Development of the electric organ in embryos and larvae of the knifefish, Brachyhypopomus gauderio.

South American Gymnotiform knifefish possess electric organs that generate electric fields for electro-location and electro-communication. Electric organs in fish can be derived from either myogenic cells (myogenic electric organ/mEO) or neurogenic cells (neurogenic electric organ/nEO). To date, the embryonic development of EOs has remained obscure. Here we characterize the development of the mEO in the Gymnotiform bluntnose knifefish, Brachyhypopomus gauderio. We find that EO primordial cells arise during embryonic stages in the ventral edge of the tail myotome, translocate into the ventral fin and develop into syncytial electrocytes at early larval stages. We also describe a pair of thick nerve cords that flank the dorsal aorta, the location and characteristic morphology of which are reminiscent of the nEO in Apteronotid species, suggesting a common evolutionary origin of these tissues. Taken together, our findings reveal the embryonic origins of the mEO and provide a basis for elucidating the mechanisms of evolutionary diversification of electric charge generation by myogenic and neurogenic EOs.

Ontogeny of the electric organs in the electric eel, Electrophorus electricus: physiological, histological, and fine structural investigations.

This study attempts to clarify the controversy regarding the ontogenetic origin of the main organ electrocytes in the electric eel, Electrophorus electricus. The dispute was between an earlier claimed origin from a skeletal muscle precursor [Fritsch, 1881], or from a distinct electrocyte-generating matrix, or germinative zone [Keynes, 1961]. We demonstrate electrocyte formation from a metamerically organized group of pre-electroblasts, splitting off the ventralmost tip of the embryonic trunk mesoderm at the moment of hatching from the egg. We show details of successive stages in the development of rows of electric plates, the electrocytes, by means of conventional histology and electron microscopy. The membrane-bound pre-electroblasts multiply rapidly and then undergo a specific mitosis where they lose their membranes and begin extensive cytoplasm production as electroblasts. Electrical activity, consisting of single and multiple pulses, was noticed in seven-day-old larvae that began to exhibit swimming movements. A separation of discharges into single pulses and trains of higher voltage pulses was seen first in 45-mm-long larvae. A lateralis imus muscle and anal fin ray muscles, implicated by earlier investigators in the formation of electrocytes, begin developing at a time in larval life when eight columns of electrocytes are already present. Axonal innervation is seen very early during electrocyte formation.

Electrosensory ampullary organs are derived from lateral line placodes in cartilaginous fishes.

Ampullary organ electroreceptors excited by weak cathodal electric fields are used for hunting by both cartilaginous and non-teleost bony fishes. Despite similarities of neurophysiology and innervation, their embryonic origins remain controversial: bony fish ampullary organs are derived from lateral line placodes, whereas a neural crest origin has been proposed for cartilaginous fish electroreceptors. This calls into question the homology of electroreceptors and ampullary organs in the two lineages of jawed vertebrates. Here, we test the hypothesis that lateral line placodes form electroreceptors in cartilaginous fishes by undertaking the first long-term in vivo fate-mapping study in any cartilaginous fish. Using DiI tracing for up to 70 days in the little skate, Leucoraja erinacea, we show that lateral line placodes form both ampullary electroreceptors and mechanosensory neuromasts. These data confirm the homology of electroreceptors and ampullary organs in cartilaginous and non-teleost bony fishes, and indicate that jawed vertebrates primitively possessed a lateral line placode-derived system of electrosensory ampullary organs and mechanosensory neuromasts.

Lateral line receptors: where do they come from developmentally and where is our research going?

The lateral line system is composed of both mechanoreceptors, which exhibit little variation in structure between taxonomic groups, and electroreceptors, which exhibit considerably more variation. Cathodally sensitive ampullary electroreceptors are the primitive condition and are found in agnathans, chondrichthyans, and most osteichthyans. Aquatic amphibians also have ampullary electroreceptors for at least part of their life cycle. The more recently evolved anodally sensitive ampullary electroreceptors and tuberous electroreceptors are only found in four groups of teleost fishes. The basic ontogenetic unit of lateral line development is the dorsolateral placode. Primitively, there are six pairs of placodes, which pass through sequential stages of development into lateral line receptors. There is no question about the origin of primitive mechanoreceptors or electroreceptors, however, we do not have a good understanding of the origin of teleost mechanoreceptors and their ampullary or tuberous electroreceptors; do they come exclusively from dorsolateral placodes or from neural crest or even general ectoderm? A second intriguing lateral line question is how certain teleost fish groups evolved tuberous electroreceptors. Electroreception appears to have re-evolved at least twice in teleosts after being lost during the neopterygian radiation. It has been suggested that the development of tuberous electroreceptors might be due to changes in placodal patterning or a change in the general ectoderm that placodes arise from. Unfortunately, our understanding of lateral line origins in fishes is very sketchy, and, if we are to answer such an evolutionary question, we first need more complete information about lateral line development in a variety of fishes, which can then be combined with gene expression data to better interpret lateral line receptor development.

