Journal of Neuroscience Research 27:136-143 (1990)
Immunohistochemical Localisation of Nerve Growth Factor in a Subpopulation of Chick Spinal Ganglion Neurons A. Keller, R. Williams, J. Vahaviolos, C. Auffray, and R.A. Rush Institut d’embryologic ccllulairc et molkculaire du CNRS et du Collbge de France, Nogcnt-s-Marne, 94130, France (A.K., C.A.); School of Medicine, Flindcrs University of South Australia, Bedford Park, South Australia, 5042 (R. W . , J . V . , K.A. R.)
We have previously isolated and sequenced the chicken nerve growth factor (NGF) gene and now, from the deduced amino acid sequence, selected and produced peptides suitable for use as antigens. Antisera raised against these peptides inhibit the biological activity of a partially purified preparation of native chicken NGF and, when used in immunocytochemical studies, allow the visualisation of sensory neurons accumulating endogenous NGF. Immunoreactive cells form a distinct population of small neurons which may correspond to the well-described neurons generated in the dorsomedial area of spinal ganglia. We conclude that two subpopulations of neurons exist within dorsal root ganglia, whose requirements for, and use of, NGF may be quite distinct. Key words: amino acid sequence, DRG, NGF
development,
INTRODUCTION It is now well documented that nerve growth factor
(NGF) plays an important role in the development of the nervous system, both in the periphery (Levi-Montalcini, 1987) and in the brain (Large et al., 1989). NGF has long been established as a molecule essential for the development and maintenance of sympathetic and sensory neurons. Embryonic sensory neurons placed in culture have been shown to be dependent on NGF for their survival (Levi-Montalcini and Hamburger, 1951). Administration of exogenous NGF to embryos has been reported to rescue, for a few days, neurons destined to die (Levi-Montalcini and Hamburger, 1951; Strasnicky and Rush, 1985). Whether all sensory neurons require NGF is not clear, since NGF administration has been shown either to affect only the dorso-medial population (Levi-Montalcini and Hamburger, 1951; Dimberg and Ebendal, 1987) or both the dorso-medial and the ventro-lateral neurons (Straznicky and Rush: 1985; Hamburger et al., 1981). The avian embryo presents many advantages for embry0 1990 Wiley-Liss, Inc.
ological studies, being particularly amenable to experimental manipulation. Although carly experiments defining the action of NGF were performed on developing chickens, this species was soon abandoned in favour of mammals. This was the consequence of the discovery of a very rich source of the factor in the male mouse submandibular gland (Levi-Montalcini, 1987). Antisera obtained after injection of pure murine NGF were not suitable for the study of chicken NGF. Recently, a few laboratories have succeeded in producing antibodies to mouse NCF that cross-react with the chicken molecule, allowing experimenters to return to the use of the avian embryo (Abraharnson et al., 1987; Dimberg and Ebendal, 1988; Rohrer et al., 1988). However, the usefulness of antisera obtained by this method and their further use is restricted by limited cross-reactivity . The production of antisera directed against native chicken NGF has so far proved impossible due to the limiting amounts of chicken NGF present in various tissues. We have previously isolated and sequenced the chicken NGF gene (Wion et al., 1986). Analysis of the predicted NGF amino acid sequence led to the choice of two sequences as antigens for chemical synthesis. From hydrophilicity and hydropathic index analyses, we anticipated that these synthetic peptides would elicit production of antibodies likely to recognize native chick NGF. We now describe the characterization of antisera obtained in such a way and their further use in an immunocytochemical study. In
Received January 5 . 1990; revised April 24, 1990: accepted April 25, 1990. Address reprint requests to A . Keller’s present address: College de France, Laboratoire de Biochirnie Cellulaire, 11 PI. Marcelin Berthclot. 75005, Paris, France. Abbreviations used: nerve growth factor (NGF); dorsal root ganglion (DKG); phosphate buffered saline (I’BS); Eagle’s basal medium (EBM); fetal calf serum (FCS); normal sheep serum (NSSj; Tris buffered d i n e (TBS); brain derived neurotrophic factor (BDNF); dorsomedial IDM); ventrolateral (VL).
