Ann H. Bunt

Retrograde axonal transport of horseradish peroxidase by ganglion cells of the albino rat retina

Abstract After introduction of small amounts of horseradish peroxidase (HRP) into known visual centers of the brain, retinal ganglion cells projecting to these regions were detected by the accumulation of HRP-positive granules in their somata. Control experiments indicated that the HRP-positive granules had reached the ganglion cell somata by retrograde axonal transport, and did not represent blood-borne or endogenous peroxidase. Using this technique, it has been determined that axons of both large and medium-sized neurons in the ganglion cell layer of adult and immature rat retinae terminate in the superior colliculus and lateral geniculate body, and that characteristic displaced ganglion cells with axonal connections to these visual centers occur regularly in these retinae. In addition, certain small cells in the retinal ganglion cell layer are described which may represent glia or interneurons, or ganglion cells which lack the ability to transport peroxidase or which lack central connections to these visual centers of the brain.

Enzymatic digestion of synaptic ribbons in amphibian retinal photoreceptors.

Abstract Enzymatic digestion of photoreceptor terminals in the frog retina has been performed on ultrathin sections of both Epon and glycol-methacrylate embedded tissue. The synaptic ribbons are extracted following incubation with pronase, protease, α-chymotrypsin, or pepsin, but are unaffected by treatment with trypsin, DNAse, RNAse, or perchloric acid. Other components of the synaptic apparatus, including the arciform and postsynaptic densities, cleft filaments and synaptic vesicles, appear unaffected by these procedures. It is concluded that the ribbons probably do not contain appreciable amounts of DNA or RNA, and may be composed, at least in part, of a protein or polypeptide which is rich in aromatic amino acids. This observation is correlated with previously described staining characteristics of the ribbons, and two new functions of the ribbons are proposed: structural support for the synaptic ridge specialization and participation in a ‘sliding vesicle’ mechanism for transmitter release.

Formation of coated and “synaptic” vesicles within neurosecretory axon terminals of the crustacean sinus gland

A “coated” vesicle type has been found in neurosecretory axon terminals of the crustacean sinus gland, in addition to the previously described dense-cored granules and “synaptic” vesicles. After either in vivo or in vitro exposure of the terminals to a colloidal suspension of Thorotrast, incorporation of tracer particles into both “synaptic” and coated vesicles is observed, suggesting origin of one or both vesicle types from the plasma membrane by micropinocytosis. Relevant consequences of such a process in axon terminals are discussed, including retrieval of membrane material following exocytotic release of hormone containing granules, absorption of nutrients, and resorption of transmitter products to the interior of the axon.

Features of foreign proteins affecting their retrograde transport in axons of the visual system

SummaryThe retrograde axoplasmic transport of foreign proteins in the rat visual system shows certain specificities. The molecular features of these proteins which may underlie their entry into the retrograde phase have been examined using biochemical and morphologic techniques. Of the isoenzymes of horseradish peroxidase (HRP), the basic isoenzyme C is strongly transported, while the acidic isoenzymes Aβ and Aα are transported weakly and not at all, respectively. Decreasing the isoelectric point (pI) of isoenzyme C from 8.2 to 4.4 decreases its transport, but a basic pI is not the sole requisite for transportability since two other basic peroxidases (turnip isoenzyme P7 and lactoperoxidase) are not transported in retrograde. The sugar component as a whole of isoenzyme C does not appear to be required for determining transport. Isoenzyme C and the other proteins which are transported enter multivesicular bodies in axons and axon terminals, as well as synaptic and coated vesicles and fine tubules in axon terminals. The non-transported proteins enter only the vesicular organelles thought to be involved in neurotransmitter recycling in axon terminals and do not enter multivesicular bodies. Thus the two systems of axonal membraneous compartments involved in local synaptic recycling versus the retrograde phase of transport do not show the same specificity of uptake of extracellular tracers and can be dissociated by the experimental use of these peroxidases.

Comparative study of3H-fucose incorporation into vertebrate photoreceptor outer segments

Abstract The pattern of incorporation of 3 H-fucose into photoreceptor outer segments has been analysed by light and electron microscopic radioautography in twelve different species spanning the animal kingdom. This sugar is incorporated in a diffuse pattern into cone outer segments in all animals examined and is incorporated into a basal band in rod outer segments of certain species. Differential incorporation into outer segments of red- and blue-vs green-sensitive cones was observed in fish retinae and into red but not green rods in the frog. Diffuse labeling of frog pinealocyte outer segments was found, in agreement with previous observations of their cone-like structure and function.

Fine structure of the pars distalis and interrenals of Taricha torosa after administration of metopirone (SU-4885)

Abstract Three tinctorially different cell types have been observed in the pars distalis of the newt, Taricha torosa , by light microscopy, whereas five granulated cell types and one nongranulated stellate cell have been revealed in this gland by electron microscopy. The adrenalectomy cells of this species, observed after treatment with the adrenocortical inhibitor. Metopirone, contain secretory granules with maximum diameters of 200 mμ and appear to be derived from the type II cells. The release of secretory granules from the adrenalectomy cells is by exocytosis, a mechanism observed frequently in the pars distalis of higher vertebrates but not previously described in the amphibian pituitary. Fine structural alterations of the mitochondria and lipid droplets of the interrenal cells after Metopirone treatment are also discussed.

Ramification patterns of ganglion cell dendrites in the retina of the albino rat

Ganglion cell morphology has been analyzed in the retina of the adult, albino rat by the Golgi technique and evidence has been obtained that the ganglion cells of this species show a far greater variety and complexity of organization than described previously. Both diffuse ganglion cells, whose dendrites ramify throughout the inner plexiform layer, and stratified ganglion cells, whose dendritic branching is restricted to one or two planes, have been identified. Evidence is also presented for distinct stratification of the inner plexiform layer in this species, based on radioautographic analysis following intravitreal injection of tritiated amino acids.

An association between mitochondria and microtubules in synaptosomes and axon terminals of cerebral cortex.

SummaryA substantial number of synaptosomes prepared from rat cerebral cortex were found by electron microscopy to contain a horseshoe-shaped mitochondrion flanked by an arc of three to ten microtubules -opposite the synaptic membrane specializations. The microtubules were replaced by characteristic paracrystals following the incubation of synaptosomes with vinblastine sulfate. A similar spatial organization of microtubules and mitochondria was also observed in some axon terminals of the cerebral cortexin situ. The significance of this novel observation is discussed with regard to the role of microtubules in axonal transport at the synaptic terminal and previous reports on the identification of tubulin in synaptosomes and subsynaptic structures.

Vinblastine-induced blockage of orthograde and retrograde axonal transport of protein in retinal ganglion cells

Abstract The right eyes of adult, albino rats were injected intravitreally with 0.1–30 μg of vinblastine sulfate, and the left eyes were similarly injected with saline. Twenty-four hours later, both eyes were injected with H3-proline and both superior colliculi with horseradish peroxidase. After 24 hr, the animals were killed and brains and retinae processed both for radioautography and the demonstration of peroxidase. Doses of 0.5 μg or more of vinblastine produced blockage of rapid orthograde transport of radioactively labeled protein within optic nerve axons without causing a significant decrease in protein synthesis in ganglion cells of the retina. A higher dose of vinblastine (10 μg or more) was required to block retrograde axonal transport of peroxidase from the superior colliculus to the ganglion cell somata. This suggests vinblastine-sensitive mechanisms for both orthograde and retrograde axonal transport of protein which may involve microtubules or free tubulin within the axoplasm, since vinblastine in low concentration binds specifically to tubulin.

Fucosylation of rabbit photoreceptor outer segments: Properties of the labeled components

Abstract Photoreceptor membranes prepared from rabbits which had received an intravitreal injection of [3H]fucose were composed of intact and broken outer segments and vesiculated membranous structures. Analogous to the pattern seen in vivo, only an occasional outer segment showed heavy and diffuse labeling with [3H]fucose in electron microscopic radio-autographs. These were probably derived from cone outer segments. Following hydrolysis and thin layer chromatography, the radioactive material obtained from [3H]fucose-labeled outer segments co-migrated with authentic fucose, showing that the sugar was incorporated without modification. SDS-polyacrylamide gel electrophoresis of solubilized outer segments showed that most of the incorporated radioactivity co-electrophoresed with opsin, the major staining material present. The remainder of the radioactivity was found in minor components of higher molecular weight than opsin. Isoelectric focusing of solubilized photoreceptor membranes produced a broad peak of radioactivity which was split into two peaks at lower concentrations. The majority of the counts were found to coincide with opsin, the major staining component, at an isoelectric point of 5·7. An additional peak of radioactivity was found at a pI of 6·3. Labeled components in solubilized photoreceptor membrane preparations were not retained during passage through a column of fucose-specific lectin (from Ulex europaeus) covalently attached to agarose, but were retained on a column of agarose-concanavalin A and eluted with α-methyl mannoside. Our evidence suggests that the major component of cone outer segments which becomes labeled with [3H]fucose is a protein with properties very similar to rod opsin.