Michio Morita

Electron microscopic studies of planarian regeneration. I. Fine structure of neoblasts in Dugesia dorotocephala.

Planarian neoblasts were studied during the course of regeneration by electron microscopy. The greatest number of neoblasts were found not in the new blastema of regeneration but in the region of old tissue near the wound surface after about 45–55 hours after decapitation. At around this time they tended to occur in small clusters in this region. The immature neoblast, in accord with previous light microscope descriptions, was found to have a large nucleus, and scanty cytoplasm with numerous free ribosomes. Contrary to previous light microscope descriptions, they contain mitochondria of conventional size and appearance but these are distributed close to the nuclear membrane. They contain little or no endoplasmic reticulum. Differentiating neoblasts show polysomal aggregations of ribosomes, prominent supernumerary nucleoli, endoplasmic reticulum forming at the nuclear membrane, and a curious kind of dense lump in close proximity to the nuclear membrane on the cytoplasmic side. These dense lumps tend to be closely associated with several mitochondria and nestled into a shallow invagination of the nuclear membrane. Fibrous strands connecting the dense lump to adjacent nuclear membrane pores were observed. Annulate lamellae were observed in the differentiating neoblast. The significance of these structural components of the neoblast is discussed.

Electron microscopic studies on planaria: II. Fine structure of the neurosecretory system in the planarian Dugesia dorotocephala

The neurosecretory system in the head of the planarian Dugesia dorotocephala was studied by electron microscopy. Most of the nerve fibers in the neuropil of the brain are unmyelinated and contain organelles such as neurofilaments, synaptic vesicles, and mitochondria. Some of these contain round, electron dense granules (400–1100 A in diameter), with a limiting membrane, that are apparently neurosecretory granules. These were also found in a number of the end bulbs as well as in the perikaryon of the soma of some of the neurons. There is considerable evidence that these are formed in the soma of the neurosecretory cells and migrate out along the nerve fibers to the terminal bulbs. The neurosecretory granules seem to be organized by the Golgi complex, the membrane with a portion of its dense material being formed by vesiculation and budding of the Golgi tubes with concentration of dense material inside the membrane continuing after its formation by vesiculation. The secretory neuron appears to pass from one morphological phase to another in a manner related to the formation and dissolution of Golgi complex and endoplasmic reticulum and the production of neurosecretory granules. These processes seem isomorphic with those associated with neurosecretory activity in higher animals.

Time of Origin of the Neurons in the Caudal Brain Stem of Rat

This study was designed to determine the time of origin of neurons in the caudal brain stem of rat. A single injection of [3H]-thymidine was given to a pregnant animal for each of the days

Marginal copper deficiency in rats aortal morphology of elastin and cholesterol values in first-generation adult males

A marginal, 2 parts per million (ppm) copper diet (experimental) was fed to female rats for 4 months prior to breeding, through gestation/lactation, and to the weaned offspring to determine the consequences in adult, male offspring on cholesterol values and aortal morphology. Liver copper concentrations of the dams and pups at day 21 of lactation and of the 117-day-old offspring who consumed the experimental diet were lower (P less than 0.0001) than corresponding rats fed a 10 ppm copper diet (control). However, statistically significant differences due to dietary treatments were not evident in pre- or post-weaning gain in body weight, litter size, cannibalism of pups, or total cholesterol concentrations of the serum and aorta. Ultrastructural examination of experimental offspring aortas revealed focally abnormal features of endothelial cells, the subendothelial space, collagen fibers, smooth muscle cells, and particularly elastin. The ultrastructural irregularities of elastin included discontinuous regions of the internal elastic lamina comprised of stained clumps of elastin of irregular size and shape. The results of this study suggest that a marginal copper nutriture begun in utero will elicit morphologic abnormalities of the aorta in rats that are otherwise without overt signs of copper deficiency.

Toxicology of planarians

Potential advantages of using planarians such as Dugesia dorotocephala (Woodworth) to assay a number of different kinds of toxic effects are illustrated by our experiments on neurotoxicity of nicotine, developmental neurotoxicity and behavioral teratogenesis of methylmercuric chloride (MMC), and carcinogenesis of Cd and tetradecanoyl-phorbol-acetate (TPA). Various concentrations of nicotine in the culture water in which planarians were maintained produced acute neurotoxic effects, evident as disturbances in locomotor and fissioning behavior; planarians also acquired tolerance to nicotine. Behavioral teratogenesis in planarians is illustrated by the effect of subteratogenic concentrations of MMC on regeneration of the brain and behavior of decapitated specimens. Despite normal appearance of heads and brains of MMC-treated regenerates, significant deficits were noted in righting response, locomotion, and prey-catching behavior. EM observations revealed quantitative deficits in brain synapses of MMC-treated regenerates. Exposure to the carcinogens Cd and TPA produced a single kind of neoplastic tumor, a malignant reticuloma, in 76% of specimens treated jointly with sublethal concentrations of both chemicals. Toxicological responses of planarians can be compared to those of vertebrates.

Fine structure and function of planarian goblet cells

Planarians secrete quantities of mucus that appears to contain one or more glycoproteins as its major constituent. The fine structure associated with the various maturational and functional stages of a type of secretory cell of the planarian Dugesia dorotocephala is described which appears to be isomorphic with the goblet cells involved in the mucous secretion of mammals. This type of secretory cell, which occurs in large numbers in D. dorotocephala , differs from other cells which have been implicated in planarian mucous secretion. It originates and matures in the mesenchymal regions of the planarian, then migrates through the ectodermal basement membrane into the ducts penetrating the ectodermal cell layer. In its early stages it has a pronounced laminar endoplasmic reticulum and extensive Golgi complex activity. Electron transparent vesicles bud off from the tubes of the Golgi complex. These vesicles then, by swelling or coalescing, form the large mucus-filled Golgi vacuoles which almost completely fill the cytoplasm of the mature cell.

Acute toxic responses of the freshwater planarian,Dugesia dorotocephala, to methylmercury

Toxic responses of planaria to various aquatic habitat concentrations of methylmercury chloride (MMC) were investigated. One hundred percent lethality occurred within 5 h in 2 ppM MMC, 24 h in 1 ppM MMC, and 5 days in 0.5 ppM MMC. No deaths occurred in 0.2 ppM MMC over a 10 day period, however, non-lethal toxic responses were observed. Varying degrees of head resorption, progressing caudally from the snout were observed. With continuing exposure, partial head regeneration and recovery toward more normal appearance occurred by 10 days. Teratogenic effects were observed in surgical decapitation experiments. Head regeneration was retarded in 0.1 and 0.2 ppM MMC. Malformations, visible lesions, or gross behavioral abnormalities were produced by 2 week exposure of planaria to concentrations of 20 ppB MMC or lower. (RJC)

Phagocytic response of planarian reticular cells to heat-killed bacteria

The defence mechanism of the planarian Dugesia dorotocephala (Woodworth) against invasion of foreign material was studied by inserting heat-killed bacteria into an incision and then examining the tissues around the incision by light and electron microscopy. The incision was made behind the right eye of each planarian, and a small aggregate of heat-killed Mycobacterium tuberculosis H37Ra was inserted into it using a fine needle. Samples of tissues were collected at 2, 4, 6, 8, 10, 12, 24, and 48 h after insertion of bacteria. As early as 10 h after insertion, bacteria were found in phagosomes of reticular cells, which are mesenchymal cells similar to fixed parenchymal cells and known previously to phagocytize degenerate tissues. By 12 h, aggregates of bacteria were found encapsulated in extensions of reticular cells, and by 24 h encapsulated bacteria were found between epithelial cells of the intestinal wall. These findings indicate that foreign material can be phagocytized or encapsulated by reticular cells and expelled into the intestinal cavity; it is thus conceivable that reticular cells could act as an immune surveillance system against foreign invaders.

Structure and function of the reticular cell in the planarian Dugesia dorotocephala

Structural and functional characteristics of the reticular cell in the planarian Dugesia dorotocephala were studied by light and electron microscopy. Since the reticular cells have numerous glycogen granules, lipid droplets and some lysosomes in their cytoplasm, they are easily distinguishable from other cell types. They migrate into the injured tissue, cover the injured mesenchyme, and also phagocytize debris of degenerating cells. The reticular cells also recognize foreign invaders such as bacteria. The larger aggregates of killed bacteria are encapsulated by reticular cells and eliminated into the intestine, whereas small aggregates are phagocytized by reticular cells. When cell wall extract of bacteria was inserted into the planarian body before insertion of killed bacteria, reticular cells were found to respond more quickly and vigorously to subsequent insertion of killed bacteria, indicating that the reticular cell has an immune response memory. When planarians were treated with 0.3 ppm cadmium sulfate and 0.01 ppm TPA, reticuloma tumors were induced in 76% of exposed planarians, indicating the similarity to blood cell diseases in mammals such as leukemia or lymphoma which are also induced by TPA. When these tumors were transplanted into normal hosts, the tumor cells were attacked by host reticular cells. These observations indicate that planarian reticular cells are primitive blood cells, playing important roles in nutrient transportation, homeostatic control of cells, and in defence and immune surveillance systems.

Two-stage carcinogenesis in the planarian

Healthy freshwater planarians of the species Dugesia dorotocephala (Woodworth) were exposed to 0.3 mg 1-1 cadmium and to 0.01 mg 1-1 TPA (12-O-tetradecanoyl-phorbol-13-acetate) in various combinations of order and duration. Exposure to cadmium followed by exposure to TPA induced tumors in 50% of the animals only after 12 d exposure to each carcinogen. Reversed order of exposure, i.e., to TPA followed by cadmium, produced tumors in about 18% of the planarians, a rate similar to that in animals exposed to TPA alone. Cadmium by itself, however, did not produce tumors. These findings indicate that cadmium is mutagenic and acts as an initiator, whereas TPA is epigenetic and acts as a promoter. Ultimate damage to DNA or to the DNA-repair system appears to occur during the first 12 d of cadmium treatment.