NGF Immunolocalisation in Chick DRG
particular, we sought to determine which cells within the brachial dorsal root ganglia (DRG) normally contain NGF in vivo, at various times of development. This may allow a conclusion to be reached about which neurons utilize endogenous NGF.
137
product was measured at 405 nm with a Titertek Multiskan plate reader. A similar test for detection of the mouse PNGF was performed by E. Dicou. as described (Dicou et al., 1989).
Dorsal Root Ganglia Bioassay Whole dorsal root ganglia (DRG) from E8 chicken MATERIALS AND METHODS embryos were cultured in Eagle’s basal medium (EBM) Synthesis of Chicken PNGF Peptides supplemented with 10% fctal calf serum (FCS). Culture Two peptides were selected for synthesis, by anal- dishes were preplated with polycirnithine. Ganglia were ysis of the hydrophilicity profile (Hopp and Woods, grown in presence of partially purified chicken NGF 1981) and hydropathic index (Kyte and Doolittle, 1982). (Ferguson et al., 1989), either with or without antiserum Peptides were synthesized by Neosystem Laboratoires 30 (directed against the R16K peptide) that had been (Strasbourg, France), 92-95% pure from the HPLC pro- dialyzed overnight to remove sodium azide. Negative file. controls were cultured with EBM and FCS only. After 24 Each peptide was conjugated to ovalbumin, in the hr, neurite outgrowth was scored blindly according to presence of 1% glutaraldehyde (Avrameas, 1969). Equal length and density of outgrowth and ranked on a scale of volumes (600 11.1) of the peptide and ovalbumin, I mgirnl 0 to 5 (Bclcw and Ebendal, 1987). A Mann-Whitney in sterile phosphate buffer saline (PBS), were mixed and U-test on the scores indicated a significant reduction of 60 pl of 20% glutaraldehyde were added dropwise under outgrowth in those ganglia exposed to the antiserum gentle mixing. The coupling reaction proceeded then at (P
Immunohistochemical Localisation of Nerve Growth Factor in a Subpopulation of Chick Spinal Ganglion Neurons A. Keller, R. Williams, J. Vahaviolos, C. Auffray, and R.A. Rush Institut d’embryologic ccllulairc et molkculaire du CNRS et du Collbge de France, Nogcnt-s-Marne, 94130, France (A.K., C.A.); School of Medicine, Flindcrs University of South Australia, Bedford Park, South Australia, 5042 (R. W . , J . V . , K.A. R.)
We have previously isolated and sequenced the chicken nerve growth factor (NGF) gene and now, from the deduced amino acid sequence, selected and produced peptides suitable for use as antigens. Antisera raised against these peptides inhibit the biological activity of a partially purified preparation of native chicken NGF and, when used in immunocytochemical studies, allow the visualisation of sensory neurons accumulating endogenous NGF. Immunoreactive cells form a distinct population of small neurons which may correspond to the well-described neurons generated in the dorsomedial area of spinal ganglia. We conclude that two subpopulations of neurons exist within dorsal root ganglia, whose requirements for, and use of, NGF may be quite distinct. Key words: amino acid sequence, DRG, NGF
development,
INTRODUCTION It is now well documented that nerve growth factor
(NGF) plays an important role in the development of the nervous system, both in the periphery (Levi-Montalcini, 1987) and in the brain (Large et al., 1989). NGF has long been established as a molecule essential for the development and maintenance of sympathetic and sensory neurons. Embryonic sensory neurons placed in culture have been shown to be dependent on NGF for their survival (Levi-Montalcini and Hamburger, 1951). Administration of exogenous NGF to embryos has been reported to rescue, for a few days, neurons destined to die (Levi-Montalcini and Hamburger, 1951; Strasnicky and Rush, 1985). Whether all sensory neurons require NGF is not clear, since NGF administration has been shown either to affect only the dorso-medial population (Levi-Montalcini and Hamburger, 1951; Dimberg and Ebendal, 1987) or both the dorso-medial and the ventro-lateral neurons (Straznicky and Rush: 1985; Hamburger et al., 1981). The avian embryo presents many advantages for embry0 1990 Wiley-Liss, Inc.
