DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : I. Cytological and Cytochemical Studies on the Neuromuscular Junction of Differentiating Muscle in the Regenerating Limb of the Newt Triturus

Development of the neuromuscular junction on differentiating muscle was investigated in the regenerating limb of the newt Triturus. Motor end-plate formation begins when vesicle-filled axon terminations approach differentiating muscle cells that have reached the stage of a multinucleate cell containing myofibrils. Slight ridges or elevations occur on the muscle surface, and there is an increase in density of the cytoplasm immediately beneath the plasma membrane of the elevation. The axon becomes more closely approximated to the muscle cell and comes to lie in a shallow depression or gutter on the surface of the muscle. The surface ridges increase in length and constrict at their bases to form junctional folds. In the axon terminal, focal accumulations of vesicles are found where the axon contour projects slightly opposite the secondary synaptic clefts. Cholinesterase activity in the developing junctions was demonstrated by the thiolacetic acid-lead nitrate method. Enzymatic activity is not found on intercellular nerve fibers or the muscle surface prior to close approximation of axon endings and muscle. Eserine- and DFP-sensitive activity appears concurrently with morphological differentiation. Activity occurs in membranous tubulovesicles in the sarcoplasm subjacent to the neuromuscular junction and in association with the sarcolemma. The largest reaction deposits occur at the tips of the emerging junctional folds. Smaller and less numerous localizations occur on the axon membrane and within the axoplasm. It is concluded from these studies that the nerve endings have an inductive effect on both the morphological and chemical specializations of the neuromuscular junction.     

DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : III. Degeneration of Motor End Plates After Denervation and Maintenance In Vitro by Nerve Explants

To determine the effects of nerve explants on the integrity of motor end plates in vitro, cholinesterase activity and structure of end plates were compared in newt muscle denervated in vivo, cultured in the absence of nerve explants, and cultured in the presence of sensory ganglia. In neuromuscular junctions denervated in vivo or in vitro, the synaptic vesicles become clumped and fragmented. A few intact vesicles escape into the synaptic cleft. Axon terminals degenerate until they are left as residual bodies within the Schwann cell cytoplasm. Junctional folds on the muscle surface are reduced in height and are no longer evident once traces of axoplasm within the Schwann cell disappear. End plate cholinesterase activity is reduced as junctional folds are lost. When muscle is cultured in the presence of a sensory ganglion, the terminal axoplasm degenerates in the same manner but junctional folds persist on the muscle surface. Moderately intense cholinesterase activity remains in association with the junctional folds, so that normal motor end plates are maintained in the absence of innervation. These results show that degenerative changes in the structure of the motor end plate and loss of cholinesterase activity occurring in organ culture as a result of denervation can be retarded by nerve explants that do not directly innervate the muscle.     

Localization of 5''-Ribonucleotide Phosphohydrolase in Regenerating (and Normal) Limb Tissues of the Adult Newt Notophthalmus viridescens

The regenerating forelimb of the adult newt, Notophthalmus viridescens was investigated for 5'-nucleotidase (5' ribonucleotide phosphohydrolase, 3.1.3.5) acitivity. The newt's humeri were surgically removed, and after a twenty-one-day recovery period, the forelimbs amputated above the elbows. Regenerates were sampled at predetermined times for specific phases in the progress of regeneration, frozen, sectioned in a cryostat, and the sections fixed in 10% cold formol calcium. The Wachstein and Meisel [25] lead procedure at neutral pH was used predominately in these experiments, although tests were also conducted with Gomori's [14] calcium, Allen's [21] highly alkaline procedures. The substrates used to obtain specific enzyme reactions were adenine, cytosine, guanine, uracil and inosine 5'-monophosphate nucleotides. Sodium beta-glycerophosphate served as a non-specific phosphomonoesterase substrate, distilled water replaced substrate, and inhibitors such as zinc and cyanide ions were used as control measures to assist in increasing the precision in interpreting the results obtained. The most reactive 5'-nucleotidase (5'-Nase) loci were in the walls of the blood vascular system, mysial and neural sheaths, dermis, and periosteum: the principal cells involved were macrophages, endothelium of blood vessels, and fibrocytes of connective tissues. A moderate enzyme response was elicited from secretory cells of some of the subcutaneous glands, hypertrophied chondrocytes and osteogenic centers, chondrocytes in the articular regions and within red blood cells and leucocytes. Normal, injured and degenerating, or regenerating striated muscle and nerve fibers were judged unreactive for 5'-Nase. The epidermis and wound epithelium displayed negative responses for 5'-Nase. Cells forming the regeneration blastema were 5'-Nase reactive during the early formative phase, but with growth and development of the blastema into bulb and conic forms, these cells did not respond for this enzyme-activity. One suggestion offered is that the absence of 5'-Nase in cells of the blastema may be related to the lack of an adequate blood-vascular supply. Several functions of 5'-Nase in normal and regenerating tissues are discussed. A basic conclusion reached is that 5'-nucleotidase hydrolyses may be more involved in fundamental anabolic than in catabolic metabolism.     

