Pat G. Model

Depletion of vesicles and fatigue of transmission at a vertebrate central synapse

Synapses from Mauthner to giant fibers in the hatchetfish are chemically transmitting excitatory axo-axonic synapses located in the medulla. The synapses are 4--10 mum in diameter and easily identified for electron microscopy. Presynaptic vesicles are clustered near the contact regions and are round, clear and 40-60 nm in diameter. Stimulation of the Mauthner fiber at 10/sec for 10 min greatly reduces PSP amplitude and causes profound changes in presynaptic structures. Synaptic vesicles become few in number and there is a marked accumulation of irregular membranous structures. These changes are reversible. During the recovery period, the number of synaptic vesicles progressively increases to control values, and the number of irregular membranous structures declines. Further, stimulation during cooling induces depletion of vesicles together with a great increase in the surface area of the presynaptic membrane and in the number of coated vesicles. Internal irregular membranous structures are few. Our data provide evidence for the vesicular release of transmitter and are consistent with there being a mechanism of membrane recycling in which vesicle membrane fuses with the presynaptic membrane and is reclaimed from it by coated vesicles that then coalesce to form irregular membranous structures from which new synaptic vesicles are formed.

Inter- and intramyotomal gap junctions in the axolotl embryo

Abstract In early tailbud embryos of the axolotl ( Ambystoma mexicanum ), cells of the anterior myotomes begin to elongate and align along the longitudinal axis of the animal. Soon thereafter, gap junctions appear between the differentiating myotubes. These junctions occur between adjacent cells within a myotome (intramyotomal) and between the cells of adjacent myotomes which are separated from one another by narrow connective tissue septa (intermyotomal). The latter are found at the ends of the elongating cells where muscle-tendon insertion will occur and nerve-muscle synapses will form. The gap junctions are transient: They appear with the onset of myofibrillar formation at the time that nerve fibers enter the intermyotomal septa. The junctions last until the cells have differentiated into mature striated muscle cells and neuromuscular synapses are fully developed. These gap junctions may provide a means for the direct intercellular spread of electrical excitation between the differentiating muscle cells and so account for the observed myogenic contraction of myotomes. We also suggest that these junctions may form a means for cellular communication and interaction during the development of the axial musculature.

THE PRESERVATION OF THE FINE STRUCTURE OF CRYOSTAT-SECTIONED TISSUE WITH DIMETHYLSULFOXIDE FOR COMBINED LIGHT AND ELECTRON MICROSCOPY

A method which allows the observation of identical tissue components with both the light and electron microscopes is presented. The technique is based on the preparation of cryostat sections of organs with dimethylsulfoxide. This agent appears to prevent the damage to fine structure caused by freezing and thawing. The use of plastic sheets permits flat embedding of organ sections in Epon. Examination of the embedded sections, stained or unstained, in the light microscope allows the exact selection of material to be studied further in the electron microscope.

Vestibular axons form synapses on abnormally derived Mauthner cells

Abstract Axolotl prospective forebrain-midbrain can be reprogrammed to differentiate as Mauthner cell (M-cell)-containing medulla 12 . Light and electron microscopic examination of the abnormally derived M-cells reveals vestibular club endings appropriately localized on the ventral surface of their lateral dendrites. This observation indicates that the surface of such cells is like that of ordinary M-cells insofar as it is recognized and forms synapses with vestibular axons.

The ultrastructural localization of DOPA-3H in differentiating amphibian melanophores grown in vitro☆

Abstract Explants of trunk neural crest from axolotl (Ambystoma mexicanum) embryos were grown for 7 days in hanging drop cultures. In the differentiating melanophores, the Golgi apparatus and rough-surfaced endoplasmic reticulum (RER) are prominent and well developed. The cytoplasm is further characterized by groups of free ribosomes and extensive arrays of polyribosomes, an irregular system of smooth endoplasmic reticulum, yolk platelets, lipochondria, mitochondria, microtubules, vacuoles, vesicles, and numerous pigment granules in various stages of development. Although some newly formed premelanosomes are found near the Golgi and RER, most have no special topographic relationship to either organelle. Seven-day-old cultures were treated with tritiated 3,4-dihydroxyphenylalanine (DOPA-3H) and studied by means of electron microscopic radioautography. The specific localization of radioactivity in premelanosomes shows that this organelle is the site of melanogenesis in differentiating amphibian melanophores. The time at which the premelanosome is first able to utilize exogenous DOPA coincides with the time at which the developing granule comes to contain thickened fibrils with periodic substructure. The earlier unpigmented premelanosome containing a meshwork of thin fibrils and small vesicles does not incorporate isotope. During the genesis of the melanosome there is a morphologically distinct stage at which the premelanosome acquires the capacity for melanin synthesis and deposition.

