GABAergic cell types in the lizard hippocampus.

The neurochemical classification of GABAergic cells in the lizard hippocampus resulted in a further division into four major, non-overlapping subtypes. Each GABAergic cell subtype displays specific targets on the principal hippocampal neurons. The synaptic targets of the GABA/neuropeptide subtype are the distal apical dendrites of principal neurons. Calretinin- and parvalbumin-containing GABAergic cells synapse on the cell body and proximal dendrites of principal cells. Calbindin is expressed in a distinct group of interneurons, the synapses of which are directed to the dendrites of principal neurons. Finally, another subtype displays NADPH-diaphorase activity, but its synaptic target has not been established.     

Ultrastructural contributions to an understanding of the cellular mechanisms involved in lizard skin shedding with comments on the function and evolution of a unique Lepidosaurian phenomenon

Previous reports on the fine structure of lizard epidermis are confirmed and extended by SEM and TEM observations of cell differentiation and the form of shed material from the American anole Anolis carolinensis. Attention is drawn to two issues: 1) the tips of the spinules arising from the mature oberhautchen are markedly curved; this morphology can be seen during differentiation; 2) the median keels of scales from all parts of the body show “naked” oberhautchen cells that lack characteristic spinules, but have a membrane morphology comprising a complex system of serpentine microridges. Maderson's ([1966] J. Morphol. 119:39–50) “zip-fastener” model for the role of the shedding complex formed by the clear layer and oberhautchen is reviewed and extended in the light of recent SEM data. Apparently periodic lepidosaurian sloughing permits somatic growth; understanding how the phenomenon is brought about requires integration of data from the organismic to the molecular level. The diverse forms of integumentary microornamentation (MO) reported in the literature can be understood by considering how the cellular events occurring during the renewal phase prior to shedding relate to the emergence of the form-function complex of the β-layer, which provides physical protection. Issues concerning the evolutionary origin of lepidosaurian skin-shedding are discussed. J. Morphol. 236:1–24, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructure of the cell types of the anterior hypophysis in a lizard. I. Corticotrophs

Corticotrophs of the teiid lizard Cnemidophorus lemniscatus are situated in the rostral zone of the pars distalis. In normal animals, they are usually rounded cells with slightly eccentric vesicular nuclei, especially characterized by a lucent hyaloplasm and medium-sized secretory granules of uniform high density. Granules are almost spherical, with small angular deformations, and closely bounded by a fuzzy membrane. Many cells have only a few or a moderate number of granules, with large areas of cytoplasm devoid of them; in others, granules fill the supranuclear region. The cytoplasm exhibits numerous ribosomes, often in rosettes and mostly free, a series of loosely superimposed cisternae of rough endoplasmic reticulum, small dictyosomes, and elongate mitochondria of light matrix. Metyrapone administration during 2-8 days causes dramatic alterations in corticotrophs; they become hypertrophic and extensively degranulated, with a great development of the endoplasmic reticulum and Golgi apparatus, eventually showing a row of large peripheral granules of uneven structure, enclosed in ample vesicles studded with ribosomes. A lesser degree of hypertrophy and degranulation of corticotrophs appears during the first two weeks after thyroidectomy or gonadectomy, and may be partially attributed to surgical stress. Well granulated enlarged corticotrophs, with hypertrophic endoplasmic reticulum and Golgi apparatus, are probably a result of hormonal imbalance in lizards of both sexes gonadectomized for one or two months.     

Ultrastructural localization of exocytotic release sites in immunocytochemically characterized cell types

Summary Preembedding visualization of exocytosis by tannic acid treatment and postembedding immunocytochemical identification of cell types were combined to demonstrate the release of secretory products by exocytosis of characterized cell types. Treatment with tannic acid was carried out by perfusion with Ringer containing tannic acid, followed by fixation, dehydration and embedding. For electron microscopical immunocytochemistry protein A-gold was used as marker. In this study, exocytotic release was demonstrated for prolactin by cells in the pars distalis, and for oxytocin by axon terminals in the pars nervosa of the pituitary gland of the rat.     

Immunocytochemical identification of proliferating cell types in mouse mammary gland

To study cell proliferation in different cell types and segments of the mammary gland, we devised a dual staining procedure, combining nuclear labeling by 5-bromo-2'-deoxy-uridine (BrdU) uptake (revealed by a dark-brown precipitate) and an alternative (red or blue) cytoplasmic labeling by antibodies specific for the differentiation proteins of epithelial, myoepithelial, and secretory cell types. The following markers, revealed by APAAP or beta-galactosidase procedure, were selected: alpha-smooth muscle actin for the myoepithelial cells, keratin (detected by AE1 monoclonal) for the luminal epithelial cells, alpha-lactalbumin and beta-casein for the secretory cells. To follow the full process of organogenesis, the study was conducted in mouse mammary glands from virgin, primed, and lactating animals and from glands cultured in vitro under specific hormone stimulation. Cell proliferation was localized mainly in focal areas (end buds), and mostly corresponded to "null" undifferentiated cells. Estrogen and progestin stimulation induced a relative increase of proliferating differentiated cells of either epithelial or myoepithelial type, localized in ducts and alveolar structures. Prolactin stimulation induced proliferation in secretory cells.     

Ultrastructure of the pars distalis of the lizard Anolis carolinensis with special reference to the identification of the gonadotropic cell

Summary Five categories of granulated cells were distinguished by their ultrastructural features, and quantitative analyses were made of the pars distalis cells in normal and castrated lizards. The gonadotropin-producing cell was identified on the basis of its uniform distribution in the gland as well as from cytological changes resulting from castration. The secretory granules of the gonadotropic cell vary in size (100–500 m) and density, and lipid bodies are commonly present. Following castration, the endoplasmic reticulum proliferates, forming many small, rough-surfaced, dilated cisternae which do not coalesce greatly as in other vertebrate species. Degranulation is accompanied by hypertrophy and hyperplasia of the mitochondria and by the appearance in the cytoplasm of conspicuous clusters of microfilaments. The designated gonadotropic cell was the only class of secretory cell showing consistent changes following three weeks of castration.In addition to the uniformly distributed gonadotrope cell, two secretory cells occur mainly in the rostral half of the gland, and two in the caudal half. Tentative identification of the cell types is discussed in the light of available information on the localization of the hormones in the pars distalis of this species.Grateful recognition is given to the Electron Microscope Laboratory of the University of California, Berkeley, for use of their facilities, and to Emily Reid for her assistance with the illustrations.Member of Consejo Nacional de Investigaciones Cientificas y Técnicas.     

The ultrastructural basis for the identification of cell types in the pancreatic islets

Summary The pancreatic islets of rabbit, dog and opossum have been studied by light and electron microscopy. Silver-positive cells in the rabbit are predominantly sandwiched between the peripheral A and central B cells, and by electron microscopy are identified as D cells. Pancreatic islets in the tail of the dog pancreas have A, B, and D (silver-positive) cells, but the islets in the uncinate process of the dog pancreas lack phosphotungstic acid hematoxylin-positive A cells. By electron microscopy the characteristic D cells are found in both tail and uncinate process, but A cells are confined to the tail islets, confirming the identification of cell types. A unique cell type termed the F cell is found in the dog uncinate islets and it is characterized by secretory granules of angular profiles. In the opossum, the A cells contain considerable amounts of glycogen demonstrable by both light and electron microscopy. A unique cell type is also present in the opossum islets termed an E cell (Thomas, 1937), which has large secretory granules (400–500 m). The physiological implications of a multiplicity of cell types in pancreatic islets is discussed.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases. The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donahue.     

Ultrastructural studies of the epidermal leydig cell in larvae of Salamandra salamandra (caudata,salamandrida)

Epidermal Leydig cells observed in the ventral epidermis of Salamandra salamandra larvae from birth until metamorphosis are characterized by large vesicles and a supporting Langerhans net. They decline in number after birth and disappear entirely after metamorphosis. The cells change in structure and become larger, thus indicating a process of degeneration. The Leydig cells are continuously removed by macrophagelike cells after birth.     

