Two new fluorescent dyes applicable for visualization of fungal cell walls.

Two fluorophores, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, not heretofore reported upon are described as useful dyes of fungal cell walls, septa and bud scars examined microscopically. The dyes, depending on the filter sets used, yield fluorescently stained material generally in the blue to green and yellow to red wavelengths for Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, respectively. They provide an excellent alternative to the more commonly used fluorophore, Calcofluor White M2R. The two fluorophores, in addition to being used at various spectral wavelengths from mercury arc sources, can be used with laser sources providing 488 nm and 543 nm line wavelengths, common to most scanning confocal microscopes. Unlike Calcofluor, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B do not fade quickly when exposed to selected light wavelengths; however, like Calcofluors they are compatible with living fungal cells.

Phorbol ester-stimulated stellation in primary cultures of astrocytes from different brain regions.

Stellation is the process by which astrocytes change from epithelial-like to process-bearing cells. Stellation occurs following activation of either cyclic AMP-dependent protein kinase or protein kinase C. This process occurs through tubulin-dependent rearrangement of the cytoskeleton. We have evaluated the ability of phorbol, 12-myristate, 13-acetate (PMA) to induce astrocyte stellation. Astrocytes from five brain regions (cerebellum, cerebral cortex, hippocampus, diencephalon, and brain-stem) were examined to determine if all astrocytes would exhibit similar responses to this activator of protein kinase C. Stellation was evaluated following cell fixation by either phase optics using conventional light microscopy, or scanning laser confocal light microscopy of cultures prepared using immunocytochemistry for tubulin and glial fibrillary acidic protein. Both the number of cells responding to PMA and the sensitivity to PMA varied for astrocytes from each brain region. PMA-induced stellation was most robust in cerebellar and brainstem astrocytes, with greater than 70% responding. Less than 40% of hippocampal and diencephalic astrocytes responded to PMA at the maximum dose (10(-5) M). PMA also induced different numbers of processes or branching patterns of processes on astrocytes from different brain regions. The protein kinase C induced stellation response in astrocytes supports the hypothesis that astrocytes contribute to neural plasticity.

Localizing sites of intradendritic electrophysiological recordings by confocal light microscopy.

Studies were undertaken to develop microscopic methods and imaging procedures that would permit identification of sites of intradendritic microelectrode recordings from pyramidal cells in hippocampal slice preparations. Intradendritic recordings were obtained with sharp microelectrodes filled with the dye lucifer yellow. Following a recording session a neuron was iontophoretically injected with the dye and imaged by fluorescence videomicroscopy. Images were stored on videotape for later analysis. They provided a record of the location of the microelectrode recording site. After withdrawal of the microelectrode, slices were processed histologically and imaged a second time with a Bio-Rad 600 confocal attachment on an Olympus BH-2 microscope. Confocal images provided detailed anatomical information in three dimensions. In most instances, a clear identification of the recording site was achieved by comparing video images containing the recording electrode and confocal images. Neurophysiological recordings obtained from proximal and distal apical dendrites were markedly different. Proximal dendritic recordings were similar to those obtained from pyramidal cell soma. However, distal dendrites were not electroresponsive when depolarized by intracellular current injection. The techniques described here, or variations that employ patch electrodes, could provide valuable information that should further an understanding of the properties of dendrites in the central nervous system.

Three-dimensional imaging and image analysis of hippocampal neurons: confocal and digitally enhanced wide field microscopy.

The microscopy of biological specimens has traditionally been a two-dimensional imaging method for analyzing what are in reality three-dimensional (3-D) objects. This has been a major limitation of the application of one of science's most widely used tools. Nowhere has this limitation been more acute than in neurobiology, which is dominated by the necessity of understanding both large- and small-scale 3-D anatomy. Fortunately, recent advances in optical instrumentation and computational methods have provided the means for retrieving the third dimension, making full 3-D microscopic imaging possible. Optical designs have concentrated on the confocal imaging mode while computational methods have made 3-D imaging possible with wide field microscopes using deconvolution methods. This work presents a brief review of these methods, especially as applied to neurobiology, and data using both approaches. Specimens several hundred micrometers thick can be sampled allowing essentially intact neurons to be imaged. These neurons or selected components can be contrasted with either fluorescent, absorption, or reflection stains. Image analysis in 3-D is as important as visualization in 3-D. Automated methods of cell counting and analysis by nuclear detection as well as tracing of individual neurons are presented.

Endometrial dating correlated with progesterone levels.

The aim of this work was to document the validity and usefulness of progesterone determination instead of endometrial biopsies as evidence for ovulation within a menstrual cycle. The presence of progesterone in amounts greater than 2 ng/ml of plasma was always associated with a secretory endometrium, ie, the endometrium was matured beyond cycle day 14 as diagnosed by classical histologic dating. The remarkable simplicity of quantitating luteal phase progesterone levels by this radioimmunoassay warrants their extended use for evaluating the physiology and the pathology of the corpus luteum.

Brain responses to micro-machined silicon devices.

Micro-machined neural prosthetic devices can be designed and fabricated to permit recording and stimulation of specific sites in the nervous system. Unfortunately, the long-term use of these devices is compromised by cellular encapsulation. The goals of this study were to determine if device size, surface characteristics, or insertion method affected this response. Devices with two general designs were used. One group had chisel-shaped tips, sharp angular corners, and surface irregularities on the micrometer size scale. The second group had rounded corners, and smooth surfaces. Devices of the first group were inserted using a microprocessor-controlled inserter. Devices of the second group were inserted by hand. Comparisons were made of responses to the larger devices in the first group with devices from the second group. Responses were assessed 1 day and 1, 2, 4, 6, and 12 weeks after insertions. Tissues were immunochemically labeled for glial fibrillary acidic protein (GFAP) or vimentin to identify astrocytes, or for ED1 to identify microglia. For the second comparison devices from the first group with different cross-sectional areas were analyzed. Similar reactive responses were observed following insertion of all devices; however, the volume of tissue involved at early times, <1 week, was proportional to the cross-sectional area of the devices. Responses observed after 4 weeks were similar for all devices. Thus, the continued presence of devices promotes formation of a sheath composed partly of reactive astrocytes and microglia. Both GFAP-positive and -negative cells were adherent to all devices. These data indicate that device insertion promotes two responses-an early response that is proportional to device size and a sustained response that is independent of device size, geometry, and surface roughness. The early response may be associated with the amount of damage generated during insertion. The sustained response is more likely due to tissue-device interactions.

In vivo activation and in situ BDNF-stimulated nuclear translocation of mitogen-activated/extracellular signal-regulated protein kinase is inhibited by ethanol in the developing rat hippocampus.

In order to test the hypothesis that ethanol (EtOH)-induced changes in growth factor signal transduction contribute to the teratogenic effects of EtOH in the developing brain, neonatal rat pups were administered a single dose of EtOH during the brain growth spurt (5 days of age, PN5). Hippocampal mitogen-activated/extracellular signal-regulated protein kinase (MAPK/ERK) activation was analyzed one to 6 h after exposure by electrophoretic-mobility shift assay combined with western blot. Brain-Derived Neurotrophic Factor (BDNF) was used to stimulate ERK in hippocampal slices prepared from PN5 pups and activation and cellular localization was determined with immunofluorescence combined with confocal microscopy. EtOH decreased ERK activation in vivo and decreased nuclear translocation of BDNF-stimulated ERK in situ. These data suggest EtOH-induced inhibition of growth factor signaling may contribute to the development of fetal alcohol syndrome and alcohol-related birth defects.

Application and Quantitative Validation of Computer-Automated Three-Dimensional Counting of Cell Nuclei.

: This study provides a quantitative validation of qualitative automated three-dimensional (3-D) analysis methods reported earlier. It demonstrates the applicability and quantitative accuracy of our method to detect, characterize, and count Feulgen stained cell nuclei in two tissues (hippocampus and testes). These methods can provide important insights into the interpretation of biological, pharmacological, pathological, and toxicological events. A laser-scanned confocal light microscope was used to record 3-D images in which our algorithms automatically identified individual nuclei from the optical sections given an estimate of minimum nuclear size. The hippocampal data sets were also manually counted independently by five trained observers using the STERECON 3-D image reconstruction system. The automated and manual counts were compared. The computer counts were lower ( approximately 14%) than the manual counts, mainly because the algorithms counted a nucleus only if it was present in five consecutive optical sections but the human counters included nuclei that were in fewer optical sections. A nucleus-by-nucleus comparison of the manual and automated counts verified that the automated analysis was accurate and reproducible, and permitted additional quantitative analyses not available from manual methods. The algorithms also identified subpopulations of nuclei within the hippocampal samples, and haploid and diploid nuclei in the testes. Our methods were shown to be repeatable, accurate, and more consistent than manual counting. Nuclei in regions of high (hippocampal pyramidal layer) and low (extrapyramidal layer) density were distinguished with equal ease. Haploid and diploid nuclei were distinguished in the testes, demonstrating that our automated method may be useful for ploidy analysis. The results presented here on hippocampus and testis are consistent with other qualitative results from the liver and from immunohistochemically labeled substantia nigra, demonstrating the applicability of our software across tissues and preparation methods.

Three-dimensional changes of the cytoskeleton of vascular endothelial cells exposed to sustained hydrostatic pressure.

The three-dimensional changes in the cytoskeleton and in cell proliferation of bovine pulmonary artery endothelial cells when exposed to sustained hydrostatic pressure were investigated in vitro using laser scanning confocal microscopy. Subconfluent endothelial cells on rigid substrates were exposed to 1.5, 5 and 10 cm H2O pressure under hydrostatic heads of culture medium for up to seven days. Confocal microscopic images were taken at distances of 0.4 micron through the thickness of the sample and visualised in multiplanar, stereopair and 90 degrees rotation formats. The results of the study provide evidence of: increased proliferation after exposure to 10 cm H2O pressure for five and seven days; cell bilayering after exposure to 1.5 and 5 cm H2O pressure and trilayering after exposure to 10 cm H2O pressure for seven days; and F-actin filament reorganisation into centrally located, parallel, stress fibres in confluent cells, into peripheral bands in subconfluent, multilayered cells, and into multilayers in the plane perpendicular to the applied force.

Dynamic changes in neuronal volume resulting from osmotic and sodium transport manipulations.

Electrophysiologic parameters such as input resistance and response to microperfusion of neurotransmitters vary under circumstances where neurons from isolated ganglia of Aplysia californica are subjected to either long-term (several hours) blockade of active sodium transport or to hypo- or hyperosmotic solutions. Since one of multiple possible events under these circumstances is neuronal volume changes, we have developed a system using cultured Aplysia neurons and confocal scanning laser microscopy to directly monitor cell volume when the osmolarity of the perfusing solution is altered and when sodium transport is blocked. Volume changes of greater than 30% were observed, accompanied by changes in surface area of greater than 15%. The volume increase secondary to sodium pump inhibition and hypotonic solutions and the volume decrease secondary to hypertonic solutions were reversible. Our results demonstrate that neuronal volume may change dramatically and raise the possibility that dynamic changes in neuronal cell volume may have physiological importance.

Automated 3-D montage synthesis from laser-scanning confocal images: application to quantitative tissue-level cytological analysis.

This paper presents a landmark based method for efficient, robust, and automated computational synthesis of high-resolution, two-dimensional (2-D) or three-dimensional (3-D) wide-area images of a specimen from a series of overlapping partial views. The synthesized image is the set union of the areas or volumes covered by the partial views, and is called the "montage." This technique is used not only to produce gray-level montages, but also to montage the results of automated image analysis, such as 3-D cell segmentation and counting, so as to generate large representations that are equivalent to processing the large wide-area image at high resolution. The method is based on computing a concise set of feature-tagged landmarks in each partial view, and establishing correspondences between the landmarks using a combinatorial point matching algorithm. This algorithm yields a spatial transformation linking the partial views that can be used to create the montage. Such processing can be a first step towards high-resolution large-scale quantitative tissue studies. A detailed example using 3-D laser-scanning confocal microscope images of acriflavine-stained hippocampal sections of rat brain is presented to illustrate the method.

Progestogen-dependent endometrial protein in women: tissue concentration in relation to developmental stage and to serum hormone levels.