THE USE OF TETRANITRO-BLUE TETRAZOLIUM FOR THE CYTOCHEMICAL LOCALIZATION OF SUCCINIC DEHYDROGENASE : Cytochemical and Cytological Studies of Sarcoma 37 Ascites Tumor Cells

Succinic dehydrogenase (SDH) was localized in the mitochondria of Sarcoma 37 ascites tumor cells by the use of tetranitro-BT (TNBT) and nitro-BT (NBT) in smear preparations. Results with each tetrazolium salt as electron acceptor were evaluated with respect to: (a) size and shape of the formazan precipitate relative to standard mitochondrial morphology; (b) crystallization phenomena of reduced dye; (c) lipid adsorption of formazan. The association of formazan- or iron hematoxylin-stained mitochondria with lipid droplets within the cells was investigated, as was also the influence of formalin fixation, with and without cold acetone pretreatment, on mitochondrial morphology and enzymatic staining. Data from these studies appear to indicate that TNBT is more suitable than NBT for use as a cytochemical reagent in oxidative and/or dehydrogenase enzyme histochemistry and cytochemistry.     

Cytological Studies of the Nematocysts of Hydra : II. The Stenoteles

Entire hydras or tentacles were prepared for electron microscopy as described in the preceding paper. The stenotele capsule has been observed to be composed of an external membrane, a thick chitinous or keratin layer, and an inner membrane. A sac-like extension of the capsular wall into the capsule bears spines and stylets on its inner surface and evagination of this structure occurs on discharge. Profiles of tubular or membranous structures often are seen within the capsules of resting stenoteles. These structures are presumably related to the external filament. The spines often reveal a flattened aspect which suggests that at least some of them might more accurately be called "vanes." A cnidocil has been found to accompany each stenotele. This study revealed several aspects of the developmental stages of stenoteles: A vacuole is formed which is nearly surrounded by the nematocyte nucleus. The vacuole content changes in density and a capsular wall is formed at the periphery of the vacuole. Tubules differentiate from the capsular matrix, and spines and stylets develop somewhat later. An operculum is formed from the nematocyte cytoplasm.     

STUDIES OF EXCITABLE MEMBRANES : I. Macromolecular Specializations of the Neuromuscular Junction and the Nonjunctional Sarcolemma

The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100–110-Å particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110–140-Å particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-Å particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol. 63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.     

Growth and Development in the Young Tomato: II. THE EFFECT OF DEFOLIATION ON THE DEVELOPMENT OF THE SHOOT APEX

In tomato seedlings the effects of high temperature in delayingthe enlargement of the shoot apex and of increasing the numberof leaves produced before flowering, were counteracted by removalof the first two leaves during the early vegetative phase. Inplants grown at 25° C. defoliation was followed by rapidenlargement of the apex and earlier flower initiation, the numberof leaves produced before flowering being reduced to that inplants grown at 15° C. Defoliation of plants grown at 15°C. resulted in only a slight increase in the rate of apicalenlargement, and the time to flowering and number of leaveswere unaffected. Growth analysis showed that at the time ofremoval, the first two leaves were not self-supporting and at25° C. were utilizing a much higher proportion of assimilatetranslocated from the cotyledons than at 15° C. These resultsare considered to support the view that at higher temperatures,the first two leaves compete strongly with the shoot apex forsupplies of assimilate.     

The Cloaca and Spermatheca of the Female Smooth Newt, Triturus vulgaris L. (Amphibia: Urodela: Salamandridae)

The anatomy and histology of the cloaca and spermatheca of 13 female smooth newts ( Triturus vulgaris ) were studied by light microscopy. The cloaca consists of a short, anterior tube (which receives the oviducts), which opens into a larger, more posterior chamber. The spermatheca, which is the only gland in the cloaca, consists of a mass of exocrine, acinar tubules which empty individually and dorsolaterally into the posterior part of the cloacal tube and all but the most posterior part of the cloacal chamber. Stored sperm are most abundant in the spermathecal tubules during the peak of the breeding season (in May and June); during their period of storage, the sperm do not appear to make intimate contact with the epithelial cells lining the spermathecal tubules. Longterm storage of viable sperm from one breeding season to the next is probably absent in the smooth newt. Sperm storage by and multiple insemination of females both occur in this species and are necessary preconditions for competition between the sperm of different males for the fertilization of eggs. However, sperm competition has not been demonstrated in the smooth newt.     

Studies on Isolated Protoplasts and Vacuoles: II. THE ACTION OF GROWTH SUBSTANCES

The response of isolated protoplasts to indol-3-yl acetic acidwas investigated, and they were found to undergo a rapid water-uptakewith ultimate rupture of the plasmalemma and release of thelarge central vacuole. The use of a photomicrographic methodshowed that this response was optimal at 10^-5 M indol-3-yl aceticacid. No such response could be detected for isolated vacuoles.¹⁴C-labelled indol-3-yl acetic acid was used to obtain furtherinsight into the site of action of this growth substance. Evidenceis presented which suggests that the site of action of indol-3-ylacetic acid, for this response, is the plasmalemma, where itfacilitates an increased uptake of solutes which is followedby an osmotic water uptake.     

A Biochemical and Cytochemical Study of Peroxidase Activity in Roots of Pisum sativum: II. DISTRIBUTION OF ENZYMES IN RELATION TO ROOT DEVELOPMENT

Changes in the specific activity and multiple forms of peroxidasealong the differentiating root of Pisum sativum have been examined.The specific activity of the total homogenate increased steadilyalong the root, although that of the low speed supernatant fractionshowed a fall in the first 20 mm. This finding supports previouscytochemical studies which showed low activity in the meristemand increased cell wall activity in the older regions. Acidphosphatase also increased gradually along the root, althoughphenylalanine ammonia lyase (PAL) showed a peak in the 20–40mm region which corresponded with the first positive stainingfor lignin. When stelar and cortical tissues were compared,peroxidase specific activity was highest in a cortical supernatantfraction while PAL was highest in the stelar tissues. The separationof peroxidase by gel electrophoresis showed the presence ofa number of bands'which varied both along the root and betweenstele and cortex. The results are discussed in relation to thepossible functions of peroxidase and, in particular, to itsproposed role in lignification.     

Studies in Stomatal Behaviour: II. THE ROLE OF STARCH IN THE LIGHT RESPONSE OF STOMATA

The relationship between starch-content and aperture in thestomata of Pelargonium has been investigated by a quantitativetechnique. Heath's suggestion of an inherent diurnal rhythmin starch-content is confirmed, and the light effect which hasbeen the subject of previous contradictory reports is foundto be dependent on external humidity. When humidity is high,light (which in these experiments is confounded with reducedC0₂-content) causes a striking reduction in stomatal starch;when humidity is low, light has no effect on starch, but itseffect on aperture is unchanged. No evidence for any dependenceof aperture on carbohydrate status was obtained, and it is suggestedthat the function of carbohydrate changes in stomata is, asearlier suggested by Kisselew, the amplifying and stabilizingof changes primarily controlled by other factors.     