Electrophysiological identification of cell types in cortical collecting duct monolayers.

Double-barrel microelectrodes were used to determine membrane voltages and the intracellular pH (pHi) in primary cultures of cortical collecting duct cells (CCD) grown in the absence of aldosterone. Electrophysiologically, two main cell types were identified. In cell type 1, the apical membrane voltage (Va) was -60 +/- 5 mV. The fractional resistance of the apical membrane (fRa) was 0.40 +/- 0.03, and pHi was 7.21 +/- 0.04. Exposure to 50 mM K+ on the apical side depolarized Va by 21 +/- 4 mV. When Cl- was replaced by cyclamate two types of responses were observed: (a) depolarization of Va by 26 +/- 3 mV while pHi remained unchanged, and (b) no change in Va. In cell type 2, Va was -36 +/- 5 mV, fRa was 0.91 +/- 0.03 and increasing apical [K+] from 5 to 50 mM did not change Va. Two subpopulations were distinguished by the response of pHi to lowering apical [Cl-]. In one of them pHi increased from 6.99 +/- 0.05 to 7.11 +/- 0.07. In the other, pHi was significantly decreased from 7.16 +/- 0.08 to 7.03 +/- 0.07. These results are compatible with the conclusion that about 50% of the impaled cells type 2 have a Cl-/HCO-3 exchanger at the apical membrane. In summary, two different cell types can be identified electrophysiologically in CCD monolayers. Cell type 1 has the electrical characteristics of principal cells. Cell type 2 resembles the intercalated cells. The cell alkalinization observed in approximately 50% of the cells type 2 in response to Cl- removal suggests the presence of an apical Cl-/HCO-3 exchanger. Thus, these cells should be the bicarbonate-secreting cells. The remaining cells should correspond to the acid-secreting cells.     

Ultrastructural studies of lizard (Anolis carolinensis) myogenesis in vitro

In vitro differentiation of lizard (Anolis carolinensis) skeletal muscle cells was studied by electron microscopy. Myogenesis was studied under conditions in which large numbers of postmitotic prefusion myoblasts accumulate (Growth Medium) and under conditions which are permissive for myotube formation (Fusion Medium). In Growth Medium, myogenic cells proliferate, then assume a characteristic spherical morphology which permits definitive identification of prefusion myoblasts. During the early stages of culture, these round myoblasts resemble myoblasts described in other systems; ultrastructural similarities and differences are discussed. After longer periods of culture in Growth Medium, a continuum of differentiation from isolated myofilaments to assembled myofibrils was seen in these mononucleated cells. These observations confirm the dissociability of contractile protein assembly and myoblast fusion Cultures maintained in Fusion Medium or transferred from Growth Medium to Fusion Medium form multinucleated myotubes on a predictable time scale. Myogenesis was followed in these cultures with particular reference to the early events in myofilament assembly and myofibril formation.     

Quantifying the relative contributions of divisive and subtractive feedback to rhythm generation

Biological systems are characterized by a high number of interacting components. Determining the role of each component is difficult, addressed here in the context of biological oscillations. Rhythmic behavior can result from the interplay of positive feedback that promotes bistability between high and low activity, and slow negative feedback that switches the system between the high and low activity states. Many biological oscillators include two types of negative feedback processes: divisive (decreases the gain of the positive feedback loop) and subtractive (increases the input threshold) that both contribute to slowly move the system between the high- and low-activity states. Can we determine the relative contribution of each type of negative feedback process to the rhythmic activity? Does one dominate? Do they control the active and silent phase equally? To answer these questions we use a neural network model with excitatory coupling, regulated by synaptic depression (divisive) and cellular adaptation (subtractive feedback). We first attempt to apply standard experimental methodologies: either passive observation to correlate the variations of a variable of interest to system behavior, or deletion of a component to establish whether a component is critical for the system. We find that these two strategies can lead to contradictory conclusions, and at best their interpretive power is limited. We instead develop a computational measure of the contribution of a process, by evaluating the sensitivity of the active (high activity) and silent (low activity) phase durations to the time constant of the process. The measure shows that both processes control the active phase, in proportion to their speed and relative weight. However, only the subtractive process plays a major role in setting the duration of the silent phase. This computational method can be used to analyze the role of negative feedback processes in a wide range of biological rhythms.     

