal investment. In amphibians, provisioning eggs with yolk is the main source of maternal investment. While larger eggs generally result in larger, higher-quality offspring, the relationship between egg size and offspring phenotype is complicated because offspring can evolve to be more or less responsive to variation in yolk provisions. Previous studies of several ambystomatid salamanders suggest that the effects of embryonic yolk reserve reduction on hatchling life history traits increase with egg size. In this study, a similar controlled experimental yolk removal technique in Ambystoma mexicanum was used to determine the effects of reduced yolk reserves on phenotypes including hatching time and stage, hatchling and larval size and performance in predation trials with fish. Surprisingly, yolk reduction revealed no effects on any traits. These findings suggest that larval morphology in A. mexicanum is highly canalized and larval phenotypes are decoupled from yolk reserve variation. This surprising lack of yolk removal effects in hatchling and larval axolotls illustrates the evolutionary flexibility of early life history traits. Traits can evolve to increase or decrease their response to resources and can even become completely unresponsive. Since we found no effects in early life history, we hypothesize that domestication of the axolotl may have altered yolk properties or allocation dynamics and that maternal investment in yolk reserves may manifest at later life stages by reducing the time to reproductive maturity or increasing fecundity.

Analysis of signal processing in vestibular circuits with a novel light-emitting diodes-based fluorescence microscope.

Optical visualization of neural network activity is limited by imaging system-dependent technical tradeoffs. To overcome these constraints, we have developed a powerful low-cost and flexible imaging system with high spectral variability and unique spatio-temporal precision for simultaneous optical recording and manipulation of neural activity of large cell groups. The system comprises eight high-power light-emitting diodes, a camera with a large metal-oxide-semiconductor sensor and a high numerical aperture water-dipping objective. It allows fast and precise control of excitation and simultaneous low noise imaging at high resolution. Adjustable apertures generated two independent areas of variable size and position for simultaneous optical activation and image capture. The experimental applicability of this system was explored in semi-isolated preparations of larval axolotl (Ambystoma mexicanum) with intact inner ear organs and central nervous circuits. Cyclic galvanic stimulation of semicircular canals together with glutamate- and γ-aminobutyric acid (GABA)-uncaging caused a corresponding modulation of Ca(2+) transients in central vestibular neurons. These experiments revealed specific cellular properties as well as synaptic interactions between excitatory and inhibitory inputs, responsible for spatio-temporal-specific sensory signal processing. Location-specific GABA-uncaging revealed a potent inhibitory shunt of vestibular nerve afferent input in the predominating population of tonic vestibular neurons, indicating a considerable impact of local and commissural inhibitory circuits on the processing of head/body motion-related signals. The discovery of these previously unknown properties of vestibular computations demonstrates the merits of our novel microscope system for experimental applications in the field of neurobiology.

High-efficiency electroporation of the spinal cord in larval axolotl.

Axolotls are well known for their remarkable ability to regenerate complex body parts and structures throughout life, including the entire limb and tail. Particularly fascinating is their ability to regenerate a fully functional spinal cord after losing the tail. Electroporation of DNA plasmids or morpholinos is a valuable tool to gain mechanistic insight into the cellular and molecular basis of regeneration. It provides among other advantages a simple and fast method to test gene function in a temporally and spatially controlled manner. Some classic drawbacks of the method, such as low transfection efficiency and damage to the tissue, had hindered our understanding of the contribution of different signaling pathways to regeneration. Here, we describe a comprehensive protocol for electroporation of the axolotl spinal cord that overcomes this limitations using a combination of high-voltage and short-length pulses followed by lower-voltage and longer-length pulses. Our approach yields highly efficient transfection of spinal cord cells with minimal tissue damage, which now allows the molecular dissection of spinal cord regeneration.

Abdominal Distension Associated with Luminal Fungi in the Intestines of Axolotl Larvae

Axolotls show a remarkable regeneration capacity compared with higher vertebrates, regenerating missing appendages such as limbs and tail as well as other body parts (i.e., apex of the heart, forebrain, and jaw) after amputations which makes this animal a very interesting research model for tissue regeneration mechanisms. Larvae are individually housed in a 20% Holtfreter’s solution within clear plastic containers. The photoperiod light : darkness cycle is 12 : 12 h. Larvae with a total body length of less than 5 cm are fed once a day with large brine shrimp and blood worm. Albino larvae appeared to have a tendency to exhibit abdominal distention. No clinical signs of illness seemed to be associated with the condition; however, these animals exhibit a relatively slower growth rate. To better characterize this condition, we performed histological sectioning for cross sectional slide preparation on wild type and albino axolotl larvae following euthanasia. The only lesion seen in the albino larvae was a thickened gut wall and the presence of fungi within the intestines. We hypothesize that this may be due to a lower efficacy of the albino larvae’s immune system.

Plasticity for axolotl lens regeneration is associated with age-related changes in gene expression.

Mexican axolotls lose potential for lens regeneration 2 weeks after hatching. We used microarrays to identify differently expressed genes before and after this critical time, using RNA isolated from iris. Over 3700 genes were identified as differentially expressed in response to lentectomy between young (7 days post‐hatching) and old (3 months post‐hatching) axolotl larvae. Strikingly, many of the genes were only expressed in the early or late iris. Genes that were highly expressed in young iris significantly enriched electron transport chain, transcription, metabolism, and cell cycle gene ontologies, all of which are associated with lens regeneration. In contrast, genes associated with cellular differentiation and tissue maturation were uniquely expressed in old iris. Many of these expression differences strongly suggest that young and old iris samples were collected before and after the spleen became developmentally competent to produce and secrete cells with humoral and innate immunity functions. Our study establishes the axolotl as a powerful model to investigate age‐related cellular differentiation and immune system ontogeny within the context of tissue regeneration.

Microarray analysis of a salamander hopeful monster reveals transcriptional signatures of paedomorphic brain development

BackgroundThe Mexican axolotl (Ambystoma mexicanum) is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. Unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral (metamorphic) form. The basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae (Ambystoma tigrinum tigrinum) that typically undergo a metamorphosis.ResultsTranscript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching (42, 56, 70, 84 dph) and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. Collectively, more differentially expressed genes (DEGs) were identified as unique to the axolotl (n = 76) and tiger salamander (n = 292) than were identified as shared (n = 108). All but two of the shared DEGs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. A second, complementary analysis that directly compared the expression of 1320 genes between the species identified 409 genes that differed as a function of species or the interaction between time and species. Of these 409 DEGs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times.ConclusionsMany of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. However, the axolotl also showed unique patterns of transcription early in development. In particular, the axolotl showed a genome-wide reduction in mRNA abundance across loci, including genes that regulate hypothalamic-pituitary activities. This suggests that an axolotls failure to undergo anatomical metamorphosis late in the larval period is indirectly associated with a mechanism(s) that acts earlier in development to broadly program transcription. The axolotl hopeful monster provides a model to identify mechanisms of early brain development that proximally and ultimately affect the expression of adult phenotypes.

Functional characterization of the vertebrate primary ureter: Structure and ion transport mechanisms of the pronephric duct in axolotl larvae (Amphibia)

BackgroundThree kidney systems appear during vertebrate development: the pronephroi, mesonephroi and metanephroi. The pronephric duct is the first or primary ureter of these kidney systems. Its role as a key player in the induction of nephrogenic mesenchyme is well established. Here we investigate whether the duct is involved in urine modification using larvae of the freshwater amphibian Ambystoma mexicanum (axolotl) as model.ResultsWe investigated structural as well as physiological properties of the pronephric duct. The key elements of our methodology were: using histology, light and transmission electron microscopy as well as confocal laser scanning microscopy on fixed tissue and applying the microperfusion technique on isolated pronephric ducts in combination with single cell microelectrode impalements. Our data show that the fully differentiated pronephric duct is composed of a single layered epithelium consisting of one cell type comparable to the principal cell of the renal collecting duct system. The cells are characterized by a prominent basolateral labyrinth and a relatively smooth apical surface with one central cilium. Cellular impalements demonstrate the presence of apical Na+ and K+ conductances, as well as a large K+ conductance in the basolateral cell membrane. Immunolabeling experiments indicate heavy expression of Na+/K+-ATPase in the basolateral labyrinth.ConclusionsWe propose that the pronephric duct is important for the subsequent modification of urine produced by the pronephros. Our results indicate that it reabsorbs sodium and secretes potassium via channels present in the apical cell membrane with the driving force for ion movement provided by the Na+/K+ pump. This is to our knowledge the first characterization of the pronephric duct, the precursor of the collecting duct system, which provides a model of cell structure and basic mechanisms for ion transport. Such information may be important in understanding the evolution of vertebrate kidney systems and human diseases associated with congenital malformations.

Organophosphorus pesticides effect on early stages of the axolotl Ambystoma mexicanum (Amphibia: Caudata)

Ambystoma mexicanum is an endemic salamander of Xochimilco, a wetland of the basin of Mexico valley. Nowadays, axolotl populations are decreasing due environmental stressors. Particularly, studies about organophosphorus pesticides (OPPs; i.e. chlorpyrifos and malathion) toxicity are of great importance due to their intensive use in agricultural activities in Xochimilco. Thus, the aim of this study was to evaluate under controlled conditions the toxicity of chlorpyrifos (CPF) and malathion (MLT) on embryos and larvae (stage 44 and 54) of A. mexicanum. Embryos and larvae were exposure 96 h from 0.5 to 3 mg CPF L −1 and from 10 to 30 mg MLT L −1 in independent tests. Embryos at the end of this period were maintained 9 d without pesticide in order to identify possible recuperation. Differences obtained in mortality, hatching success, development, morphological abnormalities, behaviour and activity, suggest that toxicity of CPF and MLT differs in embryos and larval stages. Embryos were less sensitive to OPPs acute exposure than axolotl larvae; additionally, toxicity of CPF in larval stages was greater than MLT. On the other hand, data obtained in axolotl embryos during the period of recuperation to CPF in particular as delay and inhibition of development, malformations and success of hatching, indicated that these responses turned out more sensitive than mortality. This study allowed to identify the toxicological potential of OPPs on early developmental stages of A. mexicanum and it is a valuable contribution for the future management of the axolotl wild population.

Early expression of two TdT isoforms in the hematopoietic system of the Mexican axolotl. Implications for the evolutionary origin of the N-nucleotide addition.

Nontemplate (N)-nucleotide addition by the terminal dideoxynucleotidyl transferase (TdT) at the junctions of rearranging V( D) J gene segments greatly contribute to antigen-receptor diversity. TdT has been identified in several vertebrate species, where it is highly conserved. We report here the isolation of two forms of TdT mRNA in an amphibian, the Mexican axolotl. The isoform TdT1 shares all of the conserved structural motifs required for TdT activity and displays an average of 50-58% similarity at the amino acid level with TdT of other species. The second axolotl TdT variant ( TdT2) differs from TdT1 by a 57-amino acid deletion located between amino acids 165-222 of TdT1, including the first helix-hairpin-helix DNA-binding motif. During ontogeny, TdT products are first detected in the head of 6-week-old larvae and further in the head and trunk of 8-month-old larvae. These developmental stages correspond to the first detection of RAG1 and antigen-receptor (TCRbeta and IgHmicro) products in axolotl larvae. Our results suggest that in contrast to mammalian development, N diversity occurs early in axolotl development to diversify the primary repertoire. Phylogenetic analyses reveal that TdT and DNA polymerase mu(Pol mu) genes are closely related, and that both enzymes were already present in the common ancestor of jawed vertebrates.

