Effects of calcium-containing fixation solutions on cholinergic synaptic vesicles.

Calcium (Ca)-containing fixation solutions applied to slices of electric organ of the electric ray, Narcine brasiliensis, have been shown to have three distinct ultrastructural effects on cholinergic synaptic vesicles of the nerve terminals. (a) An electron-dense particle (EDS) is observed within the vesicle; the particle is seen in unosmicated, unstained tissues and can be removed from thin sections by Ca-chelating agents. It is concluded that the EDS represents Ca bound by the vesicle. It is suggested that the bound ATP of the vesicle provides anionic Ca binding sites. (b) The vesicle membrane tends to 'crinkle' or collapse depending on the concentration of the other components of the fixative solution. The 'crinkling' or collapse are largely reversed by a wash step in the absence of Ca. (c) The presence of Ca results in the appearance of a population of vesicles which form characteristic fusions or 'tight' junctions with the terminal membrane. This appears to be morphological evidence for the proposal, which has been frequently put forward, that Ca facilitates such a fusion before discharge of vesicle-bound transmitter. With the discovery that the use of Ca-containing fixatives leads to the demonstration of a subpopulation of synaptic vesicles fused to the terminal membrane, we are led to propose that this is the ultrastructural location of the newly synthesized acetylcholine which has been shown by others to be preferentially released by stimulation.

Changes in cholinergic synaptic vesicle populations and the ultrastructure of the nerve terminal membranes of Narcine brasiliensis electron organ stimulated to fatigue in vivo.

Narcine brasiliensis electric organ was stimulated to fatigue in vivo. Electrical display of organ output and biochemical assay of bound acetylcholine (ACh) and ATP in isolated vesicles were used to assess the state of fatigue relative to denervated control organs of the same fish. A morphometric analysis of the fate of the synaptic vesicle populations in the nerve terminals was carried out. Statistically significant morphological changes in vesicle populations and plasma membranes were observed between control and fatigued electroplaque stacks from individual fish. Pooled data from several fish were used to evaluate the possible role of the different vesicle types in neurotransmission. Fatigue resulted in the loss of 49% of the total vesicle population and a 76% loss of vesicles with bound calcium (Ca). An approximately equivalent increase in the nerve-terminal plasma membrane area was measured. This was predominantly in the form of fingerlike protrusions and/or invaginations of the terminals which were present in the control organs but which were significantly increased by stimulation. Vesicle attachments to the nerve terminal membrane were reduced by 90%. This suggests that the failure in transmission may be due to reduction in the number of vesicles which are loaded with transmitter and can attach to the terminal membrane. The Ca-binding capacity of the lost vesicles was not transferred to the plasma membranes. This result was interpreted as support for the hypothesis that vesicle-bound ATP provides the Ca-binding site.

ATP-dependent calcium accumulation by non-mitochondrial organelles of axoplasm isolated from Myxicola giant axons.

Axoplasm from freshly isolated Myxicola giant axons was mixed with small volumes of 'artificial axoplasm' containing 45Ca and either CaEGTA/EGTA or CaDTPA/DTPA buffers giving various nominal values of [Ca2+]. The axoplasm samples were centrifuged at 100 000 X g for 30 min to form a pellet and the percentage of 45Ca bound to the pellet was determined. The fraction of bound calcium rose with increasing values of [Ca2+] along an S-shaped curve. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to reveal the presence of mitochondrial Ca uptake. At physiological values of [Ca2+], around 100 nM, Ca uptake was insensitive to FCCP. As [Ca2+] was elevated, increasing sensitivity to FCCP was noted above [Ca2+] = 0.5 microM. At low values of [Ca2+], including the physiological range, Ca binding was significantly reduced by vanadate and quercetin, agents known to inhibit Ca uptake mediated by Ca2+-activated ATPase reactions. Inhibition of Ca binding by these agents was approximately 50% at physiological values of [Ca2+]. ATP depletion decreased the percentage of Ca binding by the pellet at physiological [Ca2+]. The results suggest that about 50% of the Ca buffering by particulate matter in axoplasm is via organelles requiring intact Ca2+-ATPase reaction at physiological values of [Ca2+].

Quick-freeze fixation and freeze-drying of isolated rat pancreatic islets: application to the ultrastructural localization of inorganic phosphate in the pancreatic beta cell.

