Det'elopmental Brain Research, 69 (1992) 261-270 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00
261
BRESD 51533
Distribution and relative density of p75 nerve growth factor receptors in the rat spinal cord as a function of age and treatment with antibodies to nerve growth factor B e t h a n y A. Urschel a a n d Claire E. H u l s e b o s c h b a Department of Neuroscience, McMaster University, Hamilton, Ont., L8N 3Z5 (Canada) and h Department of Anatomy and Neurosciences and the Marine Biomedical Institute, Unicersity of Texas Medical Branch, Gah'eston, TX 77550-2772 (USA) (Accepted 30 June 1992)
Key words: Primary afferent; Sprouting; Dorsal horn
It has been postulated that nerve growth factor (NGF) binding to the low-affinity fast-dissociating NGF receptor (p75 NGFR) on Schwann cells and growing neurites is involved with the molecular feedback necessary for continued neurite extension during development and regeneration. Since central projections of somatosensory fibers sprout into the ,~pinal cord after daily neonatal injections of antibodies to NGF (ANTI-NGF) for a one month period, it is of interest to determine if the distribution of p75 NGFR correlates with the occurrence of sprouting. Spinal cords from three groups of rats: untreated, preimmune sera treated and ANTI-NGF treated were examined on postnatal days (PD) 0, 14 and 30. The p75 NGFR distribution was determined using the monoclonal antibody 192 with standard immunohistochemical techniques and the optical density of the immunoreaction product was quantified using an Amersham image analysis system. The 192 immunoreaction product was localized to laminae l-IV, the dorsal columns, the dorsolateral funiculus, Lissauer's tract (LT) and the ventral horn on PD 0; to laminae 1-111and medial IV and LT on PD 14; and laminae !-!! and LT on PD 30. The untreated and preimmune sera treated groups show no difference in distribution. In the ANTI-NGF treated group, the 192 immunoreaction product was localized to laminae I-V and LT on PD 14 and to laminae I-!ii and medial IV and LT on PD 30. Similarly, the optical density of the ANTI-NGF treated group was significantly greater than same aged untreated and preimumne sera treated groups, but was not statistically different from these two groups examined 14 days earlier. Thus, ANTI-NGF treatment interferes with the postnatal downregulation of p75 NGFR in the dorsal horn and may provide for continued neurite growth.
INTRODUCTION Nerve growth factor (NGF) is one of a family of proteins known collectively as neurotrophins. Included in this family of related factors is brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and possibly other neurotrophins such as NT4 and N T 5 4't5'21'32'38°45'46'58. However, NGF is the most well characterized of the neurotrophins in terms of neurotrophic (neuron sun, ival) activities and neurotropic (neurite elongation) activities both in the peripheral n e r v o u s s y s t e m 13't4'42'66 and in the central nervous system 2°m. Neurons that are known to be responsive to NGF are sympathetic postganglionic neurons 13'1s'3°'39-42 a subpopulation of dorsal root ganglion neurons 13'25'30'39'41'42'54'75 and some cholinergic neurons in the C N S 36'59'7l.
The trophic and tropic effects of NGF are thought to be mediated through a process of NGF binding to a NGF receptor complex 34'75-77. The current thinking is that the NGF receptor complex is composed of two proteins: a transmembrane glycoprotein with low affinity for binding NGF, p75 NGFR 74, and a transmembrane tyrosine kinase protein, the gene product of the trk-1 proto-oncogene (p140)which, in combination with p75, is thought to confer high-affinity binding for N G F 17'33'35. It is postulated that the internalization of NGF and thus, NGF-induced biological activity, is associated with the high-affinity combined receptor complex of p75 and p140. Another speculated role of the low affinity p75 NGFR on glial cells is to provide a scaffold for neurotropic binding which provides for a continuous supply of neurotropic factors for neurite growth during development and regeneration ~9'31'76.For
Correspondence: C.E. Hulsebosch, Department of Anatomy and Neurosciences, M.B.I., University of Texas Medical Branch, 200 University Blvd., Galveston, TX 77550-2772, USA.
262 example, elongating neurites grow over the NGF-laden substrate provided by the NGF bound to the p75 NGFR on Schwann cells. The high-affinity combined complex of p75 and p140 NGFR on the neurites provides for transfer of the NGF from the p75 low affinity NGFR on the Schwann cells, to the combined NGFR complex on the neurite where either NGF is then internalized and retrogradely transported to the soma ~9 or tyrosine kinase activity is initiated ~7'67 and subsequent molecular events ensure continued elongation of the neurite. in addition to developmental and regenerative neurite growth, another paradigm in which postnatal sensory neurite growth is observed is the neonatal administration of antibodies to NGF (ANTI-NGF), which produces intraspinal sprouting and synaptogenesis of primary afferents in adult rats 22'25-27.Therefore, it was of interest to determine if the p75 NGFR changed in distribution or density which would suggest involvement in this phenomenon. To this end, we used the monoclonal antibody 192, which has been shown to recognize the rat low-affinity receptors in vitro 7, to determine the laminar distribution and relative density of the p75 NGFR in the rat spinal cord at various ages and with NGF suppression. This work was presented in preliminary form elsewhere ~'H. MATERIALS AND METHODS Pregnant Sprague-Dawl~y rats were obtained from llarlan Sprague-Dawley. All procedures involving rats were performed in compliance with USDA Animal Welfare Act and amendments, the revised Guide for the Care and Use of Laboratory Animals DHEW (NIH) and were approved by the UTMB Animal Care and Use Committee. Three groups of rats were examined: (I) rats treated with antibodies to NGF (ANTI-NGF), (2) rats treated with preimmune sera from the same rabbits used to generate the ANTI-NGF (similar injection doses and schedules to the rats treated with ANTINGF, PRP-IMM) and (3) untreated littermates (UNTR). Neonatal rats were given daily subcutaneous injections of ANTI-NGF (3 p.l of undiluted rabbit serum per gram b.wt.) or similar doses of preimmune sera near the dorsal fat pad for a period of ! month beginning on postnatal day (PD) 0 (day of birth). The ANTI-NGF was raised in rabbits which had received subcutaneous inoculations of purified /3-NGF (10-20 ng/ml for I biological unit/ml) isolated from the 7S NGF purified from mouse submaxillary glands according to the methods described in Varon ct al. 7°. The classic bioassays with extirpated embryonic chick sensory ganglia11'12'4°'41 and morphological differentiation of cultured human neuroblastoma cells ~''m were used and compared to NGF preparations whose specific biological activity was 10-20 ng/ml (I biological unit/ml) as described elsewhere'~k By both assays, the biological activity of the NGF and the ANTI-NGF used in this study was found to remain stable during the course of the experiments. An antiserum dilution of 1:1,500 blocked the effects of exogenously applied NGF at concentrations containing 1 biological unit/ml 4:' and by both assays the blocking activity of the ANTI-NGF was stable during the course of the experiments. The ANTI-NGF injections were doses of undiluted rabbit antiserum prepared against the purified/3-NGF subunit. To determine in vivo injection levels, it was determined empirically that increases in antibody concentration in the rabbit sera by ultrafiltra-
tion 2s greatly affected mortality. Consequently, the use of undiluted antisera in these experiments was deemed sufficient to block endogenous levels of NGF, which is known to be in the nanogram range. As an internal assay for the ANTI-NGF activity in vivo, wet weights were taken of the superior cervical ