Growth cone formation in cultures of sensory neurons.

Abstract

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...

GABA Regulates Dendritic Growth by Stabilizing Lamellipodia in Newly Generated Interneurons of the Olfactory Bulb

“…Because microtubules are known to play an essential role in dendritic stability (Bray et al, 1978;Dehmelt et al, 2003;Henley and Poo, 2004;Suter et al, 2004), these results nicely correlate to the observed behavior of developing dendrites in our recordings. They are also consistent with previous reports suggesting that microtubules drive growth cone steering (Suter et al, 2004) and the forward momentum of newly formed protrusions (Baas and Ahmad, 2001).…”

Localized and Transient Elevations of Intracellular Ca2+Induce the Dedifferentiation of Axonal Segments into Growth Cones

“…4, 9; see also Joshi et al, 1986). Microtubule discontinuity may lead to a local accumulation of membranous vesicles (Bray et al, 1978), thus providing an abundant source of membrane for the rapidly extending growth cone. Such vesicle accumulation was observed in several experiments (Fig.…”

“…Axonal transection is followed, in many cases, by a process in which the tip of the severed axon is transformed into a motile growth cone (Shaw and Bray, 1977;Bray et al, 1978;Wessells et al, 1978;Baas and Heidemann, 1986;Baas et al, 1987;Rehder et al, 1992;Benbassat and Spira, 1993;Ashery et al, 1996). This growth cone serves as a path-finding sensor, a cytoskeleton assembly apparatus, and a site for membrane insertion (for review, see Letourneau et al, 1992).…”

Transneuronal and peripheral mechanisms for the induction of motor neuron sprouting

“…A number of observations that were made in neuronal and other systems support this hypothesis. Low doses of antimicrotubular agents induced growth cone formation in cultured neurons (Bray et al, 1978) and locomotor activity in fibroblasts (Vasiliev et al, 1970(Vasiliev et al, , 1971. Antimicrotubular agents also modulated the rate of intact motor axon sprouting in partially denervated muscles (Hoffman, 1952); low doses of cryptopleurine accelerated sprouting, whereas high doses retarded it.…”

“…The ability of colchicine to induce neurite outgrowth, of axons that were separated from their cell bodies to elongate, and of synapses to form rapidly was described previously. A low dose of colchicine initiated growth cone formation in cultured neurons (Bray et al, 1978) and sprouting and synapse formation in the rat dentate gyrus (Goldowitz and Cotman, 1980). Axons that were separated from their cell bodies and grew before undergoing subsequent degeneration were observed in chick brain, spinal cord, and dorsal root ganglia cultures (Levi and Meyer, 1945;Hughes, 1953;Shaw and Bray, 1977).…”