Ultrastructural localization of rhodopsin in the vertebrate retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.     

Ultrastructural localization of calcium in the CNS of vertebrates

The ultrastructural localization of calcium in synaptic areas of the CNS of fish was investigated. Prefixation with phosphate-buffered glutaraldehyde followed by post-fixation with osmium/potassium-bichromate was used to precipitate and visualize endogenous calcium without the addition of external calcium. The presence of calcium in the electron-dense precipitates produced using this method was demonstrated by electron spectroscopic imaging using a Zeiss EM-902 transmission electron microscope, and in various control experiments using the calcium chelator EGTA. In the optic tectum of fish, electron dense precipitates containing calcium were found not only in intracellular compartments, e.g. the smooth endoplasmic reticulum, mitochondria and synaptic vesicles, but also at extracellular locations, particularly in synaptic clefts. In the extracellular sites, only chelate complexes of ionic calcium were found. This would seem to be in agreement with electrophysiological and biochemical data reported in earlier studies. Thus, using the present method, it should be possible to obtain further ultrastructural information concerning the mechanisms of synaptic transmission.     

Ultrastructural localization of calcium in the CNS of vertebrates

Summary The ultrastructural localization of calcium in synaptic areas of the CNS of fish was investigated. Prefixation with phosphate-buffered glutaraldehyde followed by post-fixation with osmium/potassium-bichromate was used to precipitate and visualize endogenous calcium without the addition of external calcium. The presence of calcium in the electron-dense precipitates produced using this method was demonstrated by electron spectroscopic imaging using a Zeiss EM-902 transmission electron microscope, and in various control experiments using the calcium chelator EGTA. In the optic tectum of fish, electron dense precipitates containing calcium were found not only in intracellular compartments, e.g. the smooth endoplasmic reticulum, mitochondria and synaptic vesicles, but also at extracellular locations, particularly in synaptic clefts. In the extracellular sites, only chelat complexes of ionic calcium were found. This would seem to be in agreement with electrophysiological and biochemical data reported in earlier studies. Thus, using the present method, it should be possible to obtain further ultrastructural information concerning the mechanisms of synaptic transmission.     

Ultrastructural autoradiographic localization of serotonin in the pituitary of a teleost fish,Poecilia latipinna

Summary Light- and electron-microscopic autoradiographic studies of pituitaries of the molly Poecilia latipinna, after their incubation with tritiated serotonin, revealed the presence of labelled cells in the proximal pars distalis, together with cell processes or nerve fibres throughout all regions of the gland except the prolactin cell zone. The serotonincon-centrating cells and most of the fibres contained small dense-cored vesicles, but some labelled fibres contained larger granules similar in ultrastructure to those of vasotocinergic fibres.This work was supported by a SERC studentship to DJG and SERC grant GR/6379-06 to Professor J.N. Ball     

Adrenergic neurons in teleost retina

Summary The adrenergic retinal neurons of perch and trout were studied with the fluorescence microscopical method of Falck and Hillarp. Pilot studies were also performed on pike, plaice, cod, eel, goldfish, cunner, black moor, cichlid and carp. Only minor differences were noted between the species.Adrenergic varicose terminals occur in three sublayers of the inner plexiform layer. The layer adjacent to the ganglion cells is the most elaborate. Adrenergic perikarya occur in the innermost cell rows of the inner nuclear layer, sending branches to all sublayers of the inner plexiform layer. Adrenergic perikarya also occur among the ganglion cells, sending their branches to the innermost sublayer of adrenergic fibres in the inner plexiform layer. Weakly fluorescent adrenergic fibres can be seen running through the entire depth of the inner nuclear layer. They originate from the adrenergic perikarya of the inner nuclear layer, and they end in an elaborate plexus of adrenergic terminals among the horizontal cells. Either of the horizontal cell types can be in contact with adrenergic terminals, but the middle horizontal cells have the greatest density about them, being surrounded by baskets of adrenergic terminals of presumably synaptic character. It cannot be excluded that some horizontal cells contain a catecholamine.Microspectrofluometry revealed dopamine in the perch and trout retinal neurons.The research reported in this document has been sponsored by USPHS Grant No. 06092 and by a Research Professorship from Research to Prevent Blindness, Inc. to A.M.L. and by the Swedish Medical Research Council (B69-14X-712-04C and B68-14X-2321-01).     

Histochemical and cytochemical localization of acetylcholinesterase in retina and optic tectum of teleost fish.

The activity of cholinesterase and its cellular and subcellular localization were investigated in the retina and optic tectum of Eugerres plumieri and in the retina of Carassius carassius by means of radiometric, histochemical, and cytochemical procedures. In both fishes only the presence of acetylcholinesterase could be demonstrated. This study, besides confirming previous findings that acetylcholinesterase is located in the ganglion and amacrine cells of the retina as well as in the inner plexiform layer, in addition provides evidence that the enzyme is also present at the region of photoreceptor synapses between the cell bodies and apposing extensions of the horizontal cells of the same layer. The latter localization may indicate the involvement of a cholinergic mechanism at the functional contacts (transferapses) between the horizontal cells. In the optic tectum of Eugerres plumieri, histochemistry reveals fine distinguishable bands of acetylcholinesterase activity; two of the bands are quite sharply defined, whereas three others have rather a more diffuse appearance. The presence of these bands and their distribution may suggest a widespread distribution of cholinergic elements in the optic tectum.     

