Local NGF and GDNF levels modulate morphology and function of porcine DRG neurites, In Vitro

Abstract

Nerve terminals of primary sensory neurons are influenced by their environment through target derived trophic factors, like nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF). In mice, subpopulations of DRG neurons express receptors either for NGF or GDNF and therefore differentially respond to these neurotrophic factors. We probed neurite endings from porcine DRG neurons cultured in either NGF or GDNF and examined their shape, elongation and stimulus-evoked CGRP release. A compartmentalized culture system was employed allowing spatial separation of outgrown neurites from their somata and use of different growth factors in the compartments. We show that neurites of GDNF cultured somata extend into lateral compartments without added growth factor, unlike neurites of NGF cultured ones. Neurites of NGF cultured somata extend not only into NGF- but also into GDNF-containing compartments. GDNF at the site of terminals of NGF responsive somata led to a strong neurite arborization and formation of large growth cones, compared to neurites in medium with NGF. Functionally, we could detect evoked CGRP release from as few as 7 outgrown neurites per compartment and calculated release per mm neurite length. CGRP release was detected both in neurites from NGF and GDNF cultured somata, suggesting that also the latter ones are peptidergic in pig. When neurites of NGF cultured somata were grown in GDNF, capsaicin evoked a lower CGRP release than high potassium, compared to those grown in NGF. Our experiments demonstrate that the compartmented culture chamber can be a suitable model to assess neurite properties from trophic factor specific primary sensory neurons. With this model, insights into mechanisms of gain or loss of function of specific nociceptive neurites may be achieved.

Fig 1. Schematic layout of culture chamber as top and side view and photograph.

Fig 2. Morphology of neurite endings grown under different growth factor conditions.

Top: Percentage of neurites with only thin endings (black bars), with one or more thick endings (grey bars), and undefined (white bars) under different culture conditions A—E. Insets show the allocation of growth factors in the compartments of the culture chamber. Qualitative visual determination was done from microphotographs taken after 4 to 5 days in culture. Abbreviations indicate the growth factors (GF) in the chambers, e.g., G-N-G denotes GDNF in the lateral compartments and NGF in the central, somata-containing compartment. Bottom: Examples for thin (left) and thick (right) neurite endings are shown in microphotographs. Media conditions in the compartments as indicated. Number of animals: 3 to 5 for A—E, respectively.

Fig 3. Number of lanes with outgrown neurites in the lateral compartments and total neurite length in the presence of different growth factors.

(A) Number of lanes with outgrown neurites per lateral compartment. (B) Total length of the neurites per lateral compartment measured from microphotographs. (C) Frequency distribution of total neurite length for all single lateral compartments. Single data same as in (B). Error bars indicate mean ± SEM; asterisks indicate p<0.05. Number of animals: N-N-N: 7; G-N-G: 10; Ø-G-Ø: 5.

Fig 4. Time course of potassium-induced CGRP release in medium with different growth factors.

CGRP release from potassium-stimulated neurites (A) or somata (C) and release in the adjacent soma (B) or neurite (D) compartment(s). (A) CGRP release in neurite compartment in response to potassium (60 mM) for 5 min (grey shading) and (B) concomitant CGRP release in the unstimulated adjacent soma compartment. (C) CGRP release in the soma compartment in response to potassium (60 mM) for 5 min (grey shading) and (D) concomitant CGRP release in the adjacent neurite compartment. Insets: Total CGRP release (as AUC (pg/ml)) determined from the respective time course experiments, grey columns indicate total CGRP release from the stimulated compartment(s), white columns from the adjacent one(s). Symbols and abbreviations: Circles denote NGF in the lateral and central compartments (N-N-N); squares denote GDNF in the lateral compartments and NGF in the central compartment (G-N-G); triangles denote no growth factor in the lateral compartments and GDNF in the central compartment (Ø-G-Ø). A: ● n = 11/3, ■ n = 21/4, ▲ n = 18/3, B: ● n = 4/3, ■ n = 8/4, ▲ n = 8/3, C: ● n = 4/2, ■ n = 4/3, ▲ n = 5/2, D: ● n = 9/2, ■ n = 8/3, ▲ n = 11/2,(n = number of compartments/animals used). Error bars indicate ± SEM, asterisks indicate p<0.05.

Fig 5. Total CGRP release (AUC) per mm neurite length.

(A) Values for the individual compartments following neurite stimulation (filled symbols) and soma stimulation (open symbols). Symbols indicate the added growth factors in the chambers; N-N-N denotes NGF in the lateral and central compartments (circles); G-N-G denotes GDNF in the lateral compartments and NGF in the central compartment (squares); Ø-G-Ø denotes no growth factor in the lateral compartments and GDNF in the central compartment (triangles). Data derived from the experiments presented in Fig 4. (B) Mean values from data presented in A. Error bars indicate ± SEM; asterisks indicate p<0.05.

Fig 6. CGRP release from capsaicin stimulated neurites under different growth factor conditions.

(A) Time course of CGRP release in response to capsaicin stimulation (300 nM) for 5 min (grey shading). Neurites were cultured with NGF (● n = 13/2 compartments/animals) or GDNF (■ n = 11/2). Somata were cultured with NGF in both cases. Inset: CGRP release as AUC (pg/ml) determined from the respective time course experiments. (B) Comparison between Capsaicin (300 nM, grey columns) and potassium (60mM, black columns) stimulated neurites, grown under NGF or GDNF, respectively. Somata were cultured with NGF in both cases. Data were taken from Figs 5B and 6A. Error bars indicate ± SEM. Asterisks indicate p<0.05.