Effect of NGF, BDNF, bFGF, aFGF and cell density on NPY expression in cultured rat dorsal root ganglion neurones

Abstract

The effect of neurotrophic factors on neuropeptide Y (NPY) expression was studied in adult rat dispersed dorsal root ganglion (DRG) cultures. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), acidic fibroblast growth factor (aFGF) or basic FGF was included in the culture medium during incubation for 72 h. In untreated cultures, around 18% of all neurones (visualized by antibodies to PGP 9.5) expressed NPY-like immunoreactivity (LI). In contrast, in vivo uninjured neurones do not contain detectable levels of NPY-LI. In the immunohistochemical analysis aFGF increased the percentage of NPY-immunoreactive (-IR) neurones 1.8-fold, while NGF, BDNF or bFGF had no significant effect on NPY expression. When the effect of these growth factors was monitored with non-radioactive in situ hybridization, both aFGF and bFGF caused a significant increase (2.25- and 1.8-fold, respectively), whereas, again, NGF and BDNF had no effect. The results also showed an effect of cell density on NPY expression, whereby fewer neurones expressed NPY in high than in low density cultures. This difference was seen in untreated as well as growth factor-treated cultures. The present results support the hypothesis that DRG neurones in culture are in an axotomized state, since they express NPY to about the same extent as axotomized DRG neurones in vivo. Surprisingly, two growth factors of the FGF family enhance NPY expression in DRG neurones, which is in apparent contrast to a published in vivo study [Ji, R.-R., Zhang, Q., Pettersson, R.F., Hökfelt, T., 1996. aFGF, bFGF and NGF differentially regulate neuropeptide expression in dorsal root ganglia after axotomy and induce autotomy. Reg. Pept. 66, 179–189.]. Finally, NPY expression was also influenced by cell density.

Introduction

Adult dorsal root ganglion (DRG) neurones in dissociated culture may represent a model for studying mechanisms underlying nerve injury-induced changes in peptide expression and neurite outgrowth. Thus, neurones in dissociated DRG cultures can be expected to be in an axotomized state, since the surgical manipulations associated with removing the DRGs include axotomy of both the peripheral and central branch close to the cell bodies. In fact, results from earlier analyses of such cultures have shown increased levels of vasoactive intestinal polypeptide (VIP) (Mulderry and Lindsay, 1990) and galanin (Kerekes et al., 1997), changes that have been reported to occur in DRG neurones after peripheral axotomy in vivo (Shehab and Atkinson, 1986, Hökfelt et al., 1987, Villar et al., 1989). The modulation of peptide levels, e.g., of galanin, following nerve injury has been proposed to protect the body from pain sensation (Wiesenfeld-Hallin et al., 1992), and other studies support a role of VIP (White and Mansfield, 1996) and galanin (Shadiak and Zigmond, 1998) in regenerative processes.

The 36-amino acid peptide neuropeptide (NP) tyrosine (Y) (NPY) (Tatemoto, 1982, Tatemoto et al., 1982) can normally not be detected in DRGs (Gibson et al., 1984), but after peripheral nerve transection there is a marked upregulation of this peptide, mainly in large DRG neurones (Wakisaka et al., 1991, Wakisaka et al., 1992, Noguchi et al., 1993, Zhang et al., 1993). Also this peptide may attenuate pain and influence regeneration. Thus, in vivo studies have demonstrated that NPY can inhibit the release of several excitatory neuropeptides, such as substance P and calcitonin gene-related peptide (CGRP), from sensory neurones (Giuliani et al., 1989, Duggan et al., 1991), and perhaps in this way exert an anti-nociceptive action (Hua et al., 1991). Moreover, NPY indirectly, via neurotrophin-3 (NT-3), increases neurite outgrowth of dissociated rat DRG neurones, indicating a trophic action also for this peptide (White and Mansfield, 1996, White, 1998).

The mechanisms underlying the changes in expression of neuropeptides are not fully understood. However, growth factors seem to be involved, and their effects on expression of substance P, CGRP, VIP, galanin and somatostatin have been analysed in vitro (Lindsay and Harmar, 1989, Lindsay et al., 1989, Mulderry and Lindsay, 1990, Mulderry, 1994, Kerekes et al., 1997).

The aim of the present study was to analyse to what extent NPY expression, just as that of VIP (Mulderry and Lindsay, 1990) and galanin (Kerekes et al., 1997) is increased when DRG neurones are put in culture. Moreover, we have monitored the effects of four neurotrophic factors on NPY expression in these cultures. We used two target-derived growth factors, nerve growth factor (NGF) (see Levi-Montalcini, 1987) and brain-derived neurotrophic factor (BDNF) (see Barde, 1999), as well as basic fibroblast growth factor (bFGF) (see Baird, 1994) which is known to be strongly upregulated in DRGs cells after axotomy (Ji et al., 1995), and acidic FGF (aFGF) (see Baird, 1994) which is constitutively produced in DRG neurones (Elde et al., 1991). Finally, we have studied the effects of cell density on NPY expression in the DRG cell cultures.