Responses to amputation of denervated ambystoma limbs containing aneurogenic limb grafts.

The developing neural tubes and associated neural crest cells were removed from stage 30 Ambystoma maculatum embryos to obtain larvae with aneurogenic forelimbs. Forelimbs were allowed to develop to late 3 digit or early 4 digit stages. Limbs amputated through the mid radius-ulna regenerated typically in the aneurogenic condition. Experiments were designed to test whether grafts of aneurogenic limb tissues would rescue denervated host limb stumps into a regeneration response. In Experiment 1, aneurogenic limbs were removed at the body wall and grafted under the dorsal skin of the distal end of amputated forelimbs of control, normally innervated limbs of locally collected Ambystoma maculatum or axolotl (Ambystoma mexicanum) larvae. In Experiment 1, at the time of grafting or 1, 2, 3, 4, 5, 7, or 8 days after grafting, aneurogenic limbs were amputated level with the original host stump. At 7 and 8 days, this amputation included removing the host blastema adjacent to the graft. The host limb was denervated either one day after grafting or on the day of graft amputation. These chimeric limbs only infrequently exhibited delayed blastema formation. Thus, not only did the graft not rescue the host, denervated limb, but the aneurogenic limb tissues themselves could not mount a regeneration response. In Experiment 2, the grafted aneurogenic limb was amputated through its mid-stylopodium at 3, 4, 5, 7, or 8 days after grafting. By 7 and 8 days after grafting, the host limb stump exhibited blastema formation even with the graft extending out from under the dorsal skin. The host limb was denervated at the time of graft amputation. When graft limbs of Experiment 2 were amputated and host limbs were denervated on days 3, 4, or 5, host regeneration did not progress and graft regeneration did not occur. But, when graft limbs were amputated on days 7 or 8 with concomitant denervation of the host limb, regeneration of the host continued and graft regeneration occurred. Thus, regeneration of the graft was correlated with acquisition of nerve-independence by the host limb blastema. In Experiment 3, aneurogenic limbs were grafted with minimal injury to the dorsal skin of neurogenic hosts. When neurogenic host limbs were denervated and the aneurogenic limbs were amputated through the radius/ulna, regeneration of the aneurogenic limb occurred if the neurogenic limb host was not amputated, but did not occur if the neurogenic limb host was amputated. Results of Experiment 3 indicate that the inhibition of aneurogenic graft limb regeneration on a denervated host limb is correlated with substantial injury to the host limb. In Experiment 4, aneurogenic forelimbs were amputated through the mid-radius ulna and pieces of either peripheral nerve, muscle, blood vessel, or cartilage were grafted into the distal limb stump or under the body skin immediately adjacent to the limb at the body wall. In most cases, peripheral nerve inhibited regeneration, blood vessel tissue sometimes inhibited, but other tissues had no effect on regeneration. Taken together, the results suggest: (1) Aneurogenic limb tissues do not produce the neurotrophic factor and do not need it for regeneration, and (2) there is a regeneration-inhibiting factor produced by the nerve-dependent limb stump/blastema after denervation that prevents regeneration of aneurogenic limbs.

Opioid receptors mediate a postsynaptic facilitation and a presynaptic inhibition at the afferent synapse of axolotl vestibular hair cells