Prospective forebrain-midbrain from axolotl neurulae can be reprogrammed to differentiate as mauthner cell-containing medulla

In premetamorphic amphibians, the Mauthner cells (M-cells), a single pair of large neurons, are present in the medulla at ear level. M-cells are easily identified morphologically. Lability of the major axes of the CNS in the axolotl (Ambystoma mexicanum) through midneural plate stages suggests that regionalization of the CNS does not occur prior to that time. Thus, prospective forebrain-midbrain from early midneurulae was unilaterally substituted for prospective hindbrain in hosts of the same stages. Light microscopic examination of feeding larvae showed that the implanted tissue developed as hindbrain and, in addition, produced an M-cell. Proof that the graft itself differentiated as medulla was obtained through implantation of [3H]-thymidine labeled tissue into unlabeled host embryos and through implantation of pigmented tissue into albinos. The competence of prospective forebrain-midbrain taken from early midneurulae to produce medullae as well as M-cells indicates that specification of spatial pattern in the developing CNS has not yet occurred and that very precise regulating factors from the surrounding host tissues can override the original fate of the graft to bring it into accord with the system as a whole. The data also provide the first unequivocal demonstration of the generation of an identified central neuron from foreign tissue in a vertebrate.

Development of the sympathetic system in the Mexican axolotl, Ambystoma mexicanum.

Abstract Migration of trunk neural crest cells in axolotl embryos has been followed by autoradiography using grafts of [3H]thymidine-labeled neural folds. Crest cells form melanocytes, dorsal fin mesenchymal cells, spinal ganglion cells, and reach the sympathetic region. Sympathetic neurons, however, are not identifiable morphologically until about 6 weeks posthatching, in 24-mm larvae. At this stage, neurons, although few, have characteristic ultrastructure and receive synapses. The diffuse ganglia also contain innervated chromaffin cells; these differentiate earlier, a few days posthatching, in 14-mm larvae. A third type of cell is of morphologically indifferent appearance. Catecholamine-specific formaldehyde-induced fluorescence first appears clearly at 14 mm; with growth, the number of fluorescent cells increases. Series of larvae were injected intraperitoneally with nerve growth factor (NGF), six 30-unit injections over 2 weeks. NGF treatment increases the number of neurons apparent in 24-mm larvae. Furthermore, differentiated neurons occur in NGF-treated 20-mm larvae (about 4 weeks posthatching), when there are none in controls. The early appearance of differentiated chromaffin cells and the relatively late appearance of differentiated sympathetic neurons suggest that adrenergic functions during the first few weeks of larval life are controlled humorally by the chromaffin cells, and that at 24 mm, neurons begin to provide faster, finer control.

Thyroxin-stimulated ultrastructural changes in ependymoglia of thyroprivic amphibian larvae

Abstract Ependymal cells represent a major portion of the neuroglial population of the brain in lower vertebrates. Earlier investigations have suggested that thyroxin-dependent neuronal growth and maturation in the brain of metamorphosing amphibians are accompanied by proliferation and metabolic activation of the neuroglia. Electron microscopic studies of brains of Rana pipiens larvae reveal that, in the absence of the thyroid gland, ependymal cells lining ventricles of the developing amphibian brain display sparsely distributed ribosomes, few polyribosomes, and relatively simple profiles of rough-surfaced endoplasmic reticulum. When thyroxin is administered to such animals, free ribosomes and polyribosomes become numerous and stacks of lamellar ribosome-studded endoplasmic reticulum predominate. The observed changes are interpreted as reflecting an increased neuroglial capacity to synthesize export protein.