Characterization of the turning response of dorsal root neurites toward nerve growth factor.

Abstract

This study reports that chick dorsal root ganglion neurites undergo a rapid (20 min) reorientation of their direction of growth in response to nerve growth factor (NGF) concentration gradients in vitro. Dorsal root ganglia from chick embryos were explanted onto a collagen-poly-L-lysine substrate. After 24-48 h in culture, NGF gradients were applied to individual growth cones via a micropipette containing 50 biological units NGF/ml. The growth cones turned and grew toward these NGF sources. This turning response was not caused by the trophic effects of NGF on neurite initiation, survival, or growth rate. Dorsal root neurites also grew toward sources of mono- and dibutyryl cyclic adenosine monophosphate (dB cAMP), cyclic guanosine monophosphate (cGMP), and elevated calcium in the presence of the calcium ionophore A23187. These results are consistent with the hypothesis that intracellular levels of cAMP and /or cGMP and calcium may play a role in the turning response of dorsal root neurites toward NGF, but do not establish a causal relationship between the mechanisms of action of NGF, cyclic nucleotides and calcium. Total growth cone adherence to the substrate was measured using a timed microjet of perfusion medium. NGF increased the adherence of growth cones to the substrate, but caffeine and dB cAMP which also elicit the positive turning response, decreased growth cone adherence. Calcium, which did not elicit the positive turning response, produced a greater growth cone adherence to the substrate than that observed with NGF. Although these results do not rule out a role of adhesion changes in axonal turning to NGF, they show that a general increase in adherence does not correlate well with the rapid turning response observed in this study.

14-3-3 Proteins Regulate Protein Kinase A Activity to Modulate Growth Cone Turning Responses

“…S5, available at www.jneurosci.org as supplemental material) (Nagata-Ohashi et al, 2004). We confirmed that application of an NGF gradient results in an attractive mean turning angle when applied to E8 chick DRG growth cones (Gundersen and Barrett, 1980;Gehler et al, 2004) and we find that this response is unaffected by HSV-R18-GFP (12.6 Ϯ 5.8°; Fig. 2a,d).…”

Defining the concentration gradient of nerve growth factor for guided neurite outgrowth

“…While previous studies demonstrated neurite outgrowth towards a neurotrophic factor source, the concentration pro®le was ill-de®ned. For example, neurite outgrowth towards a point source was studied, with the source created either by micropipetting factors at a speci®c location in culture media 18,19,38 or using explants which released the factors of interest. 28 In both examples, the concentration pro®le was either poorly de®ned 29 or Gaussian, 22 and depended upon the experimental geometry and other parameters used.…”

“…This may explain the discrepancy in the literature between cellular response to different concentrations and concentration gradients. 18,28 To create a linear, stable, homogeneous concentration gradient throughout the agarose gel, a constant concentration of NGF was provided in both source and sink compartments while the steady-state diffusion was established. To achieve constant concentrations of NGF in both source and sink compartments, the NGF solutions in both compartments were replenished every 6 h during the course of the experiment; this ensured a concentration difference¯uctuation of less than 1% between each solution replenishment, according to our calculation (cf.…”

Differences between the effect of anisotropic and isotropic laminin and nerve growth factor presenting scaffolds on nerve regeneration across long peripheral nerve gaps

“…NGF has been shown to prevent the complete death of axotomized sensory neurons following exogenous administration [58]. There is enough evidence to show that gradients of neurotrophins help in guiding developing as well as regenerating axons in a wide variety of neuronal systems [59,60]. Although many in vitro studies have shown influence of gradients of LN-1 and NGF for neurite extension, to our knowledge, there are no in vivo studies with gradients, due to the difficulties in making and sustaining gradients in vivo.…”