Photopigment gene expression and rhabdom formation in the crayfish (Procambarus clarkii).

This study examines the expression of the photopigment gene in the developing retina of the freshwater crayfish Procambarus clarkii(Crustacea, Malacostraca, Decapoda). Both sense and anti-sense RNA probes were used for in situ hybridization (ISH) of whole embryos collected at various stages during development. A characteristic of retinal development is the formation of screening pigment in the retinular cells of the retinal ommatidia. This pigmentation is seen as a band that begins at the lateral side of the retinal field and progresses medially. At hatching the retina is approximately 50% pigmented. ISH of whole embryos shows that expression of the photopigment gene by the retinular cells correlates with the extent of the screening pigment band in the retina and with the presence of rhabdoms within the ommatidia. Sections taken through embryos after being hybridized indicate that staining is localized in the cytoplasm of the retinular cells and in the axonal region below the basement membrane. No staining reaction was seen in the rhabdoms of older ommatidia. ISH staining was also seen at the anterior midline of the protocerebrum where extraretinal photoreceptors have been reported. The data presented here show a close correlation of opsin expression within the retinular cells of the ommatidia and the formation of the very early rhabdoms, similar to Drosophila. The results will be discussed in relation to recent studies in Drosophila that suggest rhodopsin plays a role in effecting the organization of the terminal web-like cytoskeleton at the base of the developing rhabdom microvilli.

Retinal development in the lobster Homarus americanus. Comparison with compound eyes of insects and other crustaceans.

Pattern formation and ommatidial differentiation were examined in the developing retina of the lobster Homarus americanus using light and electron microscopy. In the lobster the retina differentiates from the surface ectoderm that covers the optic primordia. Initially a single band of proliferation moves across this surface ectoderm. Immediately following this wave of proliferation, rows of ommatidial cell clusters appear. The earliest cell clusters are often seen adjacent to dividing cells of the proliferation band. The changing organization of the first seven rows of ommatidial clusters, visible at the surface of the retina, reveals events in early ommatidial differentiation. A rosette-like cluster of 18 cells forms the first row. Each stage following the rosette clusters occurs in a separate staggered row. Developing ommatidia have a central cluster of retinula cells, whose organization changes at each stage. Four cone cells enclose the retinula cells in each cluster and extend to the surface. In the seventh row, rhabdome formation begins and the retinula cells recede, leaving only cone cells visible at the retinal surface. This change initiates the two-tiered organization of the adult ommatidium. In 70% embryos, asymmetries in the position of the R8 axon around R7 create an equatorial line separating the dorsal and ventral halves of the retina. Possible mechanisms for the formation of these asymmetries are discussed. Postembryonic growth of the retina continues in stage VI juvenile animals along the ventral edge of the retina.

Assessment of the musculoskeletal load of the trapezius and deltoid muscles during hand activity.

The purpose of the study was to analyse the influence of the precision of a task on tension and fatigue of the trapezius and deltoid muscles. Ten young men took part in experiments. Different levels of force and different frequencies of pressing a button defined the precision of the task. Surface electromyography (EMG) was used. Muscle tension and fatigue were reflected by 2 parameters of the EMG signal: the Root Mean Square amplitude related to the maximum value and changes in the Median Power Frequency. The results showed that hand activities influence the descending part of the trapezius muscle and do not influence the deltoid muscle, and that the precision of work can influence the examined muscles of the arm and shoulder even during work in which only the hand is involved in a performed task.

Characterization of cells collected from the normal human ocular surface by contact lens cytology.

PURPOSE: In this investigation, we characterized cells collected from the normal human ocular surface using contact lens cytology (CLC). METHODS: Cells were characterized over the course of a day in three different ways. In experiment 1, we collected samples from 10 subjects six times during 1 day. Cell viability was determined by a calcein-ethidium assay. The same collection methods were used in experiment 2, but cell types were identified by fluorescent probes AE5 (corneal epithelium), AE1, AE3 (all epithelium), and T200 (all inflammatory cells). In experiment 3, cells were collected from five subjects two times in 1 day and characterized by fluorescent probes CD3 (T cells) and CD19 (B cells). For morning samples, we used an HLA-DR probe and transmission electron microscopy to examine the inflammatory activation of collected cells. RESULTS: We found viable and nonviable cells in all CLC samples, as well as an intermediate cell type that stained with both calcein and ethidium. There was a diurnal variation in cell numbers over the course of I day in viability and cell type assays, with greater cell numbers collected in the morning and evening, and fewest at midday. In the late afternoon and evening, there was an increase in corneal epithelial cell counts. There were more inflammatory cells in morning collections that included polymorphonuclear cells (PMNs). Many of these were HLA-DR+ and actively phagocytic, reflecting the immune activation of cells. CONCLUSION: The ocular surface is a dynamic environment characterized by cyclical shedding of the epithelium and active monitoring by immune cells.

Practical application of ergonomic settings of typical computerised workstations.

The goal of the study was to check, with regard to ergonomics, workstations equipped with visual display terminals in selected enterprises. Over 180 workstations were tested in 3 enterprises. Most workstations were equipped with computers. The ergonomics of both the parameters of the basic components of the workstation (i.e., a chair and a desk, and the position of the computer at the workstation and its screen with respect to windows) and lighting fittings were analysed. Typical mistakes in the layout of a workstation were chairs inappropriate for computer work, as well as broken chair adjustment mechanisms, which qualified chairs for repair or replacement. Wrong positioning of monitors on the desk and with regard to windows and lighting fittings was also noted.

Assessment of psychophysical abilities of people with disabilities during occupational rehabilitation.

Designing objective and subjective methods of studying the abilities of disabled people is one of the most important things to be done before employing them. That is why, new assessment methods are proposed. Evaluation as a testing procedure used for defining the abilities of disabled people general capacity, range, and strength of the main groups of muscles and extremities, psychological tests, and many others were developed by an interdisciplinary team. In total, 48 testing procedures, including physiological, psychological, and biomechanical ones, were prepared. We also propose another subjective assessment method because it is also very important to know what an individual person with disability needs, wants to do, or what kind of job he or she prefers. The subjective evaluation of abilities, possibilities, and needs might be done by using a questionnaire. These testing procedures offer a chance to use the same methods in all diagnostic centres so the criteria of health and work ability will be the same and easily comparable. Using the same tests for assessment will be helpful in observing the results and progress of medical, social, and vocational rehabilitation, too.

Morphogenesis and pattern formation in the retina of the crayfish Procambarus clarkii.

Pattern formation and ommatidial differentiation in the crayfish retina were analyzed using confocal, light and electron microscopy. Optic primordia first appear in the embryo as round elevations covered by a surface epithelial layer. Retinal differentiation begins with a wave of mitotic activity that moves across this epithelium from lateral to medial. Ommatidial cell clusters are visible at the surface along a transition zone, which lies at the interface of the medial undifferentiated retina and the lateral patterned retina. This zone is 8-10 cells wide and composed of small uniform cell profiles. Lateral to the transition zone the initial ommatidial cell clusters form staggered rows across the surface. Each first row cluster contains eight retinula cells surrounded by four cone, two corneagenous and two distal pigment cells. Ommatidial clusters in the first nine rows show significant changes in their organization, which are visible at the surface of the retina. In row 10 the retinula cells recede from the surface and the cone cells close in above them creating a constant cell pattern at the surface. Rhabdome development begins distally and extends downward as the retinula cluster recedes from the surface. Movement of the retinula cells inward and enlargement of the cone and corneagenous cells at the surface creates a two-tiered organization characteristic of each ommatidium. Comparison of retinal pattern formation and differentiation in the crayfish with retinal morphogenesis in Drosophila and other insects show several similarities between the two arthropod groups.

The effects of paroxetine given repeatedly on the 5-HT receptor subpopulations in the rat brain.

Effects of paroxetine (10 mg/kg PO, twice daily, 14 days) on 5-HT receptor subpopulations in the brain were evaluated pharmacologically, electrophysiologically and biochemically in male Wistar rats. Imipramine was used for comparison. Repeated paroxetine antagonized the 8-OH-DPAT-induced behavioural syndrome (a 5-HT1A effect); imipramine showed similar, yet weaker, activity. The 5-HT-or 8-OH-DPAT-induced inhibition of population spikes in hippocampal slices was increased by both those repeated antidepressants. Repeated (or acute) paroxetine decreased the density of and increased the affinity for 5-HT1A receptors ([3H]-8-OH-DPAT used as ligand) in the hippocampus, while imipramine induced opposite effects. m-Chlorophenyl piperazine (m-CPP)-evoked exploratory hypoactivity, a 5-HT2C effect, was reduced by repeated paroxetine, but not by imipramine. Either of the antidepressants given repeatedly antagonized TFMPP-induced hyperthermia (another putative 5-HT2C effect). 5-HTP-induced head twitches (a 5-HT2A effect) were inhibited by repeated paroxetine or imipramine. Either antidepressant given repeatedly decreased the density of 5-HT2A receptors ([3H]-ketanserin as a ligand) in the brain cortex, but did not change their affinity. The present results indicate that paroxetine given repeatedly induces secondary changes in 5-HT2 receptors, which lead to reduction of the 5-HT2 neurotransmission (reduced responsiveness of 5-HT2 postsynaptic receptors). The consequences of the secondary changes in 5-HT1A receptors, found here still await clarification.

Localization of actin in the retina of the crayfish Procambarus clarkii.

The distribution of actin in the retina of the crayfish was investigated at the LM level using FITC-phalloidin. Fluorescent staining was associated with the main rhabdom and eighth cell rhabdom, the zonula adherens junctions between retinula cells, and the basement membrane of the retina. EM and S1 decoration were used to confirm the presence of actin and identify its structural relationships. Phalloidin staining of the rhabdom and S1 decoration of actin filaments in the rhabdom microvilli confirmed earlier findings that actin is a component of the microvillus cytoskeleton in the crayfish. At the zonula adherens junctions, actin filaments, identified by S1 decoration, run longitudinally within the plaque of the junction. At the extreme proximal end of the rhabdom, actin filaments associated with the junctions fill each small area of retinula cell cytoplasm. In the basement membrane, EM and S1 decoration show that basilar cells contain large bundles of actin filaments which are associated with cell-matrix adherens junctions. Foot cells which lie immediately below the rhabdom also contain similar junctions and actin is tentatively identified in these cells. The functional role of actin at these various locations is discussed in relation to retinal organization in the crayfish and other invertebrates.

Changes in the microvillus cytoskeleton during rhabdom formation in the retina of the crayfish Procambarus clarkii.

Changes in the microvillus cytoskeleton during the formation of the light-receptive rhabdom in the crayfish retina were examined at four structurally distinct stages. The cytoskeleton of microvilli in early rhabdoms is composed of a regularly packed bundle of 12-25 actin filaments. The polarity of S1 decorated filaments indicates that the plus end of the actin filaments is located at the microvillus tip. The hexagonal packing of filaments within the bundle, their spacing, and the presence of cross-striations along the bundle in longitudinal sections indicate the filaments are held together by cross-linking proteins. Electron microscopic observations and data from three-dimensional reconstructions of individual microvilli indicate that the filaments arise from a concentration of dense material at the tip of the microvillus and extend into the cytoplasm as a rootlet. Over the four developmental stages examined there is an increase in the number of microvilli forming the rhabdomeres and a 50% decrease in the mean cross-sectional area of individual microvilli. During this same period the number of actin filaments forming the microvillus cytoskeleton also decreases. Following this decline, microvilli of late stage rhabdoms, which are structurally similar to adults, contain only two to four filaments. These changes are discussed in relation to the three phases of growth described for stereocilia and brush border microvilli.

Effect of rigid gas permeable contact lens wetting solutions on the rabbit corneal epithelium.

This investigation was designed to compare the effects of three rigid gas permeable (RGP) contact lens solutions on the rabbit corneal epithelium. Boston Advance Conditioning Solution, Boston Conditioning Solution, and Allergan Wet-N-Soak, which are preserved with 0.0015% polyaminopropyl biguanide, 0.006% chlorhexidine gluconate, and 0.003% benzalkonium chloride, respectively, were evaluated by scanning (SEM) and transmission electron microscopy (TEM). Our results show that Boston Advance Conditioning Solution is significantly more toxic to the corneal epithelium than either Boston Conditioning Solution or Allergan Wet-N-Soak Plus. This is presumably due to the presence and concentration of the preservative, 0.0015% polyaminopropyl biguanide. Although this study was conducted using rabbits, the results raise clinical concerns for human RGP contact lens wearers.

The diurnal pattern of protein and photopigment synthesis in the retina of the crayfish, Procambarus clarkii.

The interrelationship between the diurnal cycle of membrane loss and synthesis of new rhabdom components remains a key element in forming a complete picture of the turnover of photopigment-containing membrane in the crayfish photoreceptor cell. In order to examine this aspect of the turnover process, the diurnal pattern of photopigment synthesis was examined using an in vitro incubation system for incorporation of 3H-leucine into photoreceptor protein. The incorporation of 3H-leucine into total protein and photopigment specifically was measured in photoreceptors isolated from incubated retinas. The results indicate that for both total protein and photopigment there is no significant variation in the rate of synthesis during the 12-12 light-dark cycle. These data combined with earlier data on diurnal membrane loss from the rhabdom suggest that light-stimulated rhabdom membrane loss is superimposed on a diurnally constant level of synthesis and assembly of new rhabdom constituents.

Regional morphological variations within the crayfish eye.

The existence of structural asymmetries has been quantitatively demonstrated in the crayfish compound eye. Variations in the size of the rhabdomes and corneal facets, as well as the size and extent of the accessory reflecting pigment cells, have been found. It was determined that the mean rhabdome diameter within a 70 degrees arc in the dorsal quadrant of the retina is 11-19% smaller than the mean rhabdome diameter in the remaining areas of the eye. Also, the extent of the accessory reflecting pigment cells is diminished over an area corresponding generally to the dorsal region of smaller rhabdomes. Corneal facet size and shape vary over the surface of the cornea, with smaller facets occurring in the dorsal region. Both the mean rhabdome diameter and the mean corneal facet area for whole eyes increases linearly in animals ranging in size from 3.9-12 cm. The estimated number of corneal facets, and therefore the number of rhabdomes, increases from an average of 4700 in the 3-6.9 cm size range to about 6000 in 7-12 cm animals. These data indicate that structural asymmetries and various size-related parameters exist in the crayfish eye and should be considered in any quantitative analysis of this structure.

Development of the crayfish retina: a light and electron microscopic study.

The development of the crayfish retina was examined in embryos and first, second and third instars with both and light and electron microscope. Light microscopic observations indicate that differentiation begins at the posterior portion of the optic disc and progresses in an anterior direction. Development of screening pigment, dioptric elements, and rhabdoms all parallel this posterior to anterior gradient in the retina. Tracer studies in early embryos reveal that the retina is separated from the proximal neuropil regions by a distinct vascular space. This observation suggests that the source of new cells for the retina may not be the more proximal cell proliferation zone as previously indicated. It is proposed that mitotic activity within the retina and/or differentiation of cells from the anterior surface layer of the eye may be sources for addition of new cells to the retina. Proto-ommatidial clusters of seven retinula cells occur very early at the posterior region of the embryonic retina. Initially the receptor cells extend throughout the entire thickness of the retina, but later they withdraw from beneath the cornea to occupy only the proximal portion of the retina. Microvilli of the rhabdom arise from the centrally opposed membranes of the retinula cells in each cell cluster. Each new microvillus contains a core of fine filaments which extend out into the cytoplasm at its base. As development of the microvilli continues, the core filaments appear to be lost or altered, but the cytoplasmic bundles at the base of the microvilli persist.

Diurnal changes of lysosome-related bodies in the crayfish photoreceptor cells.

The effect of illumination on the degradation of microvillar membrane in the invertebrate photoreceptor cell has been correlated with the appearance in the cytoplasm of certain distinct lysosome-related bodies. Three types of organelles were distinguished in the retinula cell cytoplasm of the crayfish, multivesicular bodies (MVB), both large (.20-1.50 micron) and small (1.49-0.30 micron), combination bodies (CB), and lamellar bodies (LB). Under diurnal lighting conditions significant temporal differences were found in the appearance of these three classes of organelles in the retinula cell. Small MVB are present at a consistent level throughout most of the diurnal cycle but show peak numbers at 30 min after light onset and again after 6 h of dark adaptation. Large MVB increase significantly 1 h after light onset and remain elevated through 4 h in the light. After 4 h the large MVB decline gradually for the remaining light period. Combination bodies and LB do not begin to increase until 1 h after light onset and are at peak levels between 4 and 6 h into the light period. The minimum rhabdome diameter coincides with the peak levels of large MVB, CB, and LB. These data support support the hypothesis that light causes microvillar membrane breakdown, resulting in the initial production of MVB which in turn undergo degradation to form CB and finally LB. This primary degradative response appears to be completed within the first 8 h of the light period.