An analysis of circular intensity differential scattering measurements: studies on the sperm cell of Eledone cirrhosa.
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Cytophotometric analyses of Feulgen-stained nuclei present in homogenates of vertical and subesophageal lobes of octopus brain have shown that the latter region contains larger nuclei with up to several times the amount of DNA present in vertical nuclei. No obvious relationship was found between DNA content and nuclear size. Except for a rather small minority, nuclei of the vertical lobe have a uniform size and the expected diploid amount of DNA. These parameters are not substantially dependent on body weight. In contrast, the DNA content of subesophageal nuclei increases progressively with body weight. The amount of DNA found in subesophageal nuclei does not seem to be a simple multiple of the diploid or haploid value.
Using light and electron microscope cytochemistry and lectin blotting techniques, we have shown that the lectins concanavalin A (Con A), Ricinus communis agglutinin (RCA), and peanut agglutinin (PNA) bind to specific glycoconjugants in the adult cephalopod retina. For light microscope lectin cytochemistry, aldehyde-fixed, frozen, or Araldite-embedded, etched sections of cephalopod retinas were incubated with FITC- or TRITC-conjugated lectins and examined by using epifluorescence microscopy. Con A labeled structures in the entire retina including the inner limiting membrane (ILM), rhabdomeric membranes, interphotoreceptor matrix (IPM), and structures in the photoreceptor inner segments. RCA labeling was similar to that of Con A except that there was a decrease in the staining of the rhabdom tips near the ILM. PNA labeled only the interphotoreceptor matrix between apposing rhabdomeres. The intensity of staining of the IPM by PNA also decreased or was absent toward the rhabdom tips. None of the lectins labeled the myeloid bodies located in the photoreceptor inner segments. Electron microscope (EM) lectin cytochemistry was performed on aldehyde-fixed, LR White-embedded tissue or on Araldite-embedded, periodate-etched sections by using gold-conjugated lectins. EM results confirmed the observations made by light microscopy. Lectin blots with a retinal extract or light-sensitive membrane fraction revealed a variety of protein bands labeled by all three lectins. Con A and RCA labeled opsin and its aggregates whereas PNA did not. None of the lectins labeled retinochrome. The labeling of the cephalopod IPM by PNA suggests a structural similarity between the IPM of vertebrates and invertebrates. In other studies, we have demonstrated the presence of a retinoid binding protein in the IPM of cephalopods.(ABSTRACT TRUNCATED AT 250 WORDS)
Glial fibrillary acidic protein (GFAP) and vimentin proteins are known to be component proteins of glial filaments in the CNS of many vertebrates. The nature of the filaments present in the glial cells of the goldfish optic tectum and in the CNS of two members of the Mollusca (Helix pomatia and Octopus vulgaris) were investigated using immunocytochemical localization of monoclonal antibodies to GFAP and vimentin. Immunoblots visualized by the alkaline phosphatase method showed cross-reactive protein bands to GFAP and vimentin antibodies in total brain homogenates of the goldfish, octopus, and snail. Immunofluorescence staining of the goldfish optic tectum showed GFAP immunoreactivity, primarily in the ependymal cell processes. Immunogold labelling at the ultrastructural level verified that GFAP antibodies were bound to glial filaments. Immunolabelling of the optic lobe of Octopus vulgaris and the cerebral ganglia of Helix pomatia suggests that a protein exhibiting antigenic properties similar to GFAP is a component protein in the filaments of the protoplasmic and filamentous glia randomly distributed throughout the CNS. Unlike anti-GFAP antibodies, which stained relatively specific to filaments, vimentin staining in the CNS tissues of the three organisms studied did not appear to be exclusive to filamentous structures. As vimentin protein has been shown, in previous studies as well as our own, to exist in many tissue types, this suggests that it does not appear to be confined to glial filaments but is shared with other subcellular components. The proteins GFAP and vimentin which are thought to be well conserved in vertebrate evolution also appear to be expressed in the nervous system of some lower organisms.
The DNA content of individual sperm from populations of acriflavine-stained cells was investigated by analysis of fluorescence frequency distributions obtained with high-resolution flow-systems instruments. Sperm with spherical or cylindrical heads from three mollusk species produce narrow, symmetric fluorescence distributions. Flat sperm heads from six eutherian species produce asymmetric distributions consisting of a peak with a lateral extension to higher fluorescence values. The unexpected shape of these distributions was shown to be due to the flat geometry and high refractive index of the sperm heads in conjunction with the orthogonal axes of flow, excitation, and detection in the flow-systems instruments. The theoretical and experimeytal results indicate that the lateral extension can be eliminated either by controlling the sperm orientation with planar flow conditions or by accounting for sperm orientation by means of orientation sensing.
The morphological simplicity of the dicyemid Mesozoa is such as to allow mapping of enery individual cell during the development of the organisms. Individual cells are of a size amenable to micromanipulation and a number of potential morphogenetic makers is readily apparent. The possibility of raising the animals in vitro and obtaining developmental mutants makes these organisms excellent candidates for use as an integrated system to correlate cytodifferentiation and morphogenesis with genetic control. An axenic, nearly defined, medium in which the misozoans can be kept for over three months has been developed. Methods for isolating and maintaining the organisms are described.
Anatomical components of afferent innervation in the rim of the octopus sucker are described. In the sensory epithelium under the smooth cuticle two associated ciliated receptor cell-types (presumably chemosensitive) occur in clusters. A third ciliated receptor cell-type under the toothed cuticle may be a mechanoreceptor. A non-ciliated receptor cell-type of unknown function, under the toothed culticle, is characterized by a microvillus-lined apical canal containing dense granular material. The axons of the latter two receptors go directly into large nerve tracts which run through the infundibular muscle and on to the ganglion of the sucker. The axons of the first cell-types terminated on interneurons either in the base of the epithelium or below the epithelium. All the interneurons of the basal region of the epithelium migrate centripetally and develop into encapsulated interneurons. Within the epithelium, fine fibers provide collateral contact among cluster receptors. Collateral interaction among basal and encapsulated interneurons occur in the infundibular plexus. The microanatomy of the rim of the sucker suggests that chemosensory cues are funneled into the interneurons where they are concentrated into integrated signals, while other sensory input is probably sent directly to the ganglia of the sucker and/or arm.
Sets of animals with lesions to either the vertical lobe or median inferior frontal lobe were trained first visually and then by touch. Lesions of the vertical lobe system did not affect the increase produced by food in tendency to attack a moving figure in the visual field. Any lesion that interrupted the circuit through the vertical lobe greatly impaired the capacity to inhibit attacks on crabs when these attacks resulted in shocks. Removal of the median inferior frontal lobe did not impair this capacity to learn not to attack a crab in the octopus's visual field. The capacity to learn to respond positively to a black disc but to avoid a white one was grossly impaired by an interruption of the vertical lobe circuit. After such operations the animals showed a strong preference for white over black. The capacity to learn to discriminate between black and white was not affected by removal of the median inferior frontal lobe. Animals with interruptions of the vertical lobe circuit could learn to make discrimination between white as a positive figure and black as a negative one, but they made more mistakes than controls. Most mistakes consisted of attacks on the negative (black) figure, but there were also some failures to attack the white. In tactile discrimination between rough and smooth spheres given successively, animals with vertical lobe lesions were, under some circumstances, less accurate than controls. They took more objects than controls. They were less able than controls to reverse the the discrimination. After removal of the median inferior frontal lobe tactile discrimination was greatly impaired. The animals showed a strong preference for rough objects and could not learn to take smooth objects. However, they showed improvement in discrimination when trained with smooth negative and are therefore not wholly incapable of long-term memory storage.
Octopuses with the supraesophageal lobes split and the subesophageal centers isolated by cutting the cerebrobrachial connective on one or both sides were trained by food and shock rewards to discriminate between rough and smooth balls. Because there is a greater tendency to take the rough ball, training was done with the smooth ball positive for half the animals, and the rough ball positive for the others. In the animals with the cerebrobrachial connective cut only on one side, the subesophageal lobes showed no capacity to use the information gained by their opposite, intact, half-brains, which learned well. In animals with isolated subesophageal lobes, there was a decrease during each training session in the tendency to take both types of ball; however, this decrease did not persist from day to day. During each training session there were signs of discrimination between the balls by animals with isolated subesophageal lobes, but these also did not survive from day to day. In a series of training sessions spread over seven weeks, there was no change in results in animals with isolated subesophageal lobes when the smooth ball was positive. When the rough was positive the discrimination in its favor was slightly increased at later sessions.
Light and electron microscopy of the gravity receptor epithelia (maculae) of statocysts of normal and "spinner" juvenile Octopus maya showed differences between the structures of the hair cells, supporting cells, and afferent neurons of these cephalopods. The maculae of spinner animals were approximately 30% smaller in their surface area and had 40% fewer hair cells. Moreover, the average distance between randomly-chosen hair bundles in scanning electron micrographs of maculae of normal animals was significantly greater (4.33 +/- 6.47 microns) than those of maculae of spinner animals (3.38 +/- 4.90 microns; P less than 0.0001). The sectional area of the supporting cell's microvilli in spinner maculae was larger (0.16 +/- 0.18 microns) than those of normal (0.10 +/- 0.10 micron; P less than 0.0001) O. maya. The morphological differences observed between certain structural components of the maculae of normal and spinner O. maya may be related to the absence and/or malformation of the neuroepithelial suprastructures in spinners. This may have direct or indirect effects to their inability to orient to gravity with these organs.
