Three experimental situations have been found in which cultured sensory neurons from embryonic chicken will form growth cones from positions along the length of the neurite. If the neurons are dissected with a remaining short axonal stump and plated into serum-free medium, they can form a morphologically normal growth cone from the stump within 15 min, even in the presence of cycloheximide or puromycin. When neurites growing in culture media with low levels of serum are cut at any point with microneedles, growth cones are produced quickly from the amputated stump, usually within 20 min. Treatment of growing neurons with low concentrations of colchicine, Colcemid, or podophyllotoxin results in the progressive appearance of lateral filopodia and regions of flattened cytoplasm that closely resemble growth cones except for their preterminal positions. These observations show that the potential to form growth cones is distributed throughout the neuron and suggest that this is normally repressed in some way by the neuronal microtubules. Wherever the axons or dendrites of vertebrate nerve cells have been observed to grow in culture, they have been found to possess a terminal appendage known as the growth cone. This is distinguishable from the rest of the neuron by its numerous filopodia and flattened veil-like regions and by its continual movement (1, 2). Its special functions may include the primary attachment of the cell to the substratum (3), an active part in micropinocytosis (4), guidance of the neurite (5), and the assembly of surface membrane during growth (6).In the most extensively studied kind of nerve culture-that of embryonic sensory or sympathetic neurons growing in the presence of nerve growth factor (7)-neurite extension does not occur immediately after explantation (8). Furthermore, once initiation has occurred, growth cones are formed from existing ones rather than from other parts of the cell (3) and under the usual conditions they do not regenerate quickly upon experimental amputation (9).These observations raise the possibility that the growth cone is a complex structure whose formation is the rate-limiting step in the initiation of neurite growth. We now describe experiments that bear upon this possibility and examine the formation of growth cones from cultured sensory neurons under a variety of conditions. MATERIALS AND METHODSCultures were prepared from the dorsal root ganglia of 11-to 13-day chicken embryos by procedures that have been described (9). Cells were incubated at 370 in medium C, which is composed of Leibovitz L15 (GIBCo BioCult, Glasgow, Scotland) with 0.6% glucose, 2 mM L-glutamine, 100 units of penicillin per ml, 100 Aig of streptomycin per ml, and 0.1 jig of mouse nerve growth factor per ml purified to the DEAE-cellulose fraction described by Varon et al. (10). With a few exceptions, which are noted, this medium was supplemented with 0.6% methylcellulose and 10% fetal calf serum. All cells were grown on glass coverslips which in some experiments were built into the cultur...

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Cultures of chick skin fibroblasts were dissolved in solutions of sodium dodecyl sulphate, and their entire protein content was examined by gel electrophoresis. The most abundant species migrated in the same position as muscle actin. It gave a similar pattern of iodinated peptides after reaction with radioactive sodium iodide and digestion with proteinases, and contained comparable amounts of N-methylhistidine. Its amount was estimated by quantitative densitometry of stained gels with bovine serum albumin as an internal standard, and by radioactive assay ofcultures that had been grown in the presence of [35S]methionine. The values obtained ranged from 7 to 14% of the total cellular protein, with an average of 8.5 %. A protein band in the position of muscle myosin was also present and accounted for about 2.5 % of the total protein. Both this and the actin band increased in relative amount with the age of the cultures.

A DNA-based assay was developed to detect Aeromonas salnonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) ofA. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection ofA. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.

The mesenchymal intermediate filament protein vimentin and the 70K component of neurofilament were detected by two-dimensional gel electrophoresis in cultures of pure sensory and sympathetic neurones derived from chick embryos. The identities of these neuronal intermediate filament proteins were confirmed by comparison of their molecular weights, isoelectric points, and peptide patterns from limited papain digestions with those of the corresponding proteins from fibroblasts and brain, respectively. A specific antibody to vimentin stained filamentous structures and colcemid-induced coils in both neurones and associated satellite cells. In contrast, a specific antibody to the 70K neurofilament protein stained these structures solely in neurones. This neurone-specific staining, as well as its molecular weight and isoelectric point, distinguishes the 70K neurofilament protein from the 68K neurofilament associated protein described by others, which has been claimed to resemble the tubulin assembly protein.