The problem brought to the 2010 MPI Workshop by TIAX asked for the formulation of a mathematical model for the effect of interaction of light with metallic nanostructures, specifically the effect of surface features on the intensity of the electromagnetic field, when a metallic surface is illuminated by laser light of a given wavelength and the scale of the metallic structures is commensurate with the wavelength of the light. Devices based on this type of configuration are used in photovoltaics, catalysis, nonlinear optics, and sensors, just to name a few applications. The operation of many of these devices depends on effects which are proportional to the amplitude of the electromagnetic field in the vicinity of the metallic surface. With a solution to this problem in hand, it then becomes possible to consider optimization and design. Two examples: If a given structure is imposed, what wavelength leads to the largest field in the vicinity of the

A monoclonal antibody, mAb E40, that specifically recognizes hair cells and photoreceptors was derived from a mouse immunized with a membrane fraction prepared from the sensory maculae of the chick inner ear. In the mature chick inner ear, punctate labeling is observed along each stereocilium, but staining is mostly concentrated around the basal end of the sensory hair bundles, where it is closely associated with surface specializations known as ankle links. The epitope recognized by mAb E40 is therefore referred to as the ankle-link antigen (ALA). During early embryogenesis, the ALA is initially distributed evenly over the surface of the hair bundle. As development proceeds, it becomes more restricted to the base of the hair bundle, although a spot of the ALA remains associated with the bundle tip until just before hatching. In the eye, mAb E40 stains the calycal processes of photoreceptors. When maculae and retinae are treated with the calcium chelator BAPTA at room temperature, the ALA disappears. BAPTA-induced loss of the ALA from the hair-bundle surface is substantially reduced by lowering the temperature to 2 degrees C. The ALA and ankle links reappear on the hair-bundle surface when cells are cultured for 20 hr after BAPTA treatment. BAPTA sensitivity and recovery after BAPTA-induced loss are properties similar to those described for the tip link, a surface structure thought to gate the mechanotransducer channel. However, unlike the tip link, the ALA and ankle links are sensitive to subtilisin treatment. The results define a new component of the hair-bundle surface, with properties both common to and distinct from those of the tip link.

Naturally occurring neuronal cell death has been investigated in the electromotor system of and compared with neuronal death seen in expiant cultures of tissues from electric lobe. One objective of the study was to determine whether cell death in vitro was morphologically the same as cell death in vivo and to substantiate the validity of using in vitro models for studying naturally occurring cell death. Sequences of degeneration in vitro and in vivo have been established and compared: a single morphological sequence best represents this form of cell death in both conditions. In vivo, dying neurons are seen at all depths of the electric lobe indicating the involvement of different generations. Retrograde degeneration appears to be the first sign of cell death.

A combination of direct fluorescence and indirect immunofluorescence microscopy has been used to compare the distribution of the acetylcholine receptor with the distribution of major cytoskeletal and extracellular matrix components during electrocyte differentiation in the electric organs of Torpedo marmorata. Laminin, fibronectin and extracellular matrix proteoglycan are always more extensively distributed around the differentiating cell than the acetylcholine receptor-rich patch that forms on the ventral surface of the cell. The distribution of acetylcholinesterase within the ventral surface of the differentiating electrocyte closely resembles the distribution of the acetylcholine receptor. Areas of apparently high acetylcholine receptor density within the ventrally forming acetylcholine receptor-rich patch are always areas of apparently high extracellular matrix proteoglycan density but are not always areas of high laminin or fibronectin density. Desmin levels appear to increase at the onset of differentiation and desmin initially accumulates in the ventral pole of each myotube as it begins to form an electrocyte. During differentiation F-actin-positive filament bundles are observed that extend from the nuclei down to the ventrally forming acetylcholine receptor-rich patch. Most filament bundles terminate in the acetylcholine receptor-rich region of the cell membrane. Electron-microscopic autoradiography suggests that the filament bundles attach to the membrane at sites where small acetylcholine receptor clusters are found. The results of this study suggest that, out of the four extracellular matrix components studied, only the distribution of acetylcholinesterase (which may be both matrix- and membrane-bound at this stage) closely parallels that of the acetylcholine receptor, and that F-actin filament bundles terminate in a region of the cell that is becoming an area of high acetylcholine receptor density.

Explant cultures of electric lobe from 45-60 mm stage Torpedo embryos and both ganglionic and dissociated cell cultures prepared from 8-day chick ciliary ganglia have been used to determine whether the electric organs of Torpedo marmorata contain developmentally regulated neuronotrophic activity. Electric lobe explants were evaluated by measuring their neurone density, choline acetyltransferase (CAT0, and low salt, Triton X-100-soluble protein contents. Addition of soluble extracts prepared from the electric organs of late stage embryos (85-105 mm) to standard medium results in the maintenance of nearly theoretical neurone densities in electric lobe explants during a 7-day culture period. Soluble electric organ extracts from early embryonic stages (42-59 mm) do not increase neurone density relative to control cultures but cause an elevation in the CAT content of the explants over control values. On the basis of this analysis it is concluded (1) that late embryonic stage and adult electric organs contain neuronotrophic activity that allows electromotor neurones to survive in vitro and (2) that activity increases rapidly in the electric organs between the 59 nd 72 mm stages of development at a time when rapid increases in postsynaptic membrane markers in the electric organs occur and when peripheral synaptogenesis begins. The activity of late stage embryonic electric organs is heat stable and lost on dialysis. Using ciliary ganglion explants and evaluating both the initial fibre outgrowth and the CAT content after 4 days in vitro, trophic activity is found to be maximal at early embryonic stages (45-55 mm) and to decline thereafter. It is shown that the decline in activity is not due to an increase in toxicity. Using established dissociated ganglionic cell survival assays the specific activity of neuronotrophic factors allowing survival is constant between the 45 and 73 mm stages in the electric organs and then rapidly declines, but activity per electric organ increases rapidly between the 45 and 73 mm stages and then remains at a constant level. The use of poly-dl-ornithine substrates coated with heart-conditioned medium for the cell survival assay results in up to tenfold increase in the trophic titre of the electric organ extracts. The neuronotrophic activity supporting survival of ciliary motorneurones present in embryonic electric organs is heat labile and retained on dialysis. It is concluded that developing electric organs contain at least two neuronotrophic factors that have different properties and are differently regulated. Both factors may contribute during development to bringing naturally occurring electromotor neurone cell death to an end.

The fourth branchial arch of Torpedo marmorata has been examined at the light and electron microscopic level during development. Of interest was the determination of the extent of electric organ tissue reported to be present in this arch and its possible relationship to electromotoneuron cell death in the electric lobes. The main electric organ of the torpedo is derived from the hyoid and first three branchial arches and is innervated by four major electromotor nerves. Extensive electromotoneuron cell death occurs in the electric lobes and most notably in the posterior poles. This feature could be due to a tendency for these neurons to innervate the fourth branchial arch where little or no electric tissue is formed. Our findings support this conclusion but are not entirely consistent with the idea that a population mismatch has occurred. This is because cell death precedes the genesis of the target cells. The presence of innervated differentiated electric tissue in this arch is also reported, leading to the conclusion that Torpedo marmorata possesses an accessory electric organ.

Synaptogenesis has been investigated in the electric organ of Torpedo marmorata with the objective of determining whether a bioelectric effect could be demonstrated. Answers to 3 questions were sought. (1) Are currents and/or fields present within the organ? (2) Can they be localized? (3) Are they involved with the synaptogenic process? Voltage measurements across pieces of electric organ revealed the presence of a dorsal positive potential in the low millivolt range. Injection of DC current against this dorsal positive dipole had the effect of reducing the percent of neuritic coverage on the ventral surface as measured by quantitative electron microscopy. These results indicate the presence of a field potential, dorsal positive which, when reversed, causes a retardation in the synaptogenic rate. They are consistent with published reports of neurites growing preferentially towards cathodal sources and implicate that bioelectric forces may be one component of the synaptogenesis process.

The changes that occur in the distribution and properties of the nicotinic acetylcholine receptor and acetylcholinesterase during the development of the electric organ of Torpedo marmorata have been investigated. At early stages of development, both proteins are distributed diffusely over the myotube surface and with differentiation of the myotubes into electrocytes, they become increasingly restricted to the ventral cell surface. This process occurs before axons contact the electrocytes. The concentrations of the acetylcholine receptor and acetylcholinesterase remain at rather low and stable levels during these developmental changes. The acetylcholine receptor concentration begins to increase rapidly as soon as electromotor axons begin to contact the electrocytes. No significant differences in the subunit composition, affinity for d-tubocurarine, isoelectric point or immunochemical properties of the embryonic acetylcholine receptors were detected when they were compared to the receptors of the adult electric organ. The onset of receptor accumulation occurs before the increase in the amount of 17 S acetylcholinesterase, suggesting that increases in acetylcholine receptor and in acetylcholinesterase are not regulated by the same mechanisms. The various molecular forms of acetylcholinesterase undergo characteristic changes during development. Sequential extraction of the esterase forms indicates that their interaction with cellular compartments changes during development. The solubility properties of the esterase forms suggest that most of the 17 S and 13 S acetylcholinesterase become strongly associated with cellular components via ionic interactions and that a hydrophobic 6 S form begins to accumulate at later embryonic stages when the number of mature presynaptic terminals is beginning to show its rapid phase of increase.

Electromotoneuron proliferation and cell death have been quantitatively studied in the electric lobe of Torpedo marmorata from an embryonic body-length stage of 26-mm to adult animals. These neurons project to the electric organ and form synapses with electrocytes which possess a remarkably large postsynaptic target surface. For this reason cell death would not be predicted to occur if synaptic competition were to be hypothesized as the cause. Isolated observations at the ultrastructural level suggested, however, that cell death was indeed taking place and therefore it seemed appropriate to examine this question in detail. Our findings show first that neuron production appears to be a continuous process throughout the period studied, generating totals of over 70,000 electromotoneurons per lobe by adulthood. Second, two waves of cell death were identified, one occurring early in embryogenesis (stage 30 mm), well before the onset of synaptogenesis, and a second coincident with the onset of synaptogenesis (stages 55--74 mm). It is difficult to reconcile this latter wave with the hypothesis of synaptic competition as the postsynaptic surface at this time of development is largely devoid of synaptic contacts. We conclude that in the electromotor system of Torpedo, synaptic competition is probably not the mechanism of cell death.

An explant culture system has been used to study the electric organ and electric lobe tissues of Torpedo marmorata at different stages during the development of the electromotor system.