Cyclic nucleotide metabolism during amphibian forelimb regeneration

Summary Concentrations of the cyclic nucleotides in regenerating limb tissues change in a manner which suggests that they might mediate neural or endocrine influences upon specific developmental events. Since modulation of the role of cAMP within this process can be achieved through cAMP phosphodiesterase, enzymatic activity, relative intracellular distribution, and the kinetic parameters of this enzyme were examined at several stages of limb regeneration in adultNotophthalmus viridescens. Both forms of the phosphodiesterase displayed decreased activity about the time of bud formation. Total phosphodiesterase activity was reduced between 66% and 85% (as compared to intact limbs) between wound healing and palette stages. Relative intracellular distribution (soluble vs. particulate), however, remained essentially constant, 93%–98% soluble for the highK _m form and 61%–71% soluble for the lowK _m form of the enzyme, throughout this process. The apparentK _m of the highK _m form increased more than 2-fold during wound healing then fell to approximately 10% (0.7–1.1 M) of the value of intact limbs (8.3 M) during dedifferentiation and bud formation. A return to pre-amputational levels was subsequently achieved. In contrast, the apparentK _m of the lowK _m form increased (from 0.064 to 0.86 M) during dedifferentiation and began decreasing thereafter. These results are consistent with the hypothesis that one or more mechanisms are operating to modify either the quantity, activity, or physical characteristics of the cAMP phosphodiesterases and that such changes are instrumental in regulating endogenous concentrations of cAMP in limb tissues during regeneration.     

Expression profiles of elastase1 (NvElastaseI) and secretory leukocyte protease inhibitor (NvSLPI) during forelimb regeneration in adult Notophthalmus viridescens suggest a role in epithelial remodeling and delamination

Extracellular proteases and their inhibitors may regulate a number of important processes involved in forelimb regeneration in the adult newt, including epithelial remodeling, breakdown of extracellular matrix, and dedifferentiation. We have identified a newt homologue of human ElastaseI (NvElastaseI) and its potential inhibitor, SLPI (NvSLPI), and evaluated their spatial and temporal expression during limb regeneration. NvElastaseI is upregulated early in regeneration and is associated with subdermal and wound epithelial cells, suggesting an involvement in wound healing and the generation of the wound epithelium. Up until 15 days post-amputation, NvElastaseI is also scattered throughout the developing blastema and may have a role in the dedifferentiation of stump tissues. NvSLPI is found at the interface between the intact skin and the wound epithelium, and may limit NvElastaseI activity. NvSLPI is also expressed in dermal glands, and is likely involved in anti-microbial activity or function. Quite apart from regeneration, complementary patterns of expression of NvElastaseI and NvSLPI are associated with newt epithelial sloughing.     

Limb regeneration of the adult newt (Notophthalmus viridescens) in the absence of the spleen

Summary It has been suggested that the immune system might figure prominently in the regulation of forelimb regeneration. However, neither the nature of this influence nor the aspect(s) of regeneration influenced are clearly known. The determination of which components of the immune system are indispensable for regeneration would be a logical first step in attempting to address such questions. This investigation, therefore, examined the effects of removing the spleen, a major lymphoid organ in the newt, upon the progress of regeneration. Splenectomies performed concomitantly with or after forelimb amputation failed to alter the time course of regeneration. Splenectomies, but not sham-splenectomies, performed prior to amputation reduced the time required to achieve successive stages of regeneration under some, but not all conditions, i.e., when performed 10–20 days before amputation, during the late fall and winter. Up until 35 days after amputation, no gross morphological distortions were observed as a result of splenectomy. It was concluded that the spleen is not required for regeneration to occur.Portions of this work constitute part of the thesis submitted by M.E. Fini in partial fulfillment of the requirements for the M.S. degree in Biology at Boston College     

Consequences and causes of geographic variation in the body size of a keystone predator,Notophthalmus viridescens

Two subspecies of the predatory aquatic salamanderNotophthalmus, N. viridescens viridescens andN. v. dorsalis, differ in adult body size and geographic distribution. We tested whether experimental populations of the two predator subspecies differed in their effects on prey populations ofB. americanus, and whether observed differences in predator body size were genetic and/or environmentally induced. We compared the effects of predation by bothNotophthalmus subspecies on larvalBufo americanus by experimentally manipulating the densities (0, 2, or 4 newts/m³) and subspecies ofNotophthalmus (N. v. viridescens orN. v. dorsalis) added to artificial ponds. BothNotophthalmus subspecies significantly reducedB. americanus survival, but differed significantly in this effect. FewerBufo survived with the larger subspecies,N. v. viridescens, than with the smallerNotophthalmus subspecies,N. v. dorsalis. TheNotophthalmus subspecies differed in their patterns of adult and larval growth. Adults of the smaller subspecies,N. v. dorsalis, had a significantly higher growth rate than the larger subspecies,N. v. viridescens, under common environmental conditions, suggesting that differences in predator size were partly genetic, rather than entirely environmentally induced. LarvalN. v. dorsalis metamorphosed significantly later in the season than larvae ofN. v. viridescens, suggesting that larvalN. v. dorsalis had a lower growth rate than larvalN. v. viridescens. Differences in adult and larval growth, together with differences in the minimum adult size observed in natural populations, suggest that differences in the rate or duration of pre-adult growth may contribute substantially to observed differences in size.     

Biogenesis of myeloid bodies in regenerating newt (Notophthalmus viridescens) retinal pigment epithelium

Summary Myeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 m long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.     

The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt,Notophthalmus viridescens,during limb regeneration

Summary Previous work has shown that the monoclonal antibody 22/18 identifies progenitor cells (blastemal cells) which depend on the nerve for their division in the early stages of limb regeneration in the newt,Notophthalmus viridescens. This antibody also reacts with cultured cells derived from the newt limb, and the intensity of immunoreactivity appears related to cell density and differentiation into myotubes. We report here that the monoclonal antibody 22/18 recognizes a polypeptide (22/18 antigen) which is intracellular and filamentous. Double staining of cells with 22/18 monoclonal antibody and antibodies against various cytoskeletal components indicates that the epitope is expressed on an intermediate filament component. Although this antibody is specific for blastemal cells in cryostat sections of the regenerating limb, its reactivity on immunoblots is not confined to this tissue. The 22/18 antigen is differentially affected by aldehyde fixatives distinguished by the spacing of their reactive groups. While formaldehyde fixation impairs detection of the antigen, ethylene glycol-bis[succinic acid n-hydroxysuccinimide ester] reveals the antigen in sections of normal and regenerating limbs in a distribution that is consistent with the one obtained from immunoblots. We suggest that the 22/18 monoclonal antibody detects a change in protein conformation, probably related to changes in the physiological state of the cell, that occurs transiently during regeneration and possibly during development.     

Increased prolactin binding and morphological changes in the wound epithelium of regenerating limbs of Notophthalmus viridescens

Summary Distribution of prolactin has been examined in regenerating forelimbs from the newt Notophthalmus viridescens. Specific prolactin binding was demonstrated in homogenates of unamputated tissue, and of regenerating limbs at from 3 to 21 days postamputation. Labeled prolactin that was injected intraperitoneally into animals with one regenerating limb accumulated in the most distal portion of the regenerate at 7 and 14 days postamputation. Light microscopic autoradiography demonstrated that labeled prolactin was localized most heavily in the apical, outer layer of the wound epithelium. Scanning electron microscopy demonstrated that, in addition to changes in prolactin affinity following amputation, morphological changes occurred in the apical wound epithelium as well. Cell surfaces of the stump epidermis were characterized by periodic dispersion of papillae among a network of interconnecting structures 1–2 m across. By contrast, the surfaces of cells from the area in which labeled prolactin was found to localize most intensely were characterized by lack of papillae and, depending on the stage of regeneration, a pattern of microvilli and microplicae. These morphological alterations appear to reflect functional and biochemical differences between stump epidermis and wound epithelium.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

Regulation of intracellular cyclic AMP levels in the white-rot fungus Phanerochaete chrysosporium during the onset of idiophasic metabolism