A bounce-free mechanical quick-freeze assembly and a Coulter-Terracio freeze-dry apparatus were successfully coupled to obtain high quality ultrastructural preservation of pancreatic beta cells in a simple and dependable manner. Except for obvious shrinkage spaces, morphological relationships at the tissue, cellular, and subcellular levels were all intact. Beta cell secretory granules demonstrated a dense core surrounded by an electron lucent halo as typically described in specimens after aqueous fixation. Cell membranes and intracellular membranes demonstrated a trilaminar appearance. Golgi apparatus were well preserved. Two clearly defined populations of mitochondria were found. One group of very dark mitochondria had extremely dense matrices in which cristae were barely visible. A second group of mitochondria had light matrices with prominent cristae. The combined quick-freeze fixation and freeze-drying was applied to reevaluate the ultrastructural localization of inorganic phosphate that had been precipitated with lead in the beta cells of pancreatic islets. Accumulation of inorganic phosphate adjacent to the plasma membrane and over the nucleolus of the beta cell in nonstimulated islets was documented with better detail than heretofore possible.

Novel secretory granule morphology in physically fixed pancreatic islets.

Protein A-gold immunocytochemistry has been applied to physically fixed beta cells from rat islets of Langerhans. The punctate nature of the gold particles permits improved resolution of the antigenic sites without obscuring the fine ultrastructural preservation obtained by physical fixation. There is a filamentous material within the halo of the secretory granules that is not preserved by aqueous, chemical fixation. When viewed in stereo the filaments appear as an annular cobweb or a series of wheel spokes attached to a centrally located hub (the dense core of the granule). The filaments demonstrate insulin-like immunoreactivity using the protein A-gold technique. The immunoreactivity appears to be restricted to the filaments and the surface of the dense cores. This may be a consequence of the preservation of a solid, insolubilized core state that resists penetration by the antibody and/or the protein A-gold complex. However, the evidence that there is a halo pool of insulin which is separate from the massive core aggregate suggests that i) correspondingly massive exocytotic pits may not be as mandatory for insulin release as has been assumed and ii) the complex kinetics of insulin secretion may be, in part, a reflection of multiple insulin compartments within secretory granules.

Quick-freezing and freeze-drying in preparation for high quality morphology and immunocytochemistry at the ultrastructural level: application to pancreatic beta cell.

Quick-freeze fixation and freeze-dry methods were used successfully to obtain ultrastructural localization of insulin in the pancreatic beta cell by the unlabeled antibody-enzyme technique. In unosmicated freeze-fixed and freeze-dried islets, insulin was specifically demonstrated over the dense core of the secretory granules. In osmicated freeze-fixed and freeze-dried islets, insulin antigenicity withstood the osmium tetroxide vapor treatment. In addition, the surrounding ultrastructural resolution of morphologic features was significantly improved, which allowed insulin to be localized not only in secretory granules, but also in intracellular membranous compartments, with a degree of confidence not heretofore possible. Extracellular sites of insulin positivity in the islet were also localized and possible exocytotic activity for showing insulin release was observed.

A gentle bounce-free assembly for quick-freezing tissues for electron microscopy: application to isolated torpedine ray electrocyte stacks.

Thin, isolated stacks of Narcine brasiliensis electric organ were prepared for freeze substitution using a rapid freezing device similar to that described by Van Harreveld and Crowell (1964). Although the impact stress was reduced as much as the design of the apparatus allowed, the tissue was found to become severely squashed and the first layer of nerve terminals was found to be badly damaged. However, in occasional blocks, the morphology of the deeper layers encouraged the view that the tissue could be well suited to the procedure if the impact squash could be minimized. A second problem was identified when studying the impact of various tissue holders with the freeze surface: the original type of apparatus is highly susceptible to bouce in the millisecond time range. Tissue squash was controlled by mounting the sample on a piece of dry foam fitted into a slotted striker tip. Bounce was eliminated by coupling the striker to a simple hydraulic-pneumatic damping device. When electrocytes were frozen with this apparatus and freeze-substituted, the first layer of nerve terminals was found to be intact, well frozen and well fixed.

Isolation of synaptic vesicles from Narcine brasiliensis electric organ: some influences on release of vesicular acetylcholine and ATP.

A simple density gradient centrifugation technique for separating electric organ cholinergic synaptic vesicles from other organelles and membrane fragments is described. Frozen, ground electric organ is extracted with a solution of similar density to the vesicles; during the subsequent centrifugation, vesicles remain suspended in the extraction medium and heavier contaminating structures sediment out. In confirmation of results obtained with mammalian central nervous system vesicles, a biphasic pattern of efflux of bound ACh is demonstrated. Low levels of phospholipase A2 (EC 3.1.1.4.) induce efflux of ACh from the vesicle fraction; it is shown that the concomitant fall in vesicle bound ATP is due to ATP efflux rather than ATP hydrolysis within the vesicle.

Exocytosis and nerve terminal pseudopodia.

When exocytosis of synaptic vesicles is accompanied by the accumulation of vesicle membrane in the nerve terminal membrane, the geometric shape of the terminal must alter. The details of these rearrangements vary with the anatomical site; this laboratory has reported on the responses of abutted nerve terminals in the electric ray electric organ. When they are stimulated so as to lose synaptic vesicles, they develop reciprocal pseudopodial indentations (PSIs) with each other. Assuming that direct abutment of the interacting nerve terminals was necessary for this to occur, we have examined various nuclei of the rat brain limbic system for similar configurations. PSIs are most abundant between abutted terminals synapsing with smooth dendrites in the globus pallidus and substantia nigra. In these locations, there is good reason to believe that they are forming between swellings of the gamma-aminobutyric acid (GABA) afferents from the caudate-putamen. Conservative calculations of the potential accumulation of extracellular K released by action potentials at the PSI tip suggest that 15 mM concentrations could occur at firing rates of 150 Hz. Inasmuch as the GABA projection system to these nuclei is a system of boutons en passant, in which the safety factor for action potential conduction is low, it is suggested that the formation of PSI and the frequency-dependent accumulation of K could lower the safety factor to the point of action potential block. This may affect the inhibitory tone in the substantia nigra. An understanding of how PSI generation is regulated depends in part on knowing what options are available for synaptic vesicle behavior at the moment of depolarization of a nerve terminal. In particular, we need to know whether vesicles can open and close in situ during slow firing under physiological conditions. Recent experimental results enable us to foresee how this could be tested, and the experimental design is described.

Pseudopodial interdigitations between abutted nerve terminals: diffusion traps which occur in several nuclei of the rat limbic system.

Stimulation of the Torpedine ray electric organ can cause the loss of synaptic vesicles and the growth of pseudopodia from the nerve terminals (Boyne, A. F., and S. McLeod (1979) Neuroscience 4: 615-624). The latter embed themselves in corresponding indentations in abutted terminals. The geometry of these pseudopodial indentations (PSIs) can vary: (i) in length, (ii) in the extent of constriction of the base, and (iii) through a compound interaction between different pseudopodia extending in opposite directions. Examination of six rat brain nuclei in the limbic system has shown that their neuropil can be categorized according to the prevalence of either (i) nerve terminals indented by nerve terminal outgrowths (i.e. PSIs) or (ii) nerve terminals indented by dendritic outgrowths: these have been previously termed spinules. Clusters of simple PSIs were seen in the central nucleus of the amygdala, while base-constricted and compound forms were found in the globus pallidus and substantia nigra. Dendritic spinules were prevalent in the nucleus accumbens and the molecular layer of the hippocampus. In the CA4 hilar region of the hippocampus, large nerve terminals containing PSIs were found. The caudate neuropil appeared to be of mixed character in that the small terminals often had spinules but occasionally showed PSIs. Spinules have been recognized for many years and the possibility of their plasticity has been raised previously (Tarrant, S. B, and A. Routtenberg (1977) Tissue Cell 9: 461-473). The present report appears to be first detailed description of an alternative form of invasion which is known to be plastic in the elasmobranch electric organ. It is suggested that the extracellular space between the partners of a PSI could act as variable diffusion traps. If the involved boutons carry action potentials, then nonsynaptic release and accumulation of substances such as potassium, amino acids, and nucleotides may be expected during stimulation. Consequent direct or receptor-mediated effects on the membrane potential could influence transmission through adjacent synapses.