Ultrastructural and cytochemical observation of calcium ion localization in the black-beetle pleural muscles

The authors examined the infrastructure and distribution of calcium ions in the plexural muscles of a black-beetle (Blatta orientalis L.) after being fixed in a solution of osmiumtetroxide buffered with a cacodylate buffer (pH = 7.4) and according to the method of Carasso-Favard (1966). The infrastructure of these muscles differs from the muscles of other insects, first of all, in the amount and distribution of the sarcoplasmic net (SR) and mitochondria and also in the amount and topography of the location of lead precipitations which mark the calcium ions. The authors ascertained an intensive and permanent positive reaction to the presence of calcium in the mitochondria and sarcotubular systems of the sarcoplasmic reticulum, and in the intrafibrillar spaces and in the Z and M lines of the sarcomeres calcium concentrations are not detectable. The authors checked the results by using the method of Carasso-Favard (1966).     

Ultrastructural localization of calcium deposits during in vitro culture of pig oocytes

Calcium deposits were localized using the combined oxalate-pyroantimonate technique in follicle-enclosed oocytes fixed in situ. These deposits can be observed within vacuoles, mitochondria, and on the surface of yolk granules as well as in the caryoplasm, but are absent from the endoplasmic reticulum. Isolation of the oocyte from the follicle resulted in the immediate depletion of these calcium deposits. Replenishment of these deposits started during the first 8 hr of in vitro culture of the oocyte and they were gradually replenished to the levels observed before the liberation of oocytes during in vitro maturation to the stage of metaphase II.     

Calcium stores in drosophila retina revisited: An electron-cytochemical localization of calcium

According to the commonly accepted model of phototransduction in insects, the endoplasmic submicrovillar cisternae (SMC) is the main element of Ca²⁺ homeostasis in the photoreceptor cell. It is generally believed that the light-induced inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from these putative Ca²⁺ stores is an obligatory intermediate event in the cascade of phototransduction resulting in activation of the light-sensitive channels. However, it appears that this model fits well mainly the insects with a fused rhabdom and large SMC. In this study it has been found electron-cytochemically that in Drosophila that has an open rhabdom and miniature SMC, the reaction product for Ca²⁺ (calcium oxalate) accumulates not in SMC, but in the specialized extracellular compartment formed by the distended lacunae at the bases of the rhabdomeral microvilli, which sometimes deeply invaginate into the submicrovillar cytoplasm. It is suggested that in Drosophila and probably in other insects with an open rhabdom, it is this extracellular compartment, not SMC, that together with the calphotin area of the cytoplasm functions as a key element of the Ca²⁺ homeostasis in the photoreceptor cell.     

Calcium stores in drosophila retina revisited: An electron-cytochemical localization of calcium

According to the commonly accepted model of phototransduction in insects, the endoplasmic submicrovillar cisternae (SMC) is the main element of Ca²⁺ homeostasis in the photoreceptor cell. It is generally believed that the light-induced inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from these putative Ca²⁺ stores is an obligatory intermediate event in the cascade of phototransduction resulting in activation of the light-sensitive channels. However, it appears that this model fits well mainly the insects with a fused rhabdom and large SMC. In this study it has been found electron-cytochemically that in Drosophila that has an open rhabdom and miniature SMC, the reaction product for Ca²⁺ (calcium oxalate) accumulates not in SMC, but in the specialized extracellular compartment formed by the distended lacunae at the bases of the rhabdomeral microvilli, which sometimes deeply invaginate into the submicrovillar cytoplasm. It is suggested that in Drosophila and probably in other insects with an open rhabdom, it is this extracellular compartment, not SMC, that together with the calphotin area of the cytoplasm functions as a key element of the Ca²⁺ homeostasis in the photoreceptor cell.     

Microtubules in cone myoid elongation in the teleost retina

The myoids of retinal cone cells of the blue-striped grunt (Haemulon sciurus) undergo significant elongation during dark adaptation of the retina. Longitudinally oriented microtubules are present in myoids both before and after elongation. Injection of colchicine into the vitreous of the eye in vivo disrupts the microtubules in the myoids and prevents dark-adaptive myoid elongation. Counts of microtubules in transverse sections along the lengths of elongating myoids show that there is a uniform decrease in the number of microtubules at any one point along the myoid as the myoid elongates. The magnitude of the decrease is proportional to the extent of the elogation. The product of the mean myoid microtubule number (determined from counts at progressive intervals along the myoid) and the myoid length remains essentially constant during myoid elongation, indicating that the total quantity of microtubules in the myoid does not increase with elogation. Serial section tracings of the microtubules along the myoids suggest that individual microtubules do not extend the length of the myoid and that the myoid microtubular apparatus consists of bundles of overlapping shorter microtubules. We propose that elongation of the myoid is accompanied by sliding redistribution of microtubules along the length of the myoid, and that the sliding may be generated by interaction between microtubules in regions where they closely overlap in bundles. We find no evidence for the involvement of discrete, electron-dense microtubular organizing centers in myoid elogation.     

Ultrastructural localization of intracellular calcium stores by a new cytochemical method

We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods.