An autoradiographic and electron microscopic study of retino-hypothalamic connections

Summary Retino-hypothalamic connections have been studied autoradiographically in the rat, guinea pig, rabbit, cat and monkey following the intravitreal injection of ³H-leucine or ³H-proline, and electron microscopically following unilateral eye removal in the guinea pig and monkey. In each of the species examined evidence has been found for a direct projection from the retina to the suprachiasmatic nucleus, but to no other region of the hypothalamus. The projection to the suprachiasmatic nucleus is always bilateral (even in the albino guinea pig, in which all other components of the retinal projection are crossed) but from grain counts in our autoradiographs it appears that the input to the contralateral nucleus is about twice as heavy as that on the ipsilateral side. Most of the retinal fibers appear to terminate within the ventral part of the nucleus where they form asymmetric synapses either upon small dendritic branches or dendritic spines. The possible role of this retinal projection to the suprachiasmatic nucleus in mediating a variety of light-induced neuroendocrine responses is discussed.This work was supported in part by Grants 5 PO1 EY-00491 and 5 RO1 EY-00599 from the National Eye Institute, by U.S.P.H.S. Grant HD 02274, and by Regional Primate Center Grant RR 00166.We should like to thank Mrs. Lue-Vurn Bell, Mrs. Bente Noble, Mrs. Gay Anderson and Mrs. Ludelle Moe for their excellent technical assistance, and Miss Lynn Rogers for her help with the illustrations.     

An autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage

Summary The synthesis of the proline-rich collagen component of cartilage matrix has been studied by autoradiography using both the light and electron microscope. Amblystoma maculatum larvae had their forelimbs amputated, were allowed to regenerate for 12–15 days, and then injected intraperitoneally with tritiated proline. The animals were fixed at various times (1 min. to 28 days) after the injection and sections of the developing limbs were coated for autoradiography by dipping in Ilford L 4 or Gevaert 3.07 emulsion. The sequential labeling of the organelles of the cartilage cell which occurred is illustrated in light and electron micrographs. Radioactive products first appeared in the ergastoplasm and were associated with the cisternae of the endoplasmic reticulum. Twenty to thirty minutes after the injection, labeled material began to appear in the Golgi zone. There, the newly synthesized protein accumulated within large vacuoles. The fibrillar material within the vacuoles may represent collagen and the more amorphous material, mucoprotein. The vacuoles subsequently (2 hrs. later) discharge their labeled contents into the extracellular space. The secreted protein is probably soluble collagen (tropocollagen) for it diffuses readily through the matrix to polymerize into striated collagen fibrils some distance from the cell. These findings contradict some widely held opinions that the fibrillar component of the matrix arises by excortication and appositional growth of fibrils originating from the ectoplasm of chondrocytes. It seems reasonable to conclude that the secretory pathway by which extracellular proteins are produced in cartilage is analogous to that suggested for epithelial gland cells.Supported by grants CA 05196-04S1 and GM-K3-13, 979-C1-A from the United States Public Health Service.The results reported in this paper were presented at the second annual meeting of the American Society for Cell Biology, November 6, 1962.     

Intracellular distribution of GABA in the rat anterior pituitary. An electron microscopic autoradiographic study

We studied the internalization and intracellular distribution of [3H] GABA in rat anterior pituitary cells. Electron microscopic autoradiography of anterior pituitary fragments or dispersed pituitary cells incubated with [3H] GABA showed that lactotrophs and, to a lesser extent, somatotrophs were the only cells that contained radioactive grains. Grain density analysis performed on dispersed pituitary cells after a pulse-chase experiment (10 min pulse and then change to a medium without radioactive GABA for various periods up to 2 h) revealed that GABA internalized by lactotrophs was distributed in various intracellular membranous organelles. Of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria and secretory granules had different time-dependent labeling patterns. The highest grain density values were associated with plasma membrane (at the first chase time) and the Golgi apparatus. Mitochondria and secretory granules also showed significant grain density values. A similar pattern of distribution was observed when fragments of prolactin-secreting pituitary adenomas were incubated with [3H] GABA. These results provide morphological data on the cellular specificity and intracellular distribution of GABA in anterior pituitary cells.     