[Cytoembryologic aspects of evolution of specialized electric organs of fishes]. / Tsitoémbriologicheskie aspekty évoliutsii spetsializirovannykh élektricheskikh organov ryb.

Almost all fish electric organs (EO) developed from the skeletal muscles or from its embryonic rudiments. The only exception is the definite (in contrast to larval) EO of Apteronotidae, formed by motoneurons, whose loss of relation with muscles is secondary. The main feature of all EO of the muscle genesis is cooperative morphological and electrophysiological polarity of their electrocyte cells anterioposteriorly or (in Torpedo, Uranoscopus) of the dorso-ventral axes of the body. In particular, for the EO of muscular origin, unilateral asymmetric innervation of electrocytes by electromotoneurons is characteristic. Such innervation is a prerequisite condition for the summation of electric discharges. It is one of the main distinctions of EO from definitive skeletal muscles. However, in the emryogenesis of all vertebrates the initial innervation of muscle rudiments by the so-called pioneer motoneurons occurs. In teleosts (according to data on Brachidanio rerio available) extending to every myotome are outgrowths of three pioneer motoneurons referred to after their position in the nerotubule as "rostral", "medial" and "caudal". The former two innervate dorsally with the dorsal compartment of the myotome. The third approaches the ventral compartment of the same myotome caudally. In the gymnotic fish the innervation of EO formed from the axial skeletal muscles retains the same nature. The electrocytes of EO from the dorsal and ventral compartments of the myotome, are approached by electromotoneurons, respectively, rostrally and caudally. In compartments, the antipolarity of the innervation of the dorsal and ventral EO compartments leads to a paradoxical effect of generation of anti-polar pulses. The summation of these pulses creates a very short difference electric charge. In Mormyridae the antipolarity of the innervation of the rostral and ventral compartments of EO formed from the axial muscle is not pronounced. However, electroneurons resemble pioneer motoneurons by the following characters: the large size of the bodies and their localization near the central tube, absence of dendrities, electrosynaptic connection, polar (asymmetrical) pattern of electrocyte innervation. Outside EO, the cooperative polarity of the cells is only characteristic of epithelia, particularly, ciliated. At the same time, in some electric fish, the electrogeneratory tissue is similar to epithelium in a number of morphological characters, or the genes expressed in it show the gene of keratin AE-1, typical of epithelia. The above gives grounds to believe that EO of muscle origin are a product of fixation and aggravation by natural selection of hereditary anomalies, manifested in the recovery or in the retaining of the embryonic (i.e., polar nature) of the efferent innervation of some parts of skeletal muscles. Another distinction of EO from the muscles appears to lie in the expression of some individual components of the gene epithelial complex. A method is proposed for electromyographic recording of such anomalies and molecular-genetic approachers to analysis of their nature. The causes of the absence of EO epithelial genesis are discussed and also of the fact that these organs developed only in the evolution of fish.

Developmental regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor in Torpedo electrocyte.

Tyrosine phosphorylation is thought to play a critical role in the clustering of acetylcholine receptors (AChR) at the developing neuromuscular junction. Yet, in vitro approaches have led to conflicting conclusions regarding the function of tyrosine phosphorylation of AChR beta subunit in AChR clustering. In this work, we followed in situ the time course of tyrosine phosphorylation of AChR in developing Torpedo electrocyte. We observed that tyrosine phosphorylation of the AChR beta and delta subunits occurs at a late stage of embryonic development after the accumulation of AChRs and rapsyn in the membrane and the onset of innervation. Interestingly, in the mature postsynaptic membrane, we observed two populations of AChR differing both in their phosphotyrosine content and distribution. Our data are consistent with the notion that tyrosine phosphorylation of the AChR is related to downstream events in the pathway regulating AChR accumulation rather than to initial clustering events.