An excursion through the ultrastructural world of quick-frozen pancreatic islets.

In this article we have presented a philosophical and historical perspective on quick freezing, freeze-drying, freeze-substitution, and immunocytochemical localization of pancreatic islet hormones. A compilation of our findings indicates that quick-freezing does not produce any gross distortion of islet tissue; the amount of usable islet tissue for ultrastructural analysis is approximately 13 micron deep from the frozen edge; three different cell types can be identified in quick-frozen tissue based on general morphological characteristics; freeze-substitution with tetrahydrofuran produces a unique ultrastructural appearance in which ribosomes are particularly striking; with the use of protein A-gold, insulin and glucagon can be localized immunocytochemically on silver-gray (50-nm-thick) sections treated with 1% ovalbumin at room temperature overnight; secretory granules of quick-frozen alpha and beta cells may exist in either a swollen or condensed state; swollen beta cell secretory granules contain a filamentous material that demonstrates immunogold labeling for insulin; insulin and glucagon can be localized within the cisternae of endoplasmic reticulum; our methods provide not only discrete immunocytochemical localization of hormone, but also well-preserved cellular compartments; energy electron loss spectroscopy (EELS) has shown that quantifiable nitrogen maps can be used as an index of hormone packaging in secretory granules; and the sectioning properties of secretory granules at the ultramicrotome change when islet tissue is unosmicated and sectioned on glycerol.

Ultrastructural metabolic activity following quick-freezing and freeze-substitution in tetrahydrofuran in the superior cervical ganglion.

A method of quick-freezing and freeze-substitution has been developed for localizing diffusible substances such as 2-deoxyglucose-6-phosphate (2-DG-6-P) ultrastructurally in neural tissue. Quick-freezing under pressure provides well preserved tissue down to 30-35 microns from the surface. This allows blocks of neural tissue to be quick-frozen and analysed for diffusible substances in areas removed from the freezing face. Freeze-substitution in tetrahydrofuran following quick-freezing was found to dissolve and remove 2-deoxyglucose (2-DG) but not 2-DG-6-P. Consequently, this technique extends the ability to analyse localization of glucose utilization to postsynaptic as well as presynaptic sites. We have applied the technique to isolated superior cervical ganglion while provoking selective increases in energy metabolism. Exposure to an elevated extracellular potassium (12 mM) concentration produced a pattern of metabolic activity with enhanced neuropil labelling (neuronal and glial processes). With antidromic stimulation of the external carotid nerves, deoxyglucose uptake in neuronal and glial soma in the caudal portion of the ganglion was enhanced more than neuropil labelling. This caudal region corresponds to the region of origin of the cell bodies of the external carotid nerve. Results from this technique suggest that the contribution of glia to overall rate of energy metabolism may be significant and that this is a promising method for correlating the relationship between functional activity and cellular electrical activity.

Computer treatment of gas-liquid chromatographic data, with special reference to fatty acid methyl esters.

A computer program is described which analyzes output punched directly onto paper tape from a gas-liquid chromatograph. Although this program was written specifically for samples of fatty acid methyl esters derived from adipose tissue triglycerides which are eluted within 1 hr, modification of the dimension statements in the program would enable it to deal with samples which require a longer time to come off the column. The salient features of the rationale of the program are discussed in detail, particularly the procedures for base line correction and for estimating the contributions from components which are not perfectly separated in the column. Examples are given of the program in practice, of comparing the results it gives with those obtained by manual triangulation of the areas on a recorder chart, and of indicating the range of column load over which we have found that it operates satisfactorily. A sample computer print-out from the program is presented and interpreted.

Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry.