Studies in Dormancy of Sycamore: II. THE EFFECT OF DAYLENGTH ON THE NATURAL GROWTH-INHIBITOR CONTENT OF THE SHOOT

1. An investigation was made into effect of daylength conditionsof the inhibition content of first-year seedlings of sycamore(Acer pseudoplatanus). 2. The shoot apical regions and mature leaves were extractedwith 80 per cent. Aqueous methanol, fractionated by paper chromatographyin isopropanol/ammonia and assayed by the wheat-coleptile growthtest. 3. A growth inhibitor was present in all extracts at R_f 0.7.Higher levels of inhibitor were present in both apices and matureleaves of plants transferred to short-day conditions than ofthose maintained under long-days throughout. 4. These difference in inhibitor level can be detected after2–5 days of short-day treatment, Preceding any markedeffect of daylength on growth. 5. Evidence is adduced in support of the hypothesis that theinhibitors is produced in the leaves during darkness and istransported to the apex during the photoperiod.     

Studies in Extension Growth: II. THE LIGHT-GROWTH RESPONSES OF VICIA FABA L

1. The changes of the rate of elongation of the stem of darkgrown Vicia faba seedlings, after illumination by white light,have been measured by auxanometer. 2. Illumination of the plumular hook region, including the apex,causes a reduction in the extension rate of the tissue below.This has been called the ‘primary phase’, and itis complete within three hours of illumination. In the rangeof exposure used, the new rate may be between 50 and 20 percent, of the original rate. 3. Illumination of the extending tissue alone causes a similarprimary phase, which is followed by a period of accelerationduring the third hour after illumination. This period has beencalled the ‘reaction phase’, and it reaches itspeak rate, which is of the order of 60 per cent. of the originalrate before treatment, about 4 hours after illumination. 4. Illumination of the whole plumule causes a primary phasefollowed by a reaction phase of more variable form and size. 5. The magnitude of the primary phase appears to increase withthe exposure. There is an optimum exposure for the productionof the reaction phase in the region of 200 foot-candle seconds. 6. The significance of these results in the interpretation ofetiolation phenomena is discussed.     

Studies on the Expansion of the Leaf Surface: II. THE INFLUENCE OF LIGHT INTENSITY AND DAYLENGTH

The parts played by constant amounts of visible radiation perday and its two components—daylength and intensity—ininfluencing the growth of Cucumis sativus have been investigated.The amount of radiation per day had a far greater influencethan either of its components per se. Nevertheless, small significanteffects of photoperiod were found, leaf expansion and dry weightincrease being greatest at daylengths between 10 and 15 hr.rather than with longer days which, with similar daily totals,would be expected to give the greatest amounts of assimilation. Rates of leaf production and appearance were greatest with thehighest amounts of radiation, but the rates of expansion ofindividual leaves and their maximum areas were greatest withintermediate amounts of radiation. This response resulted inan optimum curve relating the leaf surface and the dry weightattained after a given period to radiation. The amount of radiationgiving the maximum leaf surface and dry weight decreased withage and with external nutrient supply, but at any one age washigher for increase in dry weight than for increase in leafsurface; stem and root tissues responded more to high radiationthan did the leaf surface. The net assimilation rate was a linearfunction of visible radiation over the range of 15–120cal. cm.^-2 day^-1 explored, the highest value of radiation usedrepresenting the intensity at which photosynthesis would beexpected to be maximal over a 12–15 hr. day. The inhibitory effect of high radiation on leaf expansion andthe resultant influence on the growth of the plant are explainedin terms of the number and intensity of ‘sinks’for carbohydrate and mineral nutrients within the plant.     

Mechanisms of the Effect of Dimephosphone on Synaptic Transmission in the Frog Neuromuscular Junction

We studied the influence of dimephosphone, an organophosphorus drug with a broad spectrum of therapeutic effects on the peripheral and central nervous systems, on postsynaptic end-plate currents (EPC) in the frog neuromuscular junction. Dimephosphone was demonstrated to decrease in a voltage-independent manner the EPC amplitude and to prolong the EPC decay. These effects are not related to inhibition of acetylcholinesterase. We propose a theoretical interpretation of the observed phenomena based on the model of blockade of an open ion channel of the acetylcholine receptor and conclude that postsynaptic receptors are one of the most probable targets for the action of dimephosphone.