An electron microscopic autoradiographic study of proline incorporation by mouse lingual epithelium

Summary Mouse lingual epithelium incorporates significant amounts of L-proline-2, 3-H³ one hour after intraperitoneal injection of the tritiated amino acid. All viable cell strata incorporated approximately equal amounts of proline as assessed by autoradiographic techniques. Grain counts at 30 minutes, 1 hour, 4 hours and 24 hours, the four time periods studied, indicated a progressive incorporation of proline up to 4 hours following injection. Preferential incorporation of proline into any one cell structure or group of structures was not observed. Keratohyalin granules (KHG's) demonstrated incorporated proline; however, usually only one silver grain appeared over each granule, and, based on grain counts, the amount of proline incorporated by KHG's appeared slightly less than the general labeling observed in KHG-containing cells. This finding supports recent biochemical studies which have indicated a considerably lower proline content of keratohyalin than had previously been reported. Significant proline incorporation into the epithelial basal lamina was not observed during the 24 hours of this study. Thus, while recent recombination experiments have conclusively demonstrated that epithelial basal cells synthesize considerable quantities of basal lamina in a 24 hour period; it would appear that epithelial basal cells contribute little to a formed, intact basal lamina. This finding lends credence to the concept of a long basal lamina turnover time.Supported by Public Healths Service grants DE 02731, DE 03393The authors are grateful to Dr. John H. Lillie for his help in determining blood levels of proline-H³ and to Dr. V. C. Hascall for his advice on isotope selection. Mrs. K. Y. Y. Chen performed nearly all technical matters associated with this study, and made many of the original electron microscopic observations. Her assistance was invaluable.     

An electron microscopic study of mouse foldback DNA.

Foldback DNA is defined by its rapid, concentration-independent renaturation, consistent with intramolecular base pairing of inverted repeat sequences. Foldback DNA, isolated from renatured mouse main band DNA by hydroxyapatite chromatography, is spread for electron microscopy by the formamide isodenaturing technique. A large fraction of the molecules can be recognized as intramolecular "hairpins"--structures in which complementary sequences on a single DNA strand form base-paired "stem" regions analogous to tRNA stems. The stem regions of the hairpins have a wide distribution of lengths, averaging about 1000 base pairs. About 60% of the stem regions terminate in single-stranded loops, ranging from 400 to many thousands of nucleotides in length, while 40% of the hairpins do not have discernible loops. There are about 40,000 hairpin-forming sequences in the main band portion of the mouse haploid genome. They appear to be either clustered in groups or confined to about one third of the DNA, rather than uniformly or randomly distributed. Another large fraction of the molecules seen in foldback DNA consists of linear structures, some of which are probably also hairpins. The electron microscopic results, along with simple theoretical considerations, make possible a better interpretation of our previous studies of the yield and S1 nuclease resistance of mouse foldback DNA.     

Electron microscopic autoradiographic and electron microscopic immunohistochemical studies on the anti-HPO antibody-producing cells

The secondary immune responses in mouse popliteal lymph nodes to horseradish peroxidase (HPO) were studied by a combination of electron microscopic autoradiography and electron microscopic immunohistochemistry in order to clarify the relationship between antibody-producing and DNA-synthesizing capacities of the plasmacytic series. The anti-HPO antibody-containing cells, which increased in number 72 h after the secondary antigenic stimulation, were mainly immunoblasts and immature plasma cells. Immunoblasts containing anti-HPO antibody incorporated [³H]thymidine more actively than did immature plasma cells containing anti-HPO antibody. In 144 h after the secondary antigenic stimulation, antibody containing cells consisted mainly of mature plasma cells and immature plasma cells. Immature plasma cells containing the anti-HPO antibody incorporated a little [³H]thymidine, but mature plasma cells containing anti-HPO antibody did not incorporate any [³H]thymidine.     

An electron microscopic study of surface polysaccharides in Bacteroides

The electron microscopic appearance of the cell surface of Bacteroides strains and Klebsiella pneumoniae stained with ruthenium red or colloidal iron is described. The effect of polymyxin B (PMB) was also registered. It was found that all Bacteroides strains have a polysaccharide lined 'micro-capsule' external to the outer membrane which could aggregate and form blebs. The blebs so formed were distinct from other types of bleb formed in Klebsiella involving the outer membrane and induced by PMB. Such types of PMB alterations were not induced in Bacteroides.