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— The concentrations of ACh and ATP of Torpedo electric organ synaptosomes were directly measured and found to be respectively 20.0 ± 6.4 mM and 3.1 ± 0.6 mM. The synaptosomal volume was estimated by a classical space marker technique using 14C inulin and tritiated water. After counting the synaptosomes in an haemocytometer (and knowing their volume), a mean diameter of 3.5 p was calculated. The use of these classical techniques was rendered possible because of the homogeneity of the fraction and the large size of Torpedo synaptosomes.

The effects of cetiedil, a vasodilatator substance with reported anticholinergic properties, were examined on cholinergic presynaptic functions at the nerve electroplaque junction of Torpedo marmorata using either synaptosomes or slices of intact tissue. Cetiedil abolished the calcium-dependent release of acetylcholine (ACh) triggered by depolarization or by addition of A23187 ionophore, a finding localizing the site of action downstream from the calcium entry step. In addition, a direct effect on the release process itself was indicated by the observation that cetiedil blocks the release of ACh mediated by a recently isolated presynaptic membrane protein, the mediatophore, reconstituted into ACh-containing proteoliposomes. In all three preparations, ACh release was inhibited by cetiedil with a Ki of 5-8 microM. Under the conditions used in these release experiments, the synthesis of ACh and its compartmentation within the nerve terminals were not modified. However, the drug was able to reduce high-affinity choline uptake and vesicular ACh incorporation when it was given together with the radioactive precursor, a result showing that cetiedil has a broad inhibitory action on cholinergic uptake processes.

Publisher Summary Torpedo electric organs have an abundant and purely cholinergic innervations and are therefore very convenient for biochemical studies of neurotransmission at peripheral synapses. As is the case for brain, nerve terminals pinched off during tissue subfractionation can be isolated as sealed particles = synaptosomes. The main advantage of Torpedo synaptosomal fractions is that all the synaptosomes are cholinergic. This permits the study of their physiological properties without interference from noncholinergic materials. The large field electron micrograph illustrates the homogeneity and purity of the synaptosomal fraction. Freeze-fracture methods have been used to follow morphological modifications accompanying neurotransmitter release, because a large surface of the presynaptic membrane can be analyzed. Ultrarapid freezing procedures permit the avoidance of any chemical fixation and are, therefore, very suitable to catch transient membrane modifications. The study of acetylcholine (Ach) release suffers from the impossibility of controlling the intracellular composition of synaptosomes.

The mediatophore, a protein which translocates acetylcholine, has been recently purified from the plasma membrane of the Torpedo electric organ nerve terminals. The functional integrity of the mediatophore requires the presence of lipids. Removal of associated lipids led to an irreversible loss of activity when obtained by ether precipitation of the protein. A two-phase extraction procedure was developed to gently remove the lipids. Conditions were found to reactivate the protein by emulsifying it with electric organ lipids. A large-scale preparation procedure of the delipidified mediatophore in a reactive form is described.

Pure cholinergic nerve endings (synaptosomes) were isolated from the electric organ of Torpedo by a rapid procedure. These synaptosomes are approximately 3 micron in diameter. They contain an occasional mitochondrion, numerous synaptic vesicles, and sometimes an active zone is observed. No postynaptic membrane attachment is found. This nerve ending fraction is extremely pure as shown by morphological controls and biochemical data. It is rich in choline acetyltransferase (450 nmol/h per mg protein) and acetylcholine (ACh) (130 nmol/mg protein). The isolated endings retain their cytoplasmic components and they synthesize ACh and are stable in vitro for several hours, as shown by biochemical measurements and morphological analysis.

Publisher Summary In this chapter the isolation and characterization of pure cholinergic synaptosomes from the electric organ of Torpedo is described. A technique has been developed to study the release of ACh from these synaptosomes. K + depolarization triggers a Ca 2+ dependent release. The good time resolution (4-5 sec) permits further investigations on the mechanisms of ACh release. The two main characteristics of the isolation procedure are the gradual comminution of the tissue by successive filtrations, and the use of centrifugation media having an ionic composition and osmolarity close to plasma. Torpedo synaptosomes are able to synthesize ATP from extracellular adenosine. A facilitated diffusion mechanism for adenosine uptake has been described. Most of the adenosine taken up is converted to ATP. A high affinity uptake of choline in Torpedo synaptosomes is also characterized. Twenty percent of the incorporated choline is converted to ACh. Acetate that is a good precursor of ACh in Torpedo electric organ and in neuromuscular junctions is efficiently incorporated into the ACh of synaptosomes. For extracellular acetate concentrations lower than 50 μM, as much as 95% of incorporated radioactivity was found as ACh. Since all the synaptosomal radioactivity is found as ACh, it should be possible to follow the release of ACh by measuring the efflux of radioactivity.

Mediatophore is a protein purified from the nerve terminal membranes ofTorpedo electric organ. It confers to artificial membranes a calcium-dependent mechanism that translocates acetylcholine. When similar reconstitution experiments are applied to rat brain synaptosomal membranes they reveal the presence of mediatophore activity with properties close to those described for theTorpedo protein (extractability, sensitivity to calcium, and effect of the drug cetiedil). The acitivity was more abundant in synaptosomal membranes than in mitochondrial or myelinic membranes and in cholinergic areas as compared to cerebellum.

— Slices of electric organ of Torpedo marmorata were chopped and incubated in a saline-urea-sucrose medium. This preparation of minced tissue exhibited a relative enrichment in ACh and nerve endings, which was attributed to a loss of electroplaque cytoplasm. Electron microscopic controls showed nerve endings of normal morphology, some of them forming ‘chaplets’ separated from electro-plaques. Miniature endplate potentials were recorded on sealed fragments also present in this preparation. ACh levels remained unchanged during incubation periods as long as 19 h. The time course of the incorporation of [1-14C]acetate of [2-14C]pyruvate into ACh pools was studied. These incorporations were similarly affected by the choline added to the medium. In the presence of increasing choline concentrations (up to 10-4m), the incorporation of [14C]acetate or [14C]pyruvate into ACh increased. They both diminished when choline was added above 10-4M. The ACh content of the tissue was not affected by added choline. From the constancy of ACh levels in the presence of various choline concentrations and from the steady state of our preparation, we can conclude that the release of transmitter varied in parallel to the incorporation rate of the precursor of the acetyl moiety of ACh. This fact was also found using the efflux of [14C]acetate as an evaluation of ACh release. The values of release calculated by this method were in good agreement with those determined from the incorporations of acetate and pyruvate into ACh. It is suggested that the primary action of choline is on its high affinity carrier system. This triggers a secondary action on the ACh release mechanisms.

Acetylcholine release was measured on suspensions of pure cholinergic synaptosomes, isolated from torpedo electric organ. Transmitter release was triggered by two different methods: KCl depolarization, or action of a venom extracted from a polychaete annelid Glycera convoluta . This venom was known to increase considerably the miniature endplate potential frequency at neuromuscular junctions. Ultrarapid freezing of synaptosomes in suspension in the absence of fixation, followed by freeze fracture, permitted us to show: (1) That the venom does not trigger the appearance of endo-exocytotic pits in the presynaptic membrane, in contrast to KCl depolarization. (2) That both KCl depolarization and venom action lead to a decrease in the number of small P-face intramembrane particles and to an increase in the number of medium-sized E-face particles. In addition, the venom increased the number of medium-sized P-face particles. The redistribution of the intramembrane particles is discussed in relation to the release of transmitter which has been measured in parallel.

A rabbit antiserum to mediatophore, a nerve terminal membrane protein involved in calcium dependent ACh release, was raised after immunization with the purified protein. An immunological assay for mediatophore was then developed and the subcellular distribution of this protein in Torpedo electric organ fractions was studied. A good agreement was obtained between the distribution in the different fractions of the antigen and of mediatophore related acetylcholine releasing activity as determined by reconstitution in proteoliposomes. Mediatophore was highly concentrated in presynaptic plasma membranes of electric organ, while very low contents were observed in electric nerves and electric lobes. Although some mediatophore was found in synaptic vesicle fractions, this most probably resulted from presynaptic membrane contamination as evaluated with other presynaptic membrane markers. Nerve terminals of motor end-plates were strongly stained with anti-mediatophore antibodies.

In literature many different blind controller have been proposed and it has been shown in most cases, that they can provide energy savings and better comfort. In the early approaches they where based only on one state variable, but over time more variables have been taken into account and the structure got more sophisticated. With an increasing number of state variables the performance may rise but also the cost and complexity. In this paper we present a systematic way to identify the important state variables for a blind controller by comparing the performance of the same controller with different sets of state variables. Our work is based on an adaptable hierarchical fuzzy controller which gets optimized by a multi-objective genetic algorithm in terms of energy consumption and thermal comfort, and is then tested in a dynamic simulation environment. For making this more realistic, we include stochastic processes which try to mimic the state of occupancy and the behavior of occupants.

This paper describes an algorithm for the simulation of occupant presence, to be later used as an input for future occupant behaviour models within building simulation tools. By considering occupant presence as an inhomogeneous Markov chain interrupted by occasional periods of long absence, the model generates a time series of the state of presence (absent or present) of each occupant of a zone, for each zone of any number of buildings. Tested on occupancy data from private offices, the model has proven its capacity to realistically reproduce key properties of occupant presence such as times of arrival and departure, periods of intermediate absence and presence as well as periods of long absence from the zone. This model (due to related metabolic heat gains), and associated behavioural models which use occupants' presence as an input, have direct consequences for building energy consumption. (c) 2007 Elsevier B.V. All rights reserved.

The paper describes a predictive and adaptive heating controller, using artificial neural networks to allow the adaptation of the control model to the real conditions (climate, building characteristics, user's behaviour). The controller algorithm has been developed and tested as a collaborative project between the CSEM (Centre Suisse d'Electronique et de Microtechnique, Neuchâtel, Switzerland, project leader), and the LESO-PB (Solar Energy and Building Physics Laboratory, EPFL, Lausanne, Switzerland). A significant support has been provided by leading Swiss industries in control systems. The project itself has been funded by the Swiss Federal Office of Energy (SFOE). The project has allowed the development of an original algorithm, especially suited for water heating systems, and its testing both by simulation and by experimentation on an inhabited building. The experimentation has been done using a PC software implementation. A second phase of the project, currently going on, aims at building a commercial system based on the NEUROBAT algorithm.

In this work, we investigated the interplay and the influence of lighting and blind control models on the heating, cooling, and lighting energy loads of an office room. By including different stochastic models for occupancy and appliances, we built a complete simulation environment based on the building simulation program IDA ICE. For control models, we implemented different strategies, including a simple on/off scheme, a realistic model of occupants, and an optimized control. In literature, the results are often compared with simple on/off schemes, which are not hard to beat in terms of performance. With an optimal control, the real saving potential is assessed, which can be used as benchmark case for comparison with other control models. Results based on annual simulations show that active occupants can reduce energy consumption by up to 50% from a worst- case scenario, whereas advanced controllers can further reduce the consumption by another 60%.

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