Adenylate cyclase activity in Phanerochaete chrysosporium was present in cell fractions sedimenting at 1,000xg, 15,000xg, and in the 150,000xg supernatant. A small amount of activity in the 1,000xg pellet could be solubilised by treatment with Triton X-100, and the enzyme in all fractions required an ATP-Mn²⁺ substrate. Adenylate cyclase activity in the 150,000xg pellet was low (0.003 nmol/mg protein·min) and may have resulted from contamination by other fractions. Highest adenylate cyclase specific activity (0.37 nmol/mg protein ·min) was recorded in the 150,000xg supernatant at the onset of idiophasic metabolism. During this growth phase, adenylate cyclase activity also increased in the 1,000xg pellet and was maximally 4.5-fold greater than that in primary phase cultures. No significant cAMP-phosphodiesterase activity could be detected during growht in any of the cell fractions or in the growth medium with either Mn²⁺, Mg²⁺, or Ca²⁺ as added cations. The extracellular cAMP concentration increased logarithmically during primary growth; however, in cultures in idiophasic metabolism cAMP levels remained constant and relatively low. We suggest that excretion into the medium is the principal means by which intracellular cAMP levels are decreased in P. chrysosporium.Abbreviation EB extraction buffer     

Normal newt limb regeneration requires matrix metalloproteinase function

Newts regenerate lost limbs through a complex process involving dedifferentiation, migration, proliferation, and redifferentiation of cells proximal to the amputation plane. To identify the genes controlling these cellular events, we performed a differential display analysis between regenerating and nonregenerating limbs from the newt Notophthalmus viridescens. This analysis, coupled with a direct cloning approach, identified a previously unknown Notophthalmus collagenase gene (nCol) and three known matrix metalloproteinase (MMP) genes, MMP3/10a, MMP3/10b, and MMP9, all of which are upregulated within hours of limb amputation. MMP3/10b exhibits the highest and most ubiquitous expression and appears to account for the majority of the proteolytic activity in the limb as measured by gel zymography. By testing purified recombinant MMP proteins against potential substrates, we show that nCol is a true collagenase, MMP9 is a gelatinase, MMP3/10a is a stromelysin, and MMP3/10b has an unusually broad substrate profile, acting both as a stromelysin and noncanonical collagenase. Exposure of regenerating limbs to the synthetic MMP inhibitor GM6001 produces either dwarfed, malformed limb regenerates or limb stumps with distal scars. These data suggest that MMPs are required for normal newt limb regeneration and that MMPs function, in part, to prevent scar formation during the regenerative process.     

Gene activation in suspension-cultured cells of Arabidopsis thaliana during blue-light-dependent plantlet regeneration

In cell-suspension cultures of Arabidopsis thaliana (L.) Heynh., transfer to auxin-free medium initiates regeneration leading to the formation of numerous rootlets around day 5. This process is promoted by continuous irradiation of the cell cultures with blue light (400–500 nm) while red light (600–700 nm) is ineffective in this respect. During the course of this process, two mRNA species, encoding, respectively, chalcone synthase and a plasmalemma channel protein, transiently accumulate. A second temporary increase in the steady-state level of these mRNAs is correlated with the onset of chloroplast development after 13–17 d of blue-light exposure of the cell cultures. During this cellular differentiation process a number of mRNAs start to accumulate which specify prominent plastid proteins: the small and the large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU and LSU), respectively the light-harvesting chlorophyll-a/b protein II (LHCPII). These findings are in accordance with those obtained with carrot suspension cultures where a clear sequence of development, i.e. the formation of somatic embryos followed by bluelight-dependent chloroplast differentiation, has also been observed.Abbreviations AthH2 intrinsic membrane protein of Arabidopsis thaliana (gene) - CHS chalcone-synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - EFR energy fluence rate - LHCPII cab light harvesting chlorophyll-a/b protein of photosystem II (gene) - LSU rbcL large subunit of Rubisco - SSU rbcS small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenaseDedicated to Prof. Wolfhart Rüdiger on the occasion of this 60th birthdayThe research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance. For the generous gift of cloned gene sequences we thank Prof. Dr. G. Link (Pflanzliche Zellphysiologie, Bochum, Germany), Dr. A. Batschauer (Biologisches Institut II/Botanik, Freiburg, Germany) and Dr. B. Weißhaar (MPI für Züchtungsforschung, Köln, Germany).     

Morphological changes in the ultrastructure of the thyroid epithelium of Notophthalmus viridescens after hypophysectomy and TSH- and prolactin stimulation

Summary In the newt, Notophthalmus viridescens, hypophysectomy results in progressive atrophy of the thyroid cells to the point of irreversible degeneration. After exclusive TSH-stimulation of hypophysectomized newts, increased endocytotic activity of the follicular epithelium is observed. Prolactin stimulation under the same conditions prevents atrophy but does not result in increased cell activity, as expressed by the reduced amount of microvilli and the lack of endocytotic activity. Combined TSH- and prolactin stimulation also results in cell activation, but the activation level of exclusively TSH-stimulated cells is not reached. Although prolactin prevents cellular atrophy and degeneration of the follicular epithelium, it reduces the TSH-induced activation of the thyroid epithelium.     

The response of the atrium to direct mechanical wounding in the adult heart of the newt,Notophthalmus viridescens

Summary Wound repair and proliferation were examined in the injured newt atrium with light- and electron-microscopic techniques including autoradiography. Hearts were injured by removing a piece approximately 0.5 mm² of the atrial wall. The five-day wound was an endothelial and mesothelial-lined blood clot bordered by a 150-m necrotic zone. Repair progressed from the periphery inward with areas of macrophage activity replaced by fibroblasts and connective tissue. The wound at 25 days consisted of a scar with few myocytes. There was no difference in the proliferative behavior between the right and left atria. Proliferative cells were localized to a 500-m reactive zone surrounding the wound. The maximum mesothelial cell thymidine-labeling index of 20.5% and mitotic index of 1.4% were seen 5 days after injury. The peak connective tissue cell thymidine-labeling index of 10.2% and mitotic index of 0.4% were seen 10 days after wounding. The peak thymidine-labeling index of 9.8% for myocardial cells was recorded 10 days after injury with a mitotic index of 0.2%. Proliferation returned to control levels by 25 days post-injury. Electron microscopy demonstrated that myocytes engaged in DNA synthesis were indistinguishable from control myocytes. Z-band material was not observed in mitotic myocytes, but myofilaments and junctions were present.     

Myogenesis during holothurian intestinal regeneration

Echinoderms are well known as being able to regenerate body parts and thus provide excellent models for studying regenerative processes in adult organisms. We are interested in intestinal regeneration in the sea cucumber, Holothuria glaberrima, and focus here on the regeneration of intestinal muscle components. We have used immunohistochemical techniques to describe the formation of the intestinal muscle layers. Myoblasts are first observed within the regenerating structure, adjacent to the coelomic epithelia. Within a few days, these cells acquire muscle markers and form a single cell layer that underlies the epithelia. Animals injected with BrdU at various regeneration stages have been subsequently analyzed for the presence of muscle differentiation markers. BrdU-labeled muscle nuclei are observed in myocytes of 3-week regenerates, showing that these cells originate from proliferating precursors. The peak in muscle precursor proliferation appears to occur during the second week of regeneration. Therefore, new muscle cells in the regenerating intestine originate from precursors that have undergone cell division. Our results suggest that the precursor cells arise from the coelomic epithelia. We also provide a comparative view of muscle regeneration in an echinoderm, a topic of interest in view of the many recent studies of muscle regeneration in vertebrate species. This work was supported by NSF (IBN-0110692) and NIH-MBRS (S06GM08102). We also acknowledge partial support from NIH-RCMI (RRO-3641-01) and the University of Puerto Rico     

Cellular electroporation induces dedifferentiation in intact newt limbs

Newts have the remarkable ability to regenerate lost appendages including their forelimbs, hindlimbs, and tails. Following amputation of an appendage, the wound is rapidly closed by the migration of epithelial cells from the proximal epidermis. Internal cells just proximal to the amputation plane begin to dedifferentiate to form a pool of proliferating progenitor cells known as the regeneration blastema. We show that dedifferentiation of internal appendage cells can be initiated in the absence of amputation by applying an electric field sufficient to induce cellular electroporation, but not necrosis or apoptosis. The time course for dedifferentiation following electroporation is similar to that observed following amputation with evidence of dedifferentiation beginning at about 5 days postelectroporation and continuing for 2 to 3 weeks. Microarray analyses, real-time RT-PCR, and in situ hybridization show that changes in early gene expression are similar following amputation or electroporation. We conclude that the application of an electric field sufficient to induce transient electroporation of cell membranes induces a dedifferentiation response that is virtually indistinguishable from the response that occurs following amputation of newt appendages. This discovery allows dedifferentiation to be studied in the absence of wound healing and may aid in identifying genes required for cellular plasticity.     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.     