The tectorial membrane of the lizard ear: types of structure

This study is concerned with the forms of the tectorial membrane in the lizard ear and its manner of attachment to the ciliary tufts of the hair cells. These structures and their variations were observed in 20 species representing eight families of lizards. Three forms of tectorial membrane were found, a continuous form that extends throughout the length of the auditory papilla, an abbreviated form that reaches the papilla only in one region, and a dendritic form that is particularly narrow at first and then branches extensively to supply all the hair cells. Occasionally the lower edge of the tectorial membrane makes direct connections with the hair tufts. More often there are special connecting structures between the membrane and the hair tufts. Seven types of these structures were identified, as follows: (1) simple fibers, (2) open network, (3) heavy network, (4) fiber plate, (5) finger processes, (6) sallets, and (7) remote connections. These types of tectorial connections are described and illustrated.     

Ultrastructural changes in the plasma membrane of A-431 cells exposed to epidermal growth factor

The plasma membrane ultrastructural changes after the action of epidermal growth factor were studied in A-431 cells using freeze-fracture methods. The incubation with EGF (100 ng/ml, 0 degree C, 60 min) led to a decrease in density of intramembrane particles on the P surface of ventral cell membrane, while the number of coated pits increased there. The revealed effects of EGF may be related to direct consequences of EGF-receptor complex formation, because all the temperature dependent steps of its processing were blocked. The data obtained testify to an active involvement of the membrane ventral surface in the formation of cell response towards growth factors.     

Germ cell biology--from generation to generation.

Germ cells hold a unique place in the life cycle of animal species in that they are the cells that will carry the genome on to the next generation. In order to do this they must retain their DNA in a state in which it can be used to recapitulate embryonic development. In the normal life cycle, the germ cells are the only cells that retain this ability to recapitulate development, referred to as developmental totipotency. The molecular mechanisms regulating developmental potency are poorly understood. Recently its has been shown that germ cells can be turned into pluripotent stem cells when cultured in specific polypeptide growth factors that affect their survival and proliferation. The ability to manipulate developmental potency in germ cells with growth factors allows the underlying mechanisms to be dissected. Germ cells are also the only cells that undergo the unique reductive division of meiosis. This too is essential for the ability of germ cells to form the gametes that will carry the genome into the next generation. Arguably meiosis is the most important division in the life of a nascent organism. Defects in meiosis can result in embryonic or fetal loss or, if the animal survives, in the birth of an individual with chromosomal abnormalities. Recent advances in our understanding of meiosis have come from knockout mice and studies on genes identified through studies of human infertility. This review will focus on these two key aspects of germ cell biology, developmental potency and meiosis.     

Ultrastructural identification of catecholaminergic fibers in the goldfish pituitary

Summary The monoaminergic innervation of the goldfish pituitary gland was studied by means of light- and electronmicroscopic radioautography after in vitro administration of ³H-dopamine. The tracer was specifically incorporated and retained by part of the type-B fibers innervating the different lobes of the pituitary. In the rostral pars distalis labeled fibers were most frequently observed in contact with the basement membrane separating the neurohypophysis and the adenohypophysis. In the proximal pars distalis and the pars intermedia, labeled profiles were detected in the neural tissue and in direct contact with the different types of secretory cells.According to the previous data concerning the uptake and retention of tritiated catecholamines in the central nervous system, it is assumed that the labeled fibers are mainly catecholaminergic (principally dopaminergic). This study provides morphological evidence for a neuroendocrine function of catecholamines in the goldfish.