This study was undertaken to measure the effect of maximal stimulation of sodium pump activity on the rate of energy metabolism in mouse cerebral astrocytes. The rate of uptake of 3H-2-deoxyglucose (3H-2-DG) was measured in astrocyte cultures sodium-loaded either by incubation in a K+-deficient solution or by use of the carboxylic sodium ionophore monensin. Sodium-loading by the first method caused 3H-2-DG uptake to increase by 80%, but the effect was brief (about 5 min) compared with the period of uptake measurement (20 min). In contrast, the presence of monensin (20 microM) caused a sustained 3.4-fold increase in the rate of 3H-2-DG uptake. The concentration-response relationship for monensin indicated a Kd of 1.5 microM and a maximum uptake enhancement of approximately fourfold. The monensin-stimulated uptake of 3H-2-DG was totally inhibited by incubation of the cultures in either K+-free or Na+-free solutions, or in the presence of ouabain (0.4 mM), indicating that the enhancement of uptake was the result of Na+ influx and sodium pump activation. These results raise the possibility that astroglia contribute significantly to regional variations in glucose consumption associated with functional activity in the brain. Ultrastructural analysis showed that sodium-loading in K+-free solution caused swelling confined to the trans face of Golgi stacks. However, monensin (5 microM) caused swelling of the entire Golgi stack, with progressively more severe swelling from cis to trans cisternae and formation of cytoplasmic vacuoles.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles.

1. Synaptic vesicles separated by density-gradient centrifugation from extracts of the cholinergic nerve terminals of the electric organ of Torpedo marmorata were found to contain appreciable amounts of ATP as well as acetylcholine. 2. Vesicular ATP was stable in the presence of concentrations of apyrase and myokinase that rapidly destroyed equivalent amounts of endogenous or added free ATP; pre-treatment of cytoplasmic extracts of electric tissue with these enzymes destroyed endogenous free ATP, but did not affect the vesicular ATP. 3. When [U-(14)C]ATP was added to electric tissue at the time of comminution and extraction of the vesicles, all the radioactivity was associated with soluble components in the subsequent fractionation: none was associated with vesicles or membrane fragments; thus it is unlikely that vesicular ATP can be accounted for by the sequestration of endogenous free ATP within any vesicles formed during comminution and extraction of the tissue. 4. When synaptic vesicles were passed through iso-osmotic columns of Bio-Gel A-5m, which separates vesicles from soluble proteins and small molecules, all the recovered ATP and acetylcholine passed through together in the void volume. 5. Regression analysis showed that vesicular ATP content was highly correlated with vesicular acetylcholine content in different experiments, the molar ratio acetylcholine/ATP being 5.32+/-(s.e.m.) 0.45 (21 expts.) for the peak density-gradient fraction. The ratio varied, however, somewhat across the density-gradient peak suggesting some degree of chemical heterogeneity in the vesicle population.

Comparison of the ultrastructural myopathy induced by anticholinesterase agents at the end plates of rat soleus and extensor muscles.

Rats were treated with single subcutaneous injections of the irreversible AChE inhibitors, sarin (90 to 100 micrograms/kg) or soman (55 micrograms/kg), and with chronic doses of the reversible carbamate inhibitor, pyridostigmine. In surviving animals with severe behavioral symptoms, we examined the end-plate regions of the slow-twitch soleus and the fast-twitch extensor digitorum longus muscles, using the electron microscope. Within 30 min, sarin administration caused a recognizable subjunctional myopathy. The progress of morphologic damage was followed for 7 days, during which time the occurrence of damage diminished. The initial swelling of subjunctional organelles and vacuole generation progressed to the point where nerve terminals and attached postjunctional folds were lifted away from the muscle surface. This appeared to be caused by a combination of enlarging vacuoles and insertion of Schwann and macrophage cells into the lesions, and was followed by degeneration of the postjunctional folds. A new component of anti-AChE myopathy was recognized: progressive swelling of chromatin in subjunctional muscle nuclei. The soleus muscle was considerably more sensitive to these effects than the extensor muscle. Soman had a much less prominent ultrastructural effect on the muscle end plates. Chronic pyridostigmine treatment had effects similar to those of a single sarin injection on the soleus as well as a pronounced effect on the extensor muscle.

Modification, by tractive loading, of the energy cost of walking in sheep, cattle and man.

1. A study was made in sheep and cattle walking on a treadmill, of the alteration to the energy expenditure that was caused by voluntarily exerted tension in the tether between animal and treadmill. 2. An additional experiment was carried out to investigate in human subjects walking on a treadmill, the alteration to the energy expenditure as a result of positive and negative tractive loads applied at waist level through a rope parallel to the plane of the treadmill. 3. Alterations to the net energy expenditure when walking were generally similar in the three species and varied with speed and gradient up to a maximum value of approximately 0.04 J per metre walked per gram of tension or negative load.