Eric Holtzman

Stimulation-Dependent Alterations in Peroxidase Uptake at Lobster Neuromuscular Junctions

The uptake of cytochemically demonstrable horseradish peroxidase into small vesicles within nerve endings in lobster stretcher muscles can be enhanced by electrical stimulation of transmitter release by the endings. This is observed particularly if stimulation is interrupted periodically and the nerves

NOTES ON SYNAPTIC VESICLES AND RELATED STRUCTURES, ENDOPLASMIC RETICULUM, LYSOSOMES AND PEROXISOMES IN NERVOUS TISSUE AND THE ADRENAL MEDULLA

This paper reviews aspects of the origin and fate of synaptic vesicles and of the related catecholamine-containing secretion granules of the adrenal medulla. Most attention is given to evidence concerning the proposal that the membrane surrounding synaptic vesicles can originate from axonal agranular reticulum, participate in exocytosis and endocytosis and eventually undergo degradation in lysosomes of axons and perikarya. A number of relevant details of the roles of several organelles in neurons and other cells of the nervous system are discussed. The paper centers around microscopic and cytochemical work on a few experimental systems.

Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system.

A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.

Microperoxisomes in the central nervous system of the postnatal rat

The distribution of catalase-containing microperoxisomes was studied in the central nervous system of rats during the early postnatal period when the processes of myelination and cell differentiation are active. The regions selected for study included a region previously found in adult animals to contain substantial numbers of reactive microperoxisomes, as well as areas where few such bodies were seen. Microperoxisomes were relatively numerous in all areas during the first two postnatal weeks; at later times they occurred less frequently, or, in some areas, were almost entirely absent. In early postnatal CNS, catalase-positive microperoxisomes were found in all cell types in all regions studied. Neurons of both cerebrum and cerebellum contained catalase-positive microperoxisomes during the first two postnatal weeks; few such neuronal bodies are found in these areas after the third postnatal week. During the period of active myelination, catalase-positive microperoxisomes were found in oligodendrocyte cell bodies, and also in oligodendrocyte processes associated with forming myelin sheaths in all areas. In several areas during the first 3 postnatal weeks, catalase-positive bodies were seen in synaptic terminals, a location where they are seldom observed in mature tissue. Cells of postnatal choroid plexus also were found to contain modest numbers of reactive microperoxisomes.

Smooth endoplasmic reticulum and other agranular reticulum in frog retinal photoreceptors

SummaryFrog retinal photoreceptors are favourable material for studying a number of unresolved issues concerning the interconnections, three-dimensional organization and functions of intracellular membrane systems in neurons. At least two distinct regions of smooth endoplasmic reticulum (SER) are present in these cells. One region, the subellipsoid SER, is located in rod cells at the base of the mitochondria-rich ellipsoid region, and is comprised of arrays of stacked tubules which exhibit frequent continuities with the rough endoplasmic reticulum (RER). The subellipsoid SER is also present throughout the ellipsoid region and at the apex of the inner segment. The second region of SER, the axonal SER, is comprised of agranular sacs and tubules present in the axons of rod cells, the perinuclear and Golgi regions of rod and cone cells and the synaptic terminals of rod and cone cells. These sacs and tubules exhibit continuities with cisternae of RER and with the nuclear envelope. Serial section analyses indicate that this SER can extend as a continuous network along the entire length of the rod axon and throughout synaptic terminals. The axonal SER is distinct from the subellipsoid SER not only in location and morphology but also in its ability to bind divalent lead ions, a property it shares with synaptic vesicles, with agranular sacs at one face of the Golgi apparatus and with sacs extending from the Golgi apparatus toward the axon hillock. These latter sacs may serve in transport from the Golgi region to the axon.The axonal SER in the axon, terminals, and the perinuclear and Golgi regions appears to be a source of synaptic vesicles as evidenced by this lead binding capacity and by the observation of vesicles, with the size (50–75 nm) and appearance of synaptic vesicles, budding from SER in direct continuity with RER.The endoplasmic reticulum (ER) in synaptic terminals of frog photoreceptors is not continuous with endocytic structures found in the same region, such as blunt-ended tubules or anastomosing networks of tubules. Nor does the ER acquire exogenous horseradish peroxidase. These observations suggest that the ER does not play a direct role in membrane recycling in photoreceptors.

PEROXISOMES IN DORSAL ROOT GANGLIA

Bodies with the morphologic and cytochemical characteristics of peroxisomes have been identified in the satellite and Schwann cells of rat dorsal root ganglia. They are membrane-delimited, round or oval structures which contain a moderately electron-dense matrix but lack a crystalline core. On incubation of the tissue in a cytochemical medium for demonstration of peroxisomes, these bodies show heavy deposits of reaction product. The reaction is inhibited by heating the tissue or by incubation in the presence of aminotriazole or dichlorophenolindophenol. In tissue incubated for acid phosphatase activity the bodies are not reactive, although lysosomes show reaction product.

PEROXISOMES IN NERVOUS TISSUE

Enzymes of the sort found in peroxisomes of the liver and kidney are known to occur in the brain.’.‘ These include both catalase and certain of the oxidases, such as D-aminO acid oxidase (DAAO). The abundance of these enzymes varies considerably among different brain regions. For example, DAAO seems particularly concentrated in the cerebellum. Definitive demonstration that the enzymes are associated with peroxisomes has yet to be accomplished biochemically, but there has been a study showing that much of the catalase, and oxidases, are associated with sedimentable particles.‘ Interestingly, there are differences in sedimentation behavior between catalase and the oxidases suggesting that there may be significant enzymatic heterogeneity among the organelles, some being catalase-rich and oxidase-poor and others showing oxidases but not much catalase. The available data do not yet demonstrate whether some of the catalase-containing bodies lack oxidases altogether, or whether some of the oxidase-containing bodies lack catalase, but these remains open possibilities. Cytochemically. catalase and DAAO in nervous tissue are demonstrable in membrane-delimited organelles similar to the microperoxisomal variety of peroxisomes seen in other tissues. In our work, primarily on rat tissues, we find3-7: 1. Catalase-containing bodies are generally more abundant in the nonneuronal cells (glia, satellite cells, Schwann cells] of dorsal root ganglia and central nervous tissue than they are in neurons. The catalase-containing bodies in the non-neuronal cells tend to be larger (0.2 pm in diameter) than those in neurons (0.1 pm). In some of the non-neuronal cells, the peroxisomal population demonstrated with the catalase procedures is substantial. Thus, for example, in satellite cells of dorsal root ganglia there appear to be more peroxisomes than recognizable lysosomes. As estimated from thin sections, oligodendrocytes contain 30-40 peroxisomes per 100 pmz of cytoplasm whereas neurons of corresponding brain regions contain 0-7. In oligodendrocytes and satellite cells, the catalase-reactive bodies are distributed throughout the cell body; in astrocytes they tend to be present in processes and “end-feet.” Endothelial cells of blood vessels, ependymal cells lining brain ventricles, and choroid plexus epithelial cells also contain appreciable populations of catalase-positive bodies. The catalase-containing peroxisomes of nervous system cell types show frequent propinquities to mitochondria. They also show the smooth-surfaced outpocketings or tails whose nature [connections to the ER vs. connections to other peroxisomes) is currently being debated. 2. Among the neurons of mature nervous tissue, certain types contain markedly larger populations of peroxisomes with cytochemically detectable catalase than do others. Catecholaminergic neurons especially, both in the peripheral nervous system and in the central nervous system, have large populations.

Quantitative analysis of exocytosis and endocytosis in the hydroosmotic response of toad bladder.

SummaryThis study concerns the timing and magnitude of exocytosis and endocytosis in the granular cells of toad bladder during the hydroosmotic response to antidiuretic hormone. Granule exocytosis at the luminal cell surface is extensive within 5 min of the administration of a physiological dose of hormone. Hydroosmosis becomes detectable during this time period. The amount of membrane added to the luminal surface by exocytosis during 60 min of exposure to hormone can be of the same order of magnitude as the extent of the luminal plasma membrane. Endocytosis, demonstrated by peroxidase uptake from the luminal surface, becomes extensive during the period 15–45 min after hormone administration. Thus, maximal endocytic activity occurs later than the period of most extensive exocytosis and seems to correlate with the onset of the decline in water movement. The amount of membrane retrieved from the luminal surface by endocytosis during 60 min of stimulation is at least three quarters of that added by exocytosis. The bulk membrane movement in ADH stimulated preparations does not require the presence of an osmotic gradient. Colchicine inhibits the hydroosmotic response, the exocytosis of granules, and endocytosis at the luminal surface. These results strengthen our view that the bulk circulation of membrane at the cell surface, via exocytosis and endocytosis, is closely related to the permeability changes occuring at the surface.

Acidification and endosome-like compartments in the presynaptic terminals of frog retinal photoreceptors

SummaryBy using the ‘acidotropic’ vital dye, Acridine Orange, we have found that the presynaptic terminals of rod and cone photoreceptors in retinas ofRana pipiens maintain a low pH relative to the surrounding medium through an energy dependent mechanism. When this pH is raised, by exposing the retinas to weak bases like ammonium chloride, the terminals exhibit large, membrane-delimited compartments, many of which accumulate endocytic tracers. This effect is partly reversed when the weak bases are removed. We infer that among the acidified structures within the terminals are endocytic compartments with at least some of the characteristics of the endosomes that participate in receptor-mediated endocytosis in other cell types. One role of these structures in the terminals may be in the recycling of synaptic vesicles.

A phosphatase activity and a synaptic vesicle antigen in multivesicular bodies of frog retinal photoreceptor terminals

SummaryPrevious work has suggested that multivesicular bodies participate in endocytosis and membrane cycling at nerve terminals, including the presynaptic terminals of retinal photoreceptors. We now have found that multivesicular bodies located in the presynaptic terminals of photoreceptors in retinae ofRana pipiens show reaction product in preparations incubated to demonstrate phosphatase activity at pH 5, using cytidine monophosphate as the substrate. Evidently, multivesicular bodies in photoreceptors can possess at least some hydrolytic enzymes during their sojourn in the terminals. We have also found that the multivesicular bodies in frog retinal photoreceptor terminals stain, immunocytochemically, for the presence of SV2, an antigen of synaptic vesicles. This observation supports the suggestion that, along with the extensive, repeated reuse of membrane components for synaptic vesicle recycling, there is some incorporation of the components into structures that are potentially degradative.