In the posterior salivary duct and gland of Octopus vulgaris and of Eledone cirrhosa, the duct secretory nerve trunks and their ramifications in the gland tubules include many fibres that incorporate labelled serotonin. However, there are also unlabelled secretory fibres, which cannot be discriminated from incorporating fibres on morphological grounds. Neuroglandular junctions are not apparently established by incorporating fibres. In the duct, the motor nerve trunks contain a small number of labelled fibres, and nerve bundles supplying the duct muscle contain, in variable proportions, serotonin incorporating fibres. Both labelled and unlabelled nerve fibres reach the duct muscle fibres, but neuromuscular junctions involve only unlabelled presynaptic fibres. The nerve fibres which join the gland muscle are usually unlabelled, and the small quota of incorporating fibres in the motor trunks apparently supply only duct tissues. Both secretory and motor trunks, originating from different ganglia, can be considered to contain heterogeneous fibres, releasing different neurotransmitters at the terminals. Certain of these fibres could be serotoninergic.
The neuronal and synaptic organization of the sensory epithelium (macula) of the gravity receptor system of Octopus vulgaris was investigated by serial electron microscopic reconstruction. Three different types of afferent neurons, unipolar, bipolar, and multipolar, are described. Afferent synapses exist between the secondary sensory cells (hair cells) and the afferent neurons. Consequently, the neurons are first-order neurons. Two morphologically distinct types of afferent synapses could be identified: the most common type, present on every hair cell, has a finger-like postsynaptic process; the second type, which does not occur on every hair cell, has a flat or somewhat curved postsynaptic process. As a rule, the hair cells each form synapses with more than one afferent neuron. The neurons, in turn, form synapses with more than one hair cell. A complicated arrangement of efferent synapses was found at the level of both the hair cells and the neurons. The results are discussed with reference to their physiological consequences.
The presence of secondary sensory cells in the Octopus gravity receptor system has been demonstrated. In serial thin sections of the receptor cells (hair cells) no axons were found leaving the cells. Instead, synapses were observed with synaptic vesicles lying inside the receptor cells. Both data clearly indicate that the receptor hair cells represent secondary sensory cells. In addition, efferent contacts to the receptor cells could be confirmed.
A single layer of cell secrets the hard cephalopod beaks. The beccublasts are tall columnar cells that separate the beak from the surrounding buccal muscles, and must serve to attach these muscles to the beak. Within the cell layer there are three types of cells. The first, and most frequently found contain cell-long fibrils. These fibrils may have contractile and tensile properties. Complex trabeculae extend from the beccublasts into the matrix of the beak. The fibrils are attached to these trabeculae and at the other end of the cells they are anchored near to the beccublast-muscle cell interface, closely associated with the muscles that move the beak. The second group of cells contain masses of endoplasmic reticulum the cysternae of which are arranged along the long axis of the cell. These cells also contain dense granules and are probably the major source of beak hard tissue. It is probable that each cell secretes its own column of beak hard tissue. The third group of cells cells contains a mixture of fibrils and secretory tissue. In the beccublast layer there are changes in the proportion of the three types of cells depending upon the region sampled. In the region where growth is most active there are mostly secretory cells, whereas near the biting and wearing tip there are mainly anchoring type cells.
The presence of uni-, bi- and multipolar neurons beneath the hair cell epithelium of the Octopus gravity receptor system has been demonstrated by iontophoretic cobalt staining. Counts give an average number of 1,940 neurons per macula. Whether the hair cells are primary of secondary sensory cells is discussed.
The lip of Octopus joubini is a fleshy fold around the beak that is subdivided distally into finger-like papillae and overlayed by an uninterrupted noncellular cuticle. The muscular core of the lip has a high proportion of nervous tissue. The simple epithelium contains numerous ciliated sensory cells, especially in the papillae. In many of these cells the cilia lie deep within the cytoplasm and usually appear to extend toward the surface. Receptors with intracellular cilia also lie below the epithelium and send dendrites bearing cilia to the surface. Large unipolar interneurons that may receive synapses from the ciliated receptors lie in the musculature near the papillae. The sensory system of the octopus lip is more advanced than that of the squid, and it is very similar to that of Sepia. The relationship of these findings to the phylogeny and ecology of cephalopods is discussed.
Retinal bundles, connecting the retina of the octopus to the ipsilateral optic lobe, contain both retinal photoreceptor axons that terminate in the optic lobe and centrifugal axons whose cell bodies lie within the lobe. Staining axonal elements in proximal stubs of individual retinal bundles by cobalt diffusion and subsequent sulphide treatment reveals the topographic relationship between afferent terminals and centrifugal cell bodies. At the outer border of the plexiform layer, stained terminal bags (photoreceptor axon enlargements), an indicator of photoreceptor terminal spread within this layer, overlap stained centrifugal cell bodies located within the inner granule layer. The details of this overlap indicate a dorsoventral representation of each retinal bundle within the optic lobe cortex.