Miriam M. Salpeter

Monte Carlo simulation of miniature endplate current generation in the vertebrate neuromuscular junction

A Monte Carlo method for modeling the neuromuscular junction is described in which the three-dimensional structure of the synapse can be specified. Complexities can be introduced into the acetylcholine kinetic model used with only a small increase in computing time. The Monte Carlo technique is shown to be superior to differential equation modeling methods (although less accurate) if a three-dimensional representation of synaptic geometry is desired. The conceptual development of the model is presented and the accuracy estimated. The consequences of manipulations such as varying the spacing of secondary synaptic folds or that between the release of multiple quantal packets of acetylcholine, are also presented. Increasing the spacing between folds increases peak current. Decreased spacing of adjacent quantal release sites increases the potentiation of peak current.

Electron Microscope Autoradiography

Much has been written regarding standard procedures for preparing EM autoradiograms. These aspects of the technique will therefore not be dealt with in any depth in this chapter. (A few examples of recent reviews and assessments are: Caro, 1966, 1969; Salpeter, 1966; Reimer, 1966; Jacob, 1971; Budd, 1971; Bachmann and Salpeter, 1972; Salpeter and Bachmann, 1972.) Basically, one can put a section, prepared as for electron microscopy, either onto a metal microscope grid, or onto a flat substrate (we use a collodion coated slide). The section may then be stained and carbon coated. Emulsion can be applied in liquid or in pregelled form. For the former, the most common methods are either to dip the specimen into diluted emulsion or to flood it with emulsion using a medicine dropper. In either case it is then drained vertically. Partially pregelled emulsion layers are most commonly formed in wire loops and then applied to the specimen. If a flat substrate is used, the specimen is not transferred to a metal microscope grid until after photographic processing.

Endplates after esterase inactivationin vivo: correlation between esterase concentration, functional response and fine structure

SummaryMouse sternomastoid muscles were incubated with diisopropylfluorophosphate (DFP)in vivo, and the time course of recovery was studied using histochemistry, EM autoradiography and physiology. We found that: (1) the ability of the muscle to sustain tetanus in response to nerve stimulation is eliminated when the esterases at the neuromuscular junctions are saturated with DFP. This ability is regained partially when <10% of the DFP-binding sites have recovered. (2) There is a positive correlation between the frequency of stimulation at which the tetanic response can be maintained and the extent of acetylcholinesterase (AChE) recovery. (3) Tetanic responses at fusion frequency (about 100 Hz) appear indistinguishable from controls with only about 25% of normal AChE. (4) Butyrylcholinesterase (BuChE) possibly of Schwann cell origin recovers more rapidly than does AChE. (5) The muscle shows fine structural changes involving Z band dissolution and the breakdown of sarcoplasmic reticulum within hours after esterase inactivation. (6) This myopathy reaches a peak at three days after esterase inactivation and is almost fully recovered by two weeks. (7) It can be eliminated if, at the time of esterase inactivation, the nerve is cut or the acetylcholine receptors at the endplate are inactivated by α-bungarotoxin.We suggest that the myopathy, seen after DFP, is mediated by Ca2+ fluxes due to prolonged action of acetylcholine (ACh) in the absence of esterases.

Monte Carlo simulation of neuro-transmitter release using MCell, a general simulator of cellular physiological processes

Issues surrounding synaptic current efficacy, variability, plasticity, and possible crosstalk are presently of great interest and lend themselves well to computational investigations. One important factor that impacts on all of these issues is the time course of neurotransmitter exocytosis from a synaptic vesicle.1 We have recently reported excellent quantitative agreement between highly accurate recordings of the fast rising phase of miniature endplate currents (mEPCs), and Monte Carlo simulations of vesicular acetylcholine release and postsynaptic mEPC generation.2 The simulations were performed using an early version of our program MCell, * a generalized and highly optimized outgrowth of our original Monte Carlo programs specifically tailored to simulation of mEPC generation.3,4 In this paper we first briefly introduce the present version of MCell and its capabilities, and then next discuss simulation of neurotransmitter exocytosis. We focus on: (1) the theoretical and computational factors underlying simulation accuracy: (2) how inadvertent use of seemingly appropriate input parameters can lead to orders-of- magnitude errors; and (3) how some of MCell’s features can reduce the computation time required for simulations by orders of magnitude.

Calcium-mediated myopathy at neuromuscular junctions of normal and dystrophic muscle.

Abstract Previous studies showed that a myopathy which can be produced in vertebrate muscle by inactivating esterases is mimicked by exposure to carbamylcholine and requires agonist-receptor interaction and extracellular calcium. A most consistent aspect of the myopathy is dissolution of Z-disks in myofibrils near the postsynaptic membrane. Using mouse extensor digitorum longus (EDL) muscles in vitro , we found that leupeptin partially protects the Z-disks from dissolution. Chloroquine had much less, if any, effect. These data are compatible with the suggestion that prolonged agonist action at the neuromuscular junction results in the activation of the calcium-activated protease known to destroy the Z-disk protein. Because dystrophic mouse muscles reportedly have increased activities of calcium-activated proteases, we compared the response of normal and dystrophic EDL muscle. These muscles showed no significant difference after 3 h in Krebs baths, but when carbachol was added, there was a significantly greater amount of Z-disk damage in dystrophic muscles than in muscles from wild types (129 ReJ) or from albino mice. As in normal muscle, the agonist-induced myopathy in dystrophic muscle is both calcium- and protease dependent.

SENSITIVITY IN ELECTRON MICROSCOPE AUTORADIOGRAPHY I. THE EFFECT OF RADIATION DOSE

Sensitivity in electron microscope autoradiography using Ilford L4 emulsion was shown to be affected by radiation dose (i.e., number of decays in test specimen per unit surface area). The sensitivity tended to be higher with lower doses. This dose dependence was most marked with Microdol X and least with gold latensification-Elon ascorbic acid development. Possible consequences for quantitation in electron microscope autoradiography are discussed.

Struktur und Funktion des Drüsenepithels der postabdominalen Tergite von Blatta orientalis

SummaryA two-layered glandular tissue occurs on tergites V to X and on the cerci of juvenile specimens of both sexes and of adult females of Blatta orientalis, in place of the usual monolayer of epidermal cells. This gland tissue contains two cell types and secretes a viscous product of water, free amino acids (+ glutamine), oligo- and several polypeptides onto the tergal surface. The structural differentiation of the gland is correlated with secretory activity, both in different molting stages and in different tergites of an individual; maximal values are found in tergites VI und VII on last instar females. Applying quantitative radioautography on the electron microscope level, we found, that although the most common gland cell type contained an abundantly developed rough endoplasmic reticulum and Golgi-apparatus, characteristic of protein secreting cells, not all of them incorporated equally the injected amino acids. This is consistent with an asynchronous secretory cycle, also suggested by biochemical studies. Of great significance is the demonstration that the fine structural elaboration of the cellular organelles involved in protein synthesis cannot be used as a criterion for their ongoing activity. The secretion is discharged into an end-apparatus consisting of a tortuous canal with a brushborder that penetrates the whole gland cell. One unbranched chitinous duct, formed by a “duct carrying cell”, is inserted into the end-apparatus of each gland cell. Occasionally, cell processes exhibiting the typical morphological characteristics of neurosecretory cells are seen in direct contact with gland cells. A defensive function of the secretion which acts by mechanically impairing smaller predatory arthropods was ascertained. To achieve this effect and to allow the preyanimal to escape, the secretion has to be adjusted to a proper viscosity by an adequate dilution. This might be achieved by the second gland cell type, which was not selectively labelled by injected amino acids; this cell type contains an endapparatus, abundant mitochondria, Golgi-apparatuses and small vesicles, but only few profiles of rough endoplasmic reticulum.ZusammenfassungIm Bereich der abdominalen Tergite V-X und oberseits and den Zerzi liegt bei Nymphen beider Geschlechter und bei adulten Weibchen von Blatta orientalis statt des einschichtigen Epithels ein zweischichtiges Drüsengewebe vor, welches ein visköses Sekret aus Wasser, freien Aminosäuren (+ Glutamin), Oligo- und zahlreichen Polypeptiden auf die Tergitenoberfläche sezerniert. Die strukturelle Differenzierung des Drüsengewebes ist mit der Sekretionsaktivität korreliert, sowohl während der Ontogenese als auch im Bereich verschiedener Tergite (Maxima: weibliche Subimagines, Tergite VI und VII). Untersuchungen mittels hochauflösender quantitativer Autoradiographie ergaben, daß injizierte Aminosäuren im größten Teil der Drüsenzellen angereichert werden: Markierte Zellen zeigen Radioaktivität im reichlich ausgebildeten rauhen endoplasmatischen Retikulum, Golgi-Apparat, in Sekretgranula und in ihrem Endapparat. Dieser durchsetzt die Drüsenzelle als langer gewundener Kanal mit Bürstensaum, in welchen je eine darüberliegende Gangzelle einen chitinösen Ausführgang inseriert. Die gleichzeitige Anwesenheit von markierten und nicht markierten Zellen mit praktisch gleich stark entwickeltem endoplasmatischem Retikulum und Golgi-Apparat zeigt, daß die morphologische Ausbildung dieser mit der Proteinsekretion befaßten Organellen nicht unbedingt deren Aktivität reflektiert. Vereinzelt stehen Zellfortsätze mit den morphologischen Charakteristika neurosekretorischer Tätigkeit in direktem Kontakt mit Drüsenzellen. Eine Abwehrfunktion des viskösen Sekrets durch bloße mechanische Behinderung kleiner räuberischer Arthropoden wurde sichergestellt, wobei es dem Beutetier gelingt, zu flüchten. Weiters wurde ein zweiter Drüsenzelltyp beobachtet, der mit injizierten Aminosäuren nur schwach markierbar ist, ebenfalls einen Endapparat besitzt, jedoch arm an rauhem endoplasmatischem Retikulum und gleichzeitig reich an Mitochondrien, Golgi-Apparaten und kleinen Vesikeln ist. Die Funktion dieses zweiten Zelltyps ist zwar nicht sichergestellt, möglicherweise reguliert er jedoch die funktionell wichtige Viskosität des Sekretes.

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors). In neurons, the t‐SNARE SNAP‐25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP‐25 temperature‐sensitive paralytic mutant in Drosophila, SNAP‐25ts. The mutation causes a Gly50 to Glu change in SNAP‐25s first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro‐generated 70 kDa SNARE complexes containing SNAP‐25ts are thermally stable but the mutant SNARE multimers (of ∼120 kDa) rapidly dissociate at 37°C. The SNAP‐25ts mutant has two effects on neurotransmitter release depending upon temperature. At 22°C, evoked release of neurotransmitter in SNAP‐25ts larvae is greatly increased, and at 37°C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22°C the mutation causes the SNARE complex to be more fusion competent but, at 37°C the same mutation leads to SNARE multimer instability and fusion incompetence.

Acetylcholine receptors in innervated muscles of dystrophic mdx mice degrade as after denervation.

Acetylcholine receptors (AChRs) are present at the top of the postsynaptic membrane of the neuromuscular junction (NMJ) at very high density, possibly anchored to cytoskeletal elements. The present study investigated whether AChR degradation is affected in animals lacking dystrophin, a protein that is an integral part of the cytoskeletal complex and is missing in Duchenne muscular dystrophy. The animal model for Duchenne muscular dystrophy, the mutant mdx mouse, was used to determine whether disruption of the cytoskeleton, caused by the absence of dystrophin, affects AChR degradation. Of the two populations of junctional AChRs, Rs (expressed in innervated adult muscles) and Rr (expressed in embryonic or denervated muscles), only Rs are affected in mdx animals. In innervatedmdx soleus, diaphragm, and sternomastoid muscles, the AChRs have an accelerated degradation rate (t1/2 of ∼3–5 d), similar to that acquired by Rs in control muscles after denervation. The Rs inmdx NMJs do not accelerate further when the muscles are denervated. The absence of dystrophin does not affect the degradation rate of the Rr AChRs (t1/2 of 1 d), which are expressed after denervation in mdx as in control muscles. These results suggest that dystrophin or an intact cytoskeletal complex may be required for neuronal stabilization of Rs receptors at the adult neuromuscular junctions.

SIZES OF END PLATE COMPARTMENTS, DENSITIES OF ACETYLCHOLINE RECEPTOR AND OTHER QUANTITATIVE ASPECTS OF NEUROMUSCULAR TRANSMISSION

The area of the postsynaptic membrane and the volume of the synaptic cleft were calculated for the end plates of the diaphragm and sternomastoid of mouse and rat. From these dimensions we were able to extrapolate, from data given by others on acetylcholine (ACh) released during neuromuscular transmission and on ACh receptor per whole end plate, to densities in the postneural compartments. The concentration of ACh in the cleft per nerve impulse was found to be ~ 10–5 M and the density of ACh receptor was between 5 and 10 x l03/µ2 of postsynaptic membrane. (This is approximately a factor of 2 to 3 higher than that for acetylcholinesterase at this site.) From these values we conclude that the concentration of ACh receptor in the plane of the postsynaptic membrane is considerably higher than that of ACh in the cleft during neuromuscular transmission. In addition the ACh itself is present at considerably higher concentrations than necessary to give optimal response. We calculated that the acytelcholinesterase plus ACh receptor together would occupy about 25% of the surface area of the postsynaptic membrane.