Determination of the fraction of S-phase cells in root meristems using bromodeoxyuridine labeling.

The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgen-microdensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling.

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steady-state kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.

A morphometric ultrastructural study of the nucleus of cerebellar granule cells.

A quantitative approach to the nuclear ultrastructure of cerebellar granule cells is described here. The study was made using conventional electron microscopy from cerebellar cortices of adult rats by means of a semiautomatic image analyzer. The basic observation is that the nuclei of mature granule cells constitute a homogeneous population in terms of morphometric and stereologic data; in fact, the volume density of condensed chromatin within the nuclei remains practically constant in all nuclear sections. These results seem to indicate the existence of a cell-specific nuclear morphometric phenotype which might be considered as an effective criterion for the typification of this cellular lineage.

Nucleolar organization in granule cell neurons of the rat cerebellum.

The present light and electron microscopic study deals with the morphology and cytochemical properties of the nucleolus in mature granule cells of the rat cerebellum. Nucleolar organization has been examined by several different methodological approaches: (a) determination of the number of nucleoli per cell from smear preparations, (b) morphometric estimation of nucleolar size, and (c) ultrastructural and cytochemical characterization of nucleolar components. The mean number of nucleoli per cell was found to be 1.46 +/- 0.02 (mean +/- SEM). The morphometric measurement of the nucleolar area showed an average value of 0.359 +/- 0.07 microns 2 (mean +/- SDM). At the electron microscope level, most granule cell nucleoli displayed a fine texture which does not differ substantially from the 'ring-shaped' configuration usually found in cells with low levels of protein synthesis activity; i.e., a large round fibrillar centre surrounded by the dense fibrillar component, and small masses of granular component segregated from the preceding constituents. The meaning of this nucleolar configuration is discussed in the light of recent ultrastructural and biochemical data on nucleolar function. A relationship between this pattern of nucleolar organization and metabolic characteristics of the granule cells is suggested.

Proliferation of multinucleate cells with unbalanced nuclei.

When onion root meristems are treated with gamma-hexachlorocyclohexane the anaphase chromatids are distributed in discrete unbalanced groups and subsequent inhibition of cytokinesis in these cells produced a synchronous population of viable multinucleate cells with two, three of four aneuploid nuclei. When we compare the duration of G1, S and G2 periods in diploid cells with that obtained for multinucleate cells in the present study it seems clear that the differences, if they occur, are negligible. These results are consistent with the hypothesis that the cell mass/genome ratio can play an essential role in controlling cycle rate and that most of the genic requirements for interphase development must complement between the nuclei sharing a common cytoplasm, even though some factor inside every nucleus appears to be required for replicative capacity to be effective.

The "accessory body" of Cajal in the neuronal nucleus. A light and electron microscopic approach.

The present light and electron microscopic study deals with the morphology and staining properties of two intranuclear inclusions - the "accessory body" of Cajal and the "coiled body"--in the supraoptic nuclei of adult rat hypothalamus, and supports the assumption that these structures represent the same intrinsic component of the neuronal nucleus. Consequently, we propose to term it "accessory body". The structure of this body was visualized by several different staining procedures: conventional electron microscopic techniques, a silver reaction, and the regressive EDTA staining for ribonucleoproteins. The silver-impregnation method employed here, which consists of a silver development sequence on hypothalamic tissue blocks prior to plastic embedding, permitted the study of supraoptic neurons at both light and electron microscopic levels. The nature and origin of "accessory bodies" are suggested and their possible functional role is briefly discussed.

Myelin-like inclusions in maturing and senescent muscle cells of rat myocardium.

This paper deals with the ultrastructure of two types of cytoplasmic inclusions, "myelin figures" and "lipid complexes", present in muscle cells of ventricular myocardium of the rat heart. The inclusions were numerous in both the first two postnatal weeks and in the senescent stages, being rarely observed in young adult animals. The myelin figures were entirely made up of myelin-like membrane whorls, whereas the lipid complexes exhibited discrete myeloid caps surrounding a central lipid core. The origin and functional significance of such myelin-like structures are discussed on the basis of ultrastructural and biochemical studies on myocardial physiology and age-related evolution.

Silver staining of the neuronal nucleus in electron microscopy: nucleolar organization in supraoptic nuclei of the rat hypothalamus.

The present paper describes a simple, efficient method for silver impregnation of supraoptic nuclei of the rat hypothalamus using a modification of the ammoniacal silver technique of Cajal (1903). This procedure, involving a silver-developer sequence in tissue blocks prior to plastic embedding, permits the simultaneous study of Ag-impregnated supraoptic neurons at both light and electron microscopic levels. Visualization of secretory magnocellular neurons impregnated by this technique using the electron microscope reveals a good preservation of nuclear structures. A selective accumulation of silver grains was observed over heterochromatin clumps and nucleoli, which allows the identification of the nucleolar fibrillar centers and also the dense fibrillar component as the main areas involved in the silver reaction. The meaning of such a silver-distribution pattern is discussed in the light of recent ultrastructural and biochemical data.

Coiled bodies in supraoptic nuclei of the rat hypothalamus during the postnatal period.

In electron microscopic studies of the supraoptic nuclei of the rat hypothalamus, structures identified as "coiled bodies" were found in magnocellular neurons. Although they could be seen elsewhere in mature neurosecretory cells, coiled bodies were commonly encountered in developing neurons during the postnatal period in both sexes. They appeared as distinctive nuclear inclusions consisting of round-to-oval networks of short electron-dense strands embedded in a less dense, fibrillar matrix, and lacking a limiting membrane. In fine structure and stain-affinity, they bore a resemblance to the fibrillar component of the nucleolus. Coiled bodies were located either in close association with the nucleolus or free within the nucleoplasm, showing no specific relationships with the perinucleolar chromatin or with the nuclear envelope. Their origin and functional meaning is discussed in the light of recent ultrastructural and biochemical data on cellular differentiation and nucleolar behavior.

Ciliated neurons in supraoptic nucleus of rat hypothalamus during neonatal period.

Cilia 9+0 have been found in differentiating neurosecretory neurons of the hypothalamic supraoptic nuclei of Wistar rats. These ciliated neurosecretory cells were always observed during the neonatal period of the rat and no more than one cilium per cell has been encountered. Single cilia arising from typical basal bodies were usually located in deep invaginations of the neuronal perikaryon, but can also be seen in superficial positions. Their basal bodies were commonly found in the vicinity of Golgi complexes, and associated structures such as striated rootlets and alar sheets were also present. In addition, single centrioles frequently appeared in these neurons, occurring close to basal bodies but also in centrosomatic areas. The respective roles of these morphological features are suggested and their possible ontogenetic significance is briefly discussed.

Subsurface cisterns in paraventricular nuclei of the hypothalamus of the rat.

Structures identified as subsurface cisterns (SSC's) were found in neurons of the paraventricular nuclei of the rat hypothalamus. They appeared as cytoplasmic organelles consisting most often of stacks of parallel cisterns apposed to the neuronal plasmalemma. These SSC's were located in the interneurons of the parvocellular system, but not in neurosecretory cells and glial cells. SSC's were seen at zones of cytoplasm apposed to neuronal or glial cell processes, showing in some instances specific relationships with synaptic areas. The morphological features of these SSC's are described, and their possible functional significance is briefly discussed.

Intranuclear inclusions in pericytes of the hypothalamus of the rat.

This paper deals with the ultrastructure of two types of intranuclear inclusions, "nuclear bodies" and "membranous lamellar bodies", present in hypothalamic pericytes of intact adult rats. The nuclear bodies exhibited "simple" and "granular" forms, whereas the membranous lamellar bodies were entirely made up of myelin-like membrane whorls. The occurrence of these bodies in nuclei of pericytes has never been previously reported. The origin and functional meaning of such structures is discussed in the light of recent ultrastructural and biochemical studies on nuclear inclusions.