The presence of a progestogen-dependent endometrial protein (PEP) in women was previously described. The present study was undertaken to determine whether the occurrence or the concentration of PEP in endometria is related to the developmental stage of tissues and/or to the levels of endogenous progesterone (P) or 17 beta-estradiol (E) to which the tissues are exposed. Samples of serum and endometrial tissue in different developmental stages were obtained from pregnant women and women with normal menstrual cycles and were analyzed for PEP. In addition, the sera were analyzed for P and E. The occurrence of PEP in the endometrium of all of the women who were studied was found to be correlated with the heightened secretory activity and with the decidual change in the tissue. Furthermore, in women with normal cycles, the detection of PEP was associated with high serum levels of P. Comparison of serum levels of P with endometrial levels of PEP in pregnant women and in women in the mid-secretory phase of the menstrual cycle indicated that a 2.8-fold rise in serum levels of P during pregnancy was accompanied by a comparable (about threefold) rise in tissue levels of PEP. Therefore, we conclude that, in pregnant women and women with normal cycles, the levels of PEP in the endometrium reflect developmental changes within the endometrium, and that those levels are directly related to serum levels of P.

Three-day exposure to low-dose ethanol alters guanine nucleotide binding protein expression in the developing rat hippocampus.

Alcohol-related birth defects result from acute and chronic insults that perturb sequential developmental programs. The molecular targets of EtOH include G-protein coupled signal transduction pathways. In order to test the hypothesis that G-proteins are involved in EtOH-induced hippocampal teratogenesis, rat pups were administered 3.3 g/kg.day of EtOH on postnatal days (PN) 5 to 7 using the pup-in-a-cup model of third trimester "binge" exposure. This exposure paradigm produced a selective 40% decrease in the 52 kDa isoform of the stimulatory form of the heterotrimeric guanine nucleotide binding protein (G alpha s) in the hippocampus on PN 7 with no significant changes in the levels of G alpha i or G alpha o. Immunohistochemistry demonstrated that this decrease occurred in the somas of both hippocampal pyramidal cells and granule cells of the dentate gyrus. Computer-assisted cell counting indicates that this decrease was not due to pyramidal cell death on PN 7. Northern and slot blot analysis demonstrated a 30% decrease in G alpha s messenger RNA in the hippocampus. These results suggest that EtOHs teratogenic effects in the hippocampus may involve disruption of G alpha s-coupled signal transduction pathways, which are critical for normal synaptogenesis, neurotransmitter signaling and the integration of these signals with growth factor signaling pathways.

Diverse neuronal populations mediate local circuit excitation in area CA3 of developing hippocampus.

1. Studies were undertaken to better understand why the developing hippocampus has a marked capacity to generate prolonged synchronized discharges when exposed to gamma-aminobutyric acid-A (GABAA) receptor antagonists. 2. Excitatory synaptic interactions were studied in small microdissected segments of hippocampal area CA3. Slices were obtained from 10- to 16-day-old rats. Application of the GABAA receptor antagonist penicillin produced prolonged synchronized discharges in minislices that were very similar, if not identical, to those recorded in intact slices. The sizes of minislices were systematically varied. Greater than 90% of those that measured 600 microns along the cell body layer produced prolonged synchronized discharges, whereas most minislices measuring 300 microns produced only brief interictal spikes. 3. Action potentials in the majority (75%, 158 of 254) of cells impaled with microelectrodes were able to entrain the entire CA3 population. They were also able to increase (on average 26%) the frequency of spontaneous population discharges. The population discharges were followed by a refractory period that lasted 5-60 s, during which single cells were unable to initiate a population discharge. 4. The majority (87%) of neurons with intrinsic burst properties were found to entrain the CA3 population. The electrophysiological characteristics of these cells were reminiscent of recordings obtained from more mature rats. Action potentials were quite prolonged and demonstrated a secondary shoulder or hump on the down-slope of the spike. 5. When bursting cells were filled with Lucifer yellow and imaged during recording sessions by videomicroscopy and later using confocal microscopy, they showed the anatomic features of CA3 hippocampal pyramidal cells. Confocal microscopy permitted detailed characterization of individual neurons and showed substantial variation in cellular microanatomy. 6. Another class of cells that were found to entrain the CA3 population but did not demonstrate intrinsic bursts were termed regular-firing cells. These cells possessed many of the anatomic and physiological features of bursting cells with the exception of burst firing. They were rarely encountered in intracellular recordings. 7. The third physiological class of cells was termed fast-spiking cells. These had action potentials that were shorter in duration than the other two cell types. They were distinct in the rapid rate of spike repolarization. They demonstrated modest degrees of spike frequency adaptation and fired repeatedly and at relatively high frequencies. Compared with reports on fast-spiking cells in mature hippocampus and neocortex, action potentials appear to be slower and repetitive discharging appeared to be of a lower frequency.(ABSTRACT TRUNCATED AT 400 WORDS)