Growth factor modulation of substrate-specific morphological patterns in Aplysia bag cell neurons

Abstract

Components of the extracellular matrix (ECM) can act not only as passive substrates for neuronal attachment and outgrowth but also as active sites for signal transduction. Thus, specific ECM components may modulate effects of growth factors (GFs) that play an important role in structural changes in development and adult neuronal plasticity. In this study we examined the interaction of cultured Aplysia bag cell neurons (BCNs) with components of ECM and different GFs. Different ECM substrata induce a substrate-specific BCN morphology: BCNs grown on collagen or poly-L-lysine have larger soma diameter and more extensive neurite outgrowth than BCNs grown on laminin or fibronectin. BCNs also interact in a substrate-dependent way with GFs: BDNF treatment leads to a reduction of outgrowth on poly-L-lysine but an enhancement on fibronectin and laminin. CNTF reduces the soma diameter on collagen IV but enlarges it on laminin or fibronectin. In contrast, NGF induces a reduction of both soma diameter and outgrowth, on all substrata. Plating of BCNs in the presence of anti-beta1-integrin reduces adhesion to fibronectin but does not change outgrowth. In contrast, RGD peptides block adhesion to laminin and poly-L-lysine and, additionally, reduce outgrowth on laminin. These data suggest that BCNs use different beta1-integrin-dependent as well as RGD-dependent mechanisms for adhesion and outgrowth on different ECM substrata, providing possible sites of modulation by specific GFs.

Figure 1

BCNs adhere to different substrata and show substrate-dependent morphology. BCNS were plated on glass coverslips precoated with laminin (A), fibronectin (B), poly-l-lysine (C), or collagen (D). Immunostaining with anti-β-tubulin was performed after 2 days of culture and was visualized using a fluorescein-coupled secondary antibody. Photomicrographs were taken with a confocal laser scanning microscope. Scale bar, μm.

Figure 2

Soma diameter and neurite outgrowth of BCNs are modulated in a substrate-dependent fashion. BCNs were cultured on different substrata and stained as described for Fig. 1. Morphological measurements were performed on laser microscope scans (as shown in Fig. 1) using NIH Image. The soma diameter (soma) and the total branch length of neurites (outgrowth) were measured for each cell. Here and in Figs. 3–9, numbers in the histograms indicate the number of cells examined in each condition. Asterisks (*) indicate means that are significantly different from each other (P < 0.05).

Figure 3

BDNF differentially modulates soma diameter and neurite ougrowth of BCNs in a substrate-dependent fashion. BCNs were grown on different substrata for 1 day and incubated with BDNF (50 ng/ml) for another day before immunostaining. Soma diameter and total branch length of neurites were measured for each cell as described for Fig. 2. Statistical comparisons were performed for untreated cells (solid bars) and BDNF-treated cells (open bars) that were grown on the same substrate.

Figure 4

NGF leads to a reduction of soma diameter and neurite outgrowth of BCNs on all substrata. BCNs were grown on different substrata for 1 day and incubated with NGF (20 ng/ml) for another day before immunostaining. Statistical comparisons were performed for untreated cells (solid bars) and NGF-treated cells (open bars) that were grown on the same substrate. In all cases means for NGF-treated cells are significantly lower than the corresponding controls (P < 0.05).

Figure 5

CNTF modulates soma diameter in a substrate-dependent fashion but does not change neurite outgrowth. BCNs were grown on different substrata for 1 day and incubated with CNTF (1 nm) for another day before immunostaining. Statistical comparisons were performed for untreated cells (solid bars) and CNTF-treated cells (open bars) that were grown on the same substrate.

Figure 6

Anti-β1-integrin blocks adhesion to fibronectin but not to the other substrates. Anti-β1 integrin (1:200) was added to ASW in the culture dishes immediately before the plating of BCNs, which were then grown in the absence (control) or presence of anti-β1-integrin for 2 days. The number of BCNs on each coverslip was counted immediately after plating, and the number of BCNs attached to the substrate was determined 1 and 2 days after plating. The adhesion rate was calculated by dividing the number of attached cells on day 3 by the number of initially plated cells on day 1. Statistical comparisons were performed for untreated (solid bars) and anti-β1-integrin-treated (open bars) coverslips that were coated with the same substrate.

Figure 7

Anti-β1-integrin does not interfere with neurite outgrowth but modulates the soma diameter of BCN in a substrate-specific manner. Anti-β1-integrin (1:200) was added to ASW in the culture dishes immediately before the plating of BCNs. BCNs were grown in the absence (control) or presence of anti-β1-integrin for 2 days before immunostaining. Statistical comparisons were performed for untreated cells (solid bars) and anti-β1-integrin-treated cells (open bars) that were grown on the same substrate. No significant changes in neurite outgrowth were detected.

Figure 8

RGD peptides selectively reduce adhesion to laminin and poly-lysine. RGD peptides (RGD or GRGDS, both 50 μm) were added to ASW in the culture dishes immediately before the plating of BCNs. Controls were incubated in the presence of the inactive peptide GRGESP or in the absence of any peptide. The adhesion rates were determined as described for Fig. 7. No significant differences were found between BCNs treated with RGD and GRGDSP; thus, these groups were pooled in this figure (open bars). Also, adhesion of BCNs cultured in the presence of GRGESP or in the absence of any peptide was not significantly different, and these two groups were therefore pooled as controls (solid bars).

Figure 9

RGD peptides selectively inhibit neurite outgrowth on laminin. RGD peptides (RGD or GRGDS, both 50 μm) were added to ASW in the culture dishes immediately before the plating of BCNs. Controls were incubated in the presence of the inactive peptide GRGESP or in the absence of any peptide. Immunostaining was performed after 2 days. Soma diameter and total branch length of neurites were measured for each cell as described for Fig. 2. (Solid bars) Control; (open bars) RGD. No significant changes in soma diameter were detected.

Figure 10

Schematic drawing illustrating how the effects of GFs on soma diameter or neurite outgrowth may be determined by the presetting of these morphological features by the substrate itself. Two examples are depicted that illustrate one hypothesis (see text for details): (Left) BCNs grown on collagen have a significantly larger soma diameter than BCNs grown on laminin or fibronectin. CNTF now leads to an enlargement of the small soma diameter of BCNs on laminin or fibronectin, whereas it induces a reduction of the soma diameter of BCNs on collagen. (Right) Laminin and fibronectin induce only relatively little neurite outgrowth compared with poly-l-lysine. However, outgrowth on laminin and fibronectin is increased significantly in the presence of BDNF, whereas BDNF leads to an inhibition of outgrowth on poly-l-ysine. These findings suggest that there is crosstalk between substrate-induced and GF-induced intracellular signaling pathways: The substrate causes a presetting of intracellular signaling cascades and thereby modulates the signaling process activated by GFs.

Figure 11

Schematic drawing illustrating that mechanisms of BCN adhesion and neurite outgrowth depend on the substrate. Whereas adhesion to laminin is mediated by an RGD-dependent, but integrin-independent, mechanism (Left), adhesion to fibronectin requires an RGD-independent, integrin-dependent process (Right). Interestingly, in the case of laminin, an RGD-dependent mechanism is used not only for adhesion but also for neurite outgrowth. In contrast, outgrowth on fibronectin requires a mechanism (as yet uncharacterized) that is different from the adhesion mechanism and is integrin- and RGD-independent