This study was designed to determine the effects of opiate drugs on the electrical activity of afferent neurons and on the ionic currents of hair cells from semicircular canals. Experiments were done on larval axolotls ( Ambystoma tigrinum). The multiunit spike activity of afferent neurons was recorded in the isolated inner ear under both resting conditions and mechanical stimulation. Ionic currents were recorded using voltage clamp of hair cells isolated from the semicircular canal. In the isolated inner-ear preparation, microperfusion of either non-specific opioid receptor antagonist naloxone (10 nM to 1 mM), μ receptor agonist [D-Ala 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (1 pM to 10 μM), or κ receptor antagonist nor-binaltorphimine (10 nM to 100 μM) elicited a dose-dependent long-lasting (>5 min) increase of the electrical discharge of afferent neurons. The μ receptor agonist funaltrexamine (1 nM to 100 μM) and the κ receptor agonist U-50488 (1 nM to 10 μM) diminished the basal spike discharge of vestibular afferents. The δ receptor agonist D-Pen 2-D-Pen 5-enkephalin (1 nM to 10 mM) and the antagonist naltrindole (1 nM to 10 mM) were without a significant effect. The only drug that displayed a significant action on hair-cell ionic currents was trans-(±)-3,4-dichloro- N -methyl- N -(2-[1-pyrrolidinyl]-cyclohexyl) benzeneacetamide methanesulfonate (U-50488) that reduced the Ca 2+ current in a dose-dependent fashion. On its own, μ receptor agonist [D-Ala 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (0.01 and 10 μM) significantly potentiated the response of afferent neurons to the excitatory amino acid agonist (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (0.1 μM), while synaptic transmission was blocked by the use of high-Mg 2+, low-Ca 2+ solutions. Our data indicate that the activity of vestibular afferent neurons may be regulated in a complex fashion by opioid receptors: μ opioid receptors mediating an excitatory, postsynaptic modulatory input to afferent neurons, and κ receptors mediating an inhibitory, presynaptic input to hair cells.

In Vivo Imaging Indicates Muscle Fiber Dedifferentiation Is a Major Contributor to the Regenerating Tail Blastema

During tail regeneration in urodele amphibians such as axolotls, all of the tissue types, including muscle, dermis, spinal cord, and cartilage, are regenerated. It is not known how this diversity of cell types is reformed with such precision. In particular, the number and variety of mature cell types in the remaining stump that contribute to the blastema is unclear. Using Nomarski imaging, we followed the process of regeneration in the larval axolotl tail. Combining this with in vivo fluorescent labeling of single muscle fibers, we show that mature muscle dedifferentiates. Muscle dedifferentiation occurs by the synchronous fragmentation of the multinucleate muscle fiber into mononucleate cells followed by rapid cell proliferation and the extension of cell processes. We further show that direct clipping of the muscle fiber and severe tissue damage around the fiber are both required to initiate dedifferentiation. Our observations also make it possible to estimate for the first time how many of the blastema cells arise specifically from muscle dedifferentiation. Calculations based on our data suggest that up to 29% of nondermal-derived cells in the blastema come from dedifferentiation of mature muscle fibers. Overall, these results show that endogenous multinucleate muscle fibers can dedifferentiate into mononucleate cells and contribute significantly to the blastema.

Betahistine Produces Post-synaptic Inhibition of the Excitability of the Primary Afferent Neurons in the Vestibular Endorgans

Betahistine has been used to treat several vestibular disorders of both central and peripheral origin. The objective of this work was to study the action of betahistine in the vestibular endorgans. Experiments were done in wild larval axolotl ( Ambystoma tigrinum ). Multiunit extracellular recordings were obtained from the semicircular canal nerve using a suction electrode. Betahistine (10 w M to 10 mM; n = 32) inhibited the basal spike discharge of the vestibular afferent neurons with an IC 50 of 600 w M. To define the site of action of betahistine, its interactions with the nitric oxide synthase inhibitor NG-nitro-L-arginine (3 w M) and with the cholinergic antagonists atropine (10 w M; n = 3) and d-tubocurarine (10 w M; n = 3) were studied. The action of betahistine when co-administered with these drugs was the same as that in control experiments, indicating that its effects did not include nitric oxide production or the activation of cholinergic receptors. In contrast, 0.01-1 mM betahistine reduced the excitatory action of kainic acid (10 w M; n = 6) and quiscualic acid (1 w M; n = 13). These results indicate that the action of betahistine on the spike discharge of afferent neurons seems to be due to a post-synaptic inhibitory action on the primary afferent neuron response to the hair cell neurotransmitter.