[Post-embryonic differentiation of a cutaneous electro-receptor]. / Sur la différenciation postembryonnaire d'un électrorécepteur cutané.

In order to decrease the rate of postembryonic development of electroreceptor organs, excisions of epidermis and deafferentations were carried out in the gymnotid fish Eigenmannia virescens. Twenty-five days later, the epidermis showed electroreceptor organs without innervation. Some of these at the beginning of their development consisted of masses of identical cells, whereas others showed presumed sensory cells whose cytoplasm contained rudimentary synaptic structures. The epidermis also showed differentiated tuberous organs with a low number of sensory cells. In all these organs, radioactive thymidine was fixed in the nuclei of the platform accessory cells. Thirty-five-40 days after surgery, tuberous organs were identical to the functional organs, and thymidine was detected in the nuclei of the cavity accessory cells. These results show that the gymnotid electroreceptor organs can develop before any nervous contact occurs, and suggest that they might originate from epidermal cells.

Electroreceptors and mechanosensory lateral line organs arise from single placodes in axolotls.

The lateral line system in salamanders consists of mechanoreceptive neuromasts and pit organs, distributed in lines on the head and trunk, and electroreceptive ampullary organs located adjacent to the cephalic lines of mechanoreceptors. Although numerous studies have documented that neuromast and pit organs and the cranial nerves that innervate these receptors arise from a dorsolateral series of placodes, there is no agreement concerning the number of these placodes, the specific groups of receptors that arise from them, or the embryonic origin of ampullary organs. A developmental model was recently proposed (Northcutt et al., 1994) in which all these placodes, except for the most posterior one, elongate to form sensory ridges whose central zones initially form neuromast and pit organ primordia and whose lateral zones subsequently form ampullary primordia. To test this model, individual placodes were unilaterally extirpated, or placodes from pigmented wild-type axolotl embryos were homotopically or heterotopically transplanted into albino hosts. Extirpation resulted in the loss of all three receptor classes, and both homotopic and heterotopic transplants produced pigmented receptors of all three classes in albino hosts. The receptors in the heterotopic transplants still formed lines which occasionally retained their normal orientation despite differentiating in an ectopic environment. These experiments demonstrated that, as previously postulated, specific lines of neuromasts and pit organs do arise from each placode, and ampullary organs also arise from many of the same placodes. The distribution of receptors that develop following incomplete extirpation or heterotopic transplantation also indicates that each placode is patterned regarding receptor classes and orientation prior to sensory ridge formation.

Acetylcholine receptor and myogenic factor gene expression in Torpedo embryonic development.

The mRNA levels of acetylcholine receptor (AChR) and myogenic factors were followed during embryonic development of Torpedo skeletal muscle and its homologue, the electric organ. A different developmental pattern of AChR gene expression was found in these two tissues: a slight decrease in the muscle, and a marked increase, concomitant with synapse formation, in the electric organ. However, the developmental pattern of MyoD and MRF4 mRNA levels was similar in both tissues, with no significant changes during development. This is in contrast with the sharp increase in the expression of AChR in the electric organ and may suggest that the burst in the expression of AChR during the differentiation of myotubes into electrocytes is not regulated by changes in the myogenic factor mRNA levels.

Developmental changes in the subcellular distribution of the 43K (v1) polypeptides in Torpedo marmorata electrocyte: support for a role in acetylcholine receptor stabilization.

Analysis of the relative amounts of the acetylcholine receptros (AChR) and of the 43K protein present in the membrane of developing electrocyte shows that massive accumulation of 43K protein is not required for induction of early AChR clustering. Furthermore, we demonstrate the existence o of cytosol- and membrane-associated 43K polypeptide pools in Torpedo electrocyte. Epitope analysis shows that both pools of 43K protein are related to the long mRNA transcript and share similar antigenic determinants distributed throughout the protein sequence. Their partition between the cytosol and membrane fractions abruptly increases in favor of the membrane during the postsynaptic maturation phase of development, supporting a role for 43K protein in the stabilization and maintenance of the postsynaptic domain.

Developmental expression of the 43K and 58K postsynaptic membrane proteins and nicotinic acetylcholine receptors in Torpedo electrocytes.

