Fig 1.
Definition of data used for the Sholl analysis and mathematical formulas used for the estimation of neurite branching based on the Sholl profiles.
Red filled circles indicate intersections at the generic radius ri and ri+1, whereas red empty circles indicate terminating neurites between two successive radii. (j) = neuron; (tr) = treatment; (i) = generic radius number; N(ri) = number of intersections at radius ri; N(ri+1) = number of intersections at radius ri+1; (bjtr(ri)) = branching rate; (bsjtr(ri) = specific branching; (Bmaxjtr(r)) = maximum branching; (Bsmaxjtr(r)) = maximum specific branching.
Fig 2.
DRG neurons in situ and in vitro.
(A) Histological section of a bovine DRG stained with hematoxylin and eosin (HE). Satellite cells tightly cluster around large sensory neurons. (B) Immunofluorescence staining of dissociated DRG at day 3 in culture, which contains a mixed population of cells. The neuron emits a large number of neurofilament-positive neurites (green). Satellite cells, whose nuclei are stained with DAPI (blue), remain tightly clustered around the soma of the dissociated neuron. (*) Weak DAPI-staining of the neuronal nucleus. (C) A large sensory DRG neuron, stained with neurofilament (grey), showing an extensive neurite outgrowth and few smaller neurons staining intensively with NF that have no detectable neurite outgrowth at 3 days of culture (*). (D) Neuron stained with neurofilament showing filopodia like structures (*) at day 1 in cell culture next to a neuron with neurite outgrowth and neurons without neurites. (E) DRG culture at day 7. Satellite/Schwann cells expressing S100 (red) and Vimentin (green) constituting a subconfluent layer at day 7 of culture. Nuclei are stained with DAPI (blue). (F) Immunofluorescence staining showing GFAP-positive satellite cells (red) surrounding a neuron labeled with neurofilament (green). Nuclei are stained with DAPI (blue).
Fig 3.
Ratio of neurite-bearing neurons and neurons without neurons.
The soma diameter of 360 neurons was measured and the presence or absence of neurites was assessed. (A) Dot plots showing soma diameter of neurons with and without neurites. Black horizontal lines indicate the mean. Based on soma diameter two categories (large neurons >50 μm and small neurons <50 μm) were defined. The prevalence of neurites was significantly higher in the group of large neurons as in small neurons (*) (X2 test, p < 0.0001). (B) Frequency histogram showing the distribution of neurons with and without neurites in function of size. The frequency distribution of neurons with neurites (grey) is shifted to a larger soma diameter compared to neurons without neurites (white). (C) Dot plot showing the proportions of neurons with and without neurites over 7 days of culture. The proportion of neurons with and without neurites did not change significantly over the entire period of the experiment (Kruskal Wallis test, p > 0.05). Black horizontal lines indicate the mean.
Fig 4.
GF supplementation modulates but is not required for neurite outgrowth.
(A, B, C) Box-and-whisker plots (5–95 percentiles) show the maximum number of intersections over the culture time period at day 1 (A), day 3 (B) and day 7 (C). Values are compared between GF (NGF: 10, 50, 150 ng/ml; GDNF: 10, 50, 150 ng/ml; IGF: 5, 20, 50 ng/ml, NGF 50 ng/ml combined with GDNF 50 ng/ml [COMBO2]; NGF 50ng/ml combined with GDNF 50 ng/ml and IGF20 ng/ml [COMBO3]) and control (No GF). Values below and above the whiskers are shown as individual points. Horizontal black lines indicate the median. Ninety neurons were analyzed for each GF supplementation and control. GDNF50, GDNF150, IGF20, IGF50, NGF50, COMBO2, COMBO3 induced a significant increase in the maximum number of intersections at day 1. At day 3 GDNF and NGF alone or in combined formulation but not IGF increased the maximum number of intersections. At day 7 both IGF and NGF 50 treated neurons showed a significant lower number of intersections. (p<0.05—Kruskall Wallis test, results are shown in S1 File). (D) Comparison of maximum number of intersections over time between supplementations and control. The number of intersections was higher in GF supplemented neurons compared to control and increased from day 1 to 3. From day 3 to day 7, the maximum number of intersections reached a plateau phase in all treatment groups. Mean values of maximum number of intersections calculated with the Sholl analysis are shown.
Fig 5.
DRG neurons can be maintained in culture for extended periods.
(A) Viable DRG neurons after 2 weeks in culture stained with calcein (green). (B) Diffuse neurite fragmentation in a neurofilament-positive neuron (*) at day 7. In the lower left a viable neurite bearing neuron is present.