Chytridiomycosis impacts predator-prey interactions in larval amphibian communities

Despite ecologists increasingly recognizing pathogens as playing significant roles in community dynamics, few experimental studies have quantified patterns of disease impacts on natural systems. Amphibians are experiencing population declines, and a fungal pathogen (Batrachochytrium dendrobatidis; Chytridiomycota) is a suspected causal agent in many declines. We studied the effects of a pathogenic fungus on community interactions between the gray treefrog, Hyla chrysoscelis, and eastern newts, Notophthalmus viridescens. Recent studies have characterized chytridiomycosis as an emerging infectious disease, whose suspected rapid range expansion and widespread occurrence pose a significant risk for amphibian populations worldwide. We reared larvae in outdoor polyethylene experimental tanks and tested the effects of initial larval density, predator presence, and fungal exposure on Hyla recruitment and predator-prey interactions between Hyla and Notophthalmus. Newts reduced treefrog survival, and high intraspecific density decreased metamorphic body mass independent of B. dendrobatidis. The presence of fungi reduced treefrog body mass at metamorphosis by 34%, but had no significant main effect on survival or larval period length. B. dendrobatidis differentially affected larval development in the presence of predators; Hyla developed slower when reared with the pathogen, but only when newts were present. This significant predator-by-pathogen interaction suggests that the impact of chytridiomycosis on larval amphibians may be exacerbated in complex communities. Our data suggest that B. dendrobatidis effects on host life history may be complex and indirect. Direct measurements of the community-level effects of pathogens offer an important opportunity to understand a significant threat to global biodiversity—declining amphibian populations.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,protein synthesis,and growth of Pseudomonas halodurans and Alteromonas luteo-violaceus during unperturbed batch growth

Analysis of the distribution of ³⁵S-sulfate and ¹⁴C-glutamate in major biochemical components of the two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, was compared with cell density and total cellular protein during exponential growth in batch culture. For both organisms, the sulfur distribution was restricted principally to the low molecular weight organic and protein fractions, which together accounted for over 90% of the total sulfur. Carbon was more widely distributed, with these two fractions containing only 70% of the total label.Growth rate constants calculated from increases in cell numbers, protein, and ³⁵S and ¹⁴C in the various fractions indicated nearly balanced growth in A. luteo-violaceus, with constants derived from all biosynthetic parameters agreeing within 5% during the exponential phase. In contrast, protein synthesis and ³⁵S incorporation into residue protein constants were 30% higher than constants derived from cell counts and incorporation of ¹⁴C in P. halodurans. Therefore the cellular protein content P. halodurans varied over a two-fold range, with maximum protein per cell in the late exponential phase. A distinct reduction in the rate constants for total protein and ³⁵S incorporation into residue protein foreshadowed entry into the stationary phase more than one generation before other parameters.Incorporation of ³⁵S-sulfate into residue protein paralleled protein synthesis in both bacteria. The weight percent S in protein agreed well with the composition of an average protein derived from the literature. Sulfur incorporation into protein may be a useful measurement of marine bacterial protein synthesis.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid - CFU colony forming unit     

Vesicle and granule content of sympathetic ganglion cells during limb regeneration of the newt Triturus

Summary Fine structural observations were made on the vesicle and granule content of ganglion cells in the posterior subclavian ganglion and peripheral nerve fibers of the upper forelimb of the newt Triturus. The populations of vesicles and granules in normal ganglion cells and nerve fibers were compared with those observed after limb transection. In normal neurons, clear vesicles range in size from 250 to 1000 Å in diameter, but are most frequently 400–500 Å. Vesicles with dense contents (granules) also vary greatly in size, but most are 450–550 Å in diameter and correspond to dense-core vesicles. Large granules that contain acid phosphatase activity are thought to be lysosomes. During limb regeneration, in both the ganglion cells and peripheral nerves, the ratio of dense vesicles to clear vesicles increases. There is a large increase in number of dense granules with a diameter over 800 Å, particularly in the peripheral regenerating fibers. This study shows that regenerating neurons differ from normal in their content of vesicular structures, especially large, membrane-bounded granules.This work was supported by grants from the National Science Foundation (GB 7912) and from the National Cancer Institute (TICA-5055), National Institutes of Health, United States Public Health Service.