ological studies, being particularly amenable to experimental manipulation. Although carly experiments defining the action of NGF were performed on developing chickens, this species was soon abandoned in favour of mammals. This was the consequence of the discovery of a very rich source of the factor in the male mouse submandibular gland (Levi-Montalcini, 1987). Antisera obtained after injection of pure murine NGF were not suitable for the study of chicken NGF. Recently, a few laboratories have succeeded in producing antibodies to mouse NCF that cross-react with the chicken molecule, allowing experimenters to return to the use of the avian embryo (Abraharnson et al., 1987; Dimberg and Ebendal, 1988; Rohrer et al., 1988). However, the usefulness of antisera obtained by this method and their further use is restricted by limited cross-reactivity . The production of antisera directed against native chicken NGF has so far proved impossible due to the limiting amounts of chicken NGF present in various tissues. We have previously isolated and sequenced the chicken NGF gene (Wion et al., 1986). Analysis of the predicted NGF amino acid sequence led to the choice of two sequences as antigens for chemical synthesis. From hydrophilicity and hydropathic index analyses, we anticipated that these synthetic peptides would elicit production of antibodies likely to recognize native chick NGF. We now describe the characterization of antisera obtained in such a way and their further use in an immunocytochemical study. In
Received January 5 . 1990; revised April 24, 1990: accepted April 25, 1990. Address reprint requests to A . Keller’s present address: College de France, Laboratoire de Biochirnie Cellulaire, 11 PI. Marcelin Berthclot. 75005, Paris, France. Abbreviations used: nerve growth factor (NGF); dorsal root ganglion (DKG); phosphate buffered saline (I’BS); Eagle’s basal medium (EBM); fetal calf serum (FCS); normal sheep serum (NSSj; Tris buffered d i n e (TBS); brain derived neurotrophic factor (BDNF); dorsomedial IDM); ventrolateral (VL).
NGF Immunolocalisation in Chick DRG
particular, we sought to determine which cells within the brachial dorsal root ganglia (DRG) normally contain NGF in vivo, at various times of development. This may allow a conclusion to be reached about which neurons utilize endogenous NGF.
137
product was measured at 405 nm with a Titertek Multiskan plate reader. A similar test for detection of the mouse PNGF was performed by E. Dicou. as described (Dicou et al., 1989).
Dorsal Root Ganglia Bioassay Whole dorsal root ganglia (DRG) from E8 chicken MATERIALS AND METHODS embryos were cultured in Eagle’s basal medium (EBM) Synthesis of Chicken PNGF Peptides supplemented with 10% fctal calf serum (FCS). Culture Two peptides were selected for synthesis, by anal- dishes were preplated with polycirnithine. Ganglia were ysis of the hydrophilicity profile (Hopp and Woods, grown in presence of partially purified chicken NGF 1981) and hydropathic index (Kyte and Doolittle, 1982). (Ferguson et al., 1989), either with or without antiserum Peptides were synthesized by Neosystem Laboratoires 30 (directed against the R16K peptide) that had been (Strasbourg, France), 92-95% pure from the HPLC pro- dialyzed overnight to remove sodium azide. Negative file. controls were cultured with EBM and FCS only. After 24 Each peptide was conjugated to ovalbumin, in the hr, neurite outgrowth was scored blindly according to presence of 1% glutaraldehyde (Avrameas, 1969). Equal length and density of outgrowth and ranked on a scale of volumes (600 11.1) of the peptide and ovalbumin, I mgirnl 0 to 5 (Bclcw and Ebendal, 1987). A Mann-Whitney in sterile phosphate buffer saline (PBS), were mixed and U-test on the scores indicated a significant reduction of 60 pl of 20% glutaraldehyde were added dropwise under outgrowth in those ganglia exposed to the antiserum gentle mixing. The coupling reaction proceeded then at (P
