Development of the myoneural junction in the rat

Summary The structure of the myoneural junction in the striated muscle of rat embryos and postnatal rats was studied by electron microscopy in order to assess at ultrastructural level the roles of neuronal and muscular elements and the sequence of events resulting in the formation of a functionally mature synaptic organization.From the observations it is concluded that the axon terminals enveloped by Schwann cells contain vesicles prior to apposition of the prospective synaptic membranes. Subsequently, subsarcolemmal thickening of the postsynaptic membrane takes place after the synaptic gap has been formed by disappearance of the teloglial cell from between the synaptic membranes but before the primary synaptic cleft in the strict sense is formed. Secondary synaptic clefts are formed later, when the primary synaptic cleft is regular in width, by local finger-like invaginations of the postsynaptic membrane, which thereafter expand basally, in a plane transverse to the axis of the axon terminal, to resemble flattened flasks. The junction is formed between multinucleated muscle cells and multiple axons, which at first lie side by side and later, when formation of adult-type secondary synaptic clefts is in progress, become separated by folds of the sarcoplasm and the teloglia. In extraocular muscles of adult rats the sarcoplasmic reticulum is closely associated with the postjunctional sarcoplasm.In the light of earlier observations on the development of contractibility after nerve stimulation, cholinesterase histochemistry and muscle fibre physiology, these observations are interpreted to indicate that functional differentiation of the myoneural synapse results from induction by the motor axon and that the association of the sarcoplasmic reticulum with the postjunctional sarcoplasm in adult extraocular muscles is related to modified fibre physiology.The author wishes to thank Prof. Antti Telkkä, M.D., Head of the Electron Microscope Laboratory, University of Helsinki, for placing the electron microscopic facilities at his disposal.     

Localization of acetylcholinesterase in the rat myoneural junction

Summary The distribution of myoneural acetylcholinesterase (AChE; EC. 3.1.1.7) was studied electron microscopically with the copper ferricyanide method at pH 6.0, using acetylthiocholine iodide as substrate and iso-OMPA to exclude other cholinesterase activity.It was observed that the results obtained with this method are affected by changes in the reaction temperature, inhibitor concentration and fixation time. Changes which retard the rate of hydrolysis of AChE were observed to transfer the localization of the reaction endproduct in intact junctions from the extracellular side of the postsynaptic membrane to the intracellular side of the postsynaptic membrane. From the results it is concluded that the site of most intense AChE corresponds to the region of increased subsarcolemmal electron density of the postsynaptic membrane.     

Effects of colchicine and vinblastine on neurotubules of the sciatic nerve and cholinesterases in the developing myoneural junction of the rat

Summary Colchicine (0.1 M) or vinblastine (0.01 M) was locally applied on the sciatic nerves of newborn rats. Both colchicine and vinblastine caused reversible disappearance of axonal neurotubules and appearance of increased amounts of neurofilaments at the site of application. Subsequent morphogenesis of myoneural junctions in the tibialis anterior muscle was studied after histochemical demonstration of acetylcholinesterase (AChE; E.C. 3.1.1.7) and non-specific cholinesterase (Ns. ChE; E.C. 3.1.1.8) activity in the myoneural area.Development of the postsynaptic muscle plasma membrane of the myoneural junction was arrested in the ipsilateral, but not in the contralateral control side, for a period of about three weeks following treatment with the test substances. After this delay the myoneural morphogenesis continued normally and neurotubules were seen in the axoplasm.Since disruption of neurotubules is likely to cause blockage of the intratubular axoplasmic transport system, it seems possible that the neurotrophic influence responsible for the development of the postsynaptic muscle membrane is mediated through a secretory product transported along axons intratubularly to the nerve endings.     

Electron microscopic demonstration of cholinesterases in nervous tissue

Summary Acetylcholinesterase was demonstrated at ultrastructural level in the motor nerve cells of rat's spinal cord using the Karnovsky-Roots modification of Koelle's thiocholine method. Selective inhibitors were employed to check the validity of the reaction.Prolonged formaldehyde fixation improved the poor penetration of the reactive agents and diminished the relatively large crystal size of the end product, which were the two main difficulties of the method. The preservation of ultrastructure was highly improved, when thin sections were made without freezing using a tissue chopper.Acetylcholinesterase was localized in the nuclear envelope, in the rough-surfaced endoplasmic reticulum, in medium-sized vesicles of the Golgi apparatus, and around synaptic terminals. Synaptic vesicles were found negative.     

Electron microscopic localization of monoamine oxidase in the rat liver

Summary Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2′-benzothiazolyl)-5-stryl-3-(4′-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondral membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase. Supported by research grants from the National Research Council of Canada (A 3651), The Swedish Medical Research Council (4145) and M. Bergwall's Foundation, Stockholm.     

Electron microscopic localization of monoamine oxidase in the rat liver.

Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2'-benzothiazolyl)-5-stryl-3-(4'-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondral membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase.     

Electron microscopic immunohistochemical localization of alpha-msh in the rat brain.

Using an immunohistochemical technique at the electron microscopic level, it has been shown that alpha-MSH is localized within the small vesicles of a few cell bodies found in the arcuate nucleus and numerous nerve fibers widely distributed throughout the brain. These findings suggest that alpha-MSH could possibly be considered as a neurotransmitter.     

Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary

Recent immunoelectron microscopic studies have shown that immunoreactive prolactin (PRL) in rat pituitary can be detected not only in typical PRL cells, characterized by large secretory granules, but also in another type of cell, which contains small secretory granules. To determine whether or not these two cell types are involved in PRL biosynthesis, we developed a procedure to investigate PRL gene expression by using in situ hybridization at the ultrastructural level. Rat pituitary was fixed and vibratome sections were incubated with a PRL [35S]-cDNA probe and subsequently flat-embedded in Araldite. Semi-thin and ultra-thin sections were processed for autoradiography. The results indicate that only the two PRL cell types were labeled. When immunolabeling for PRL was applied to ultra-thin sections, only immunopositive cells were seen to contain silver grains. In these cells the silver grains were associated with the rough endoplasmic reticulum and nucleus. When a growth hormone (GH) [35S]-cDNA probe was used as a control, only GH-secreting cells were labeled. This study confirms that the two PRL cell types are involved in biosynthesis of PRL. Moreover, this simple in situ hybridization technique provides a new approach to accurately localize mRNA in complex tissue and to investigate the subcellular distribution of mRNA under differing experimental conditions.     

Electron microscopic localization of alkaline phosphatase in the trigeminal ganglion of the rat

Summary The electron microscopic demonstration of alkaline phosphatase (ALP) was carried out on the trigeminal ganglion of the rat using the calcium lead modification method by Gomori (Gomori, 1952; Molnar, 1952).The ALP reaction was localized on the junction of capsular cells and nerve cells, in the cytoplasm of some dark capsular cell and in that of the endothelial cell: The enzymatic reaction products (1) existed throughout the entire length of the junction of clear cells and capsular cells, (2) aggregated at some points of the junction of dark cells and capsular cells, (3) existed on the smooth and/or rough surfaced endoplasmic reticulum and on the ribosomes of some dark capsular cells.     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.     

Localization of the ruthenium red-positive substance in the myoneural junction of rat.

Ruthenium red staining of myoneural junctions was examined in the flexor digitorum brevis muscle of the rat. Ruthenium red-positive electron dense substance was observed to emerge from the outer layer of the presynaptic axolemma and post-synaptic sarcolemma towards the synaptic cleft. Also the cleft substance was intensely stained, usually consisting of a medium dense layer between pre- and postsynaptic membranes. The probable function of acid mucopolysaccharides in the neuromuscular transmission is discussed.     

Action of inhibitors at the myoneural junction

1. A study is presented of the actions of certain inhibitors on the frog rectus abdominis muscle stimulated by acetylcholine. 2. A type of analysis has been developed which provides a reliable criterion for judging whether an inhibitor is competing with acetylcholine for receptors at the myoneural junction or whether acting by a different mechanism. 3. The "curares" are shown to act by competitive inhibition at the myoneural junction, confirming earlier work of others on the mode of action of curare. 4. Atropine acts as an inhibitor at the myoneural junction. The inhibition may be non-competitive or it may be complicated by an additional effect at some point other than the myoneural junction. 5. A possible mechanism for anomalous inhibitor effects is the action of a single compound at more than one locus in the Ach mechanism. Eserine exerts such a dual effect at the end-plate. 6. Some of the available electrical and chemical data have been correlated to make possible a partial explanation of the role of Ach in transmission at the myoneural junction.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

Electron microscopic localization of hydrolytic enzymes in osteoclasts

Synopsis Acid glycerophosphatase activity (pH optimum, 5.0) has been found within osteoclasts by numerous workers but relatively few studies have been concerned with the neutral hydrolytic enzymes that have pH optima around 7.2. Evidence is presented in this paper to show that neutral enzyme activity can be demonstrated withp-nitrophenyl phosphate, ATP andp-chloranilidophosphonate as substrates. Activity against -glycerophosphate, inorganic trimetaphosphate orp-nitrocatachol sulphate as substrates was found to be limited to an acid pH range.Electron microscopic evidence indicated that many of the hydrolytic enzymes were present within single membrane-bound bodies, vacuoles, Golgi elements, and the agranular endoplasmic reticulum of the osteoclast. This reticulum was dilated to form large lysosomes. Such activity was distingly different from the function of the Golgi membrane in its formation of primary lysosomes.The relationships between lysosomes, cytoplasmic vacuoles, and extracellular release of enzyme through the specialized ruffled border are shown and discussed.     

Electron microscopic localization of the multicatalytic proteinase complex in rat liver and in cultured cells.

The multicatalytic proteinase (MCP) prosome or proteasome is a large multifunctional complex which is believed to play a major role in non-lysosomal pathways of intracellular protein degradation and has recently been implicated in antigen processing. In this study, affinity-purified antibodies against rat liver MCP were used to investigate the localization of the proteinase both in rat liver and in growing human L-132 cells in culture, using electron microscopic immunogold techniques. Quantitation of the MCP in different subcellular localizations by morphometric analysis of electron micrographs showed the proportion in the nucleus to be 17% for hepatocytes and 51% for L-132 cells, demonstrating differences in the distribution of MCP in different cell types. In hepatocytes, 14% of the total MCP was found associated with the endoplasmic reticulum. The remainder was localized in the cytoplasmic matrix. Immunofluorescence studies with L-132 cells also showed a reaction in nuclei and cytoplasm. The localization of MCP is consistent with its proposed multiple functions in protein turnover, in the production of peptides for antigen presentation, and in RNA processing.