The expression of the postsynaptic 43-kDa and 58-kDa proteins and actin during development of the Torpedo marmorata electric organ was compared to that of nicotinic acetylcholine receptors (AChRs). Western blot analysis demonstrates that AChRs and proteins of 43 kDa (43K protein) and 58 kDa (58K protein) are all present prior to synaptogenesis. Subsequently, levels of all 3 synaptic proteins increase dramatically during differentiation and innervation of electrocytes. In contrast, actin is present in relatively high concentrations at early times and decreases thereafter. The equimolar ratio of AChRs and the 43K protein found in the adult electric organ is established early in development. Furthermore, the AChR and 43K protein share a common postsynaptic localization in electrocytes following synapse formation. Aggregates of the AChR that form at the ventral pole of the oval-shaped electrocytes prior to innervation, however, show no detectable immunofluorescence staining with anti-43K monoclonal antibodies. Therefore, in some cases, aggregation of AChRs occurs without the 43K protein.

Asynchronous assembly of the acetylcholine receptor and of the 43-kD nu1 protein in the postsynaptic membrane of developing Torpedo marmorata electrocyte.

The assembly of the nicotinic acetylcholine receptor (AchR) and the 43-kD protein (v1), the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR-rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell. Intracellular pools of AchR and 43-kD protein were followed at the EM level in 80-mm electrocytes. AchR immunoreactivity was detected within membrane compartments, which include the perinuclear cisternae of the endoplasmic reticulum and the plasma membrane. On the other hand, 43-kD immunoreactivity was not found associated with the AchR in the intracellular compartments of the cell, but codistributed with the AchR at the level of the plasma membrane. The data reported in this study suggest that AchR clustering in vivo is not initially determined by the association of the AchR with the 43-kD protein, but rather relies on AchR interaction with extracellular components, for instance from the basement membrane, laid down in the tissue before the entry of the electromotor nerve endings.

An immunoglobulin M monoclonal antibody, recognizing a subset of acetylcholinesterase molecules from electric organs of Electrophorus and Torpedo, belongs to the HNK-1 anti-carbohydrate family.

An immunoglobulin M (IgM) monoclonal antibody (mAb Elec-39), obtained against asymmetric acetylcholinesterase (AChE) from Electrophorus electric organs, also reacts with a fraction of globular AChE (amphiphilic G2 form) from Torpedo electric organs. This antibody does not react with asymmetric AChE from Torpedo electric organs or with the enzyme from other tissues of Electrophorus or Torpedo. The corresponding epitope is removed by endoglycosidase F, showing that it is a carbohydrate. The subsets of Torpedo G2 that react or do not react with Elec-39 (Elec-39+ and Elec-39-) differ in their electrophoretic mobility under nondenaturing conditions; the Elec-39+ component also binds the lectins from Pisum sativum and Lens culinaris. Whereas the Elec-39- component is present at the earliest developmental stages examined, an Elec-39+ component becomes distinguishable only around the 70-mm stage. Its proportion increases progressively, but later than the rapid accumulation of the total G2 form. In immunoblots, mAb Elec-39 recognizes a number of proteins other than AChE from various tissues of several species. The specificity of Elec-39 resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1, NSP-4, as well as IgMs that occur in human neuropathies. Although some human neuropathy IgMs that recognize the myelin-associated glycoprotein did not react with Elec-39+ AChE, mAbs HNK-1, NC-1, and NSP-4 showed the same selectivity as Elec-39 for Torpedo G2 AChE, but differed in the formation of immune complexes.

Development of the electromotor system in Torpedo marmorata: cationic staining of the electric organ.

The electric organs of embryonic Torpedo marmorata have been reacted with three cationic stains to evaluate the appearance and distribution of anionic sites. Ruthenium red, alcian blue and lysozyme were used at different pHs and found to react in a time-related manner to anionic components within the interelectrocyte space. The basal lamina covering the ventral electrocyte surface possesses the greatest number of anionic sites whereas growth cone, presynaptic terminal and glial membranes displayed almost no staining. Since this lamina serves as the exclusive substrate for ingrowing neurites during synaptogenesis, the results are consistent with the idea that charge distribution on the membrane surface may provide a necessary cue for neurite motility, extension and eventual synaptogenesis.

Development of the electromotor system of Torpedo marmorata: distribution of extracellular matrix and cytoskeletal components during acetylcholine receptor focalization.

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.

Torpedo electromotor system development: biochemical differentiation of Torpedo electrocytes in vitro.

The accumulation of 2 postsynaptic proteins--the acetylcholine receptor and acetylcholinesterase, total protein and lactate dehydrogenase levels, and the evolution of the multiple molecular forms of acetylcholinesterase (exhibiting apparent sedimentation coefficients of 17, 13, 11 and 6S) have been examined in aneural cultures of embryonic Torpedo electric organ explanted before, during or after electrocyte differentiation and the onset of synaptogenesis. During electrocyte differentiation in vitro, with explants taken before the 38 mm stage, the relative proportions of the 17, 13 and 11S forms change in vitro as in vivo but the 6S form remains abnormally dominant. In tissue explants taken from 38 to 47 mm stage embryos, the 4 major molecular forms of acetylcholinesterase differentiate in a manner identical to that observed in vivo. In explants taken after the onset of synaptogenesis (55-80 mm stages), the proportions of the acetylcholinesterase forms change as in vivo only during the first week in vitro whilst accumulation is occurring at the normal in vivo rate. The switch to the high acetylcholine receptor and acetylcholinesterase accumulation rate that occurs when synaptogenesis begins in vivo is not observed after any time lag in vitro with tissue explanted before the stage (55 mm) at which synaptogenesis begins. The effects on acetylcholinesterase and acetylcholine receptor accumulation of supplementing the medium with a neural tissue extract are described. The experiments were designed to elucidate the factors and mechanisms that regulate the differentiation and formation of chemical synapses using the electric organ of Torpedo marmorata as a model system. The results demonstrate that the complex changes occurring in the multiple molecular forms of acetylcholinesterase during electrocyte differentiation are not under direct neural control but that the switch to an increased acetylcholinesterase and acetylcholine receptor accumulation rate may be triggered by an external, possible neural factor.

Creatine phosphokinase: isoenzymes in Torpedo marmorata.

Creatine phosphokinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) is the major constituent of the "low-salt-soluble" proteins of the electric organ from Torpedo marmorata. The denatured subunits of the enzyme have an apparent Mr of 43 000 and isoelectric points ranging between pH 6.2 and pH 6.5. Identical properties are found for the creatine phosphokinase from Torpedo muscle tissue. Anti-(electric organ creatine phosphokinase) antibodies are specific for the muscle-type enzyme and do not cross-react with enzymes present in Torpedo brain and electric lobe tissue. Biochemical and immunochemical properties of the enzyme associated with acetylcholine-receptor-enriched membranes show that this enzyme is as the "low-salt-soluble" electric organ enzyme of the muscle-specific type. In vitro translation of electric organ poly(A)-rich mRNA in a reticulocyte lysate reveals the abundance of mRNA specific for muscle creatine phosphokinase. During embryonic development of the electrocyte a continuous increase of translatable amounts of this mRNA is observed. No brain-type polypeptides are synthesized. The subunits of the brain-specific enzyme differ in molecular mass (Mr approximately equal to 42000) and isoelectric properties (pI approximately equal to 7.0-7.2). The unexpected finding that the brain forms are more basic than the muscle-specific enzyme is supported by agarose and cellulose acetate electrophoresis and ion-exchange chromatography properties.

Torpedo electromotor system development: developmentally regulated neuronotrophic activities of electric organ tissue.

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.

Investigations into a bioelectric component of synaptogenesis.

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.

Production and characterization of a monoclonal antibody directed against the 43,000-dalton v1 polypeptide from Torpedo marmorata electric organ.

Subsynaptic membrane fragments prepared from Torpedo marmorata electric organ contain, in addition to the acetylcholine receptor polypeptides, a major protein band of apparent molecular mass 43,000 daltons. On two-dimensional gels, this band yields three spots referred to as v1, v2, and v3. Monoclonal antibodies against the 43,000-dalton proteins were developed in CBA mice. One of them reacted exclusively with the v1 polypeptide but not with v2 and v3. Staining by the "immunogold" reaction followed by observation by electron microscopy showed that this antibody exclusively labeled the innervated membrane of T. marmorata electroplaque on its cytoplasmic face. Electroblots of one-dimensional gels of membrane preparations from 80-mm embryo electric organ were prepared. After reaction with the anti-v1 monoclonal antibody, a strongly stained 43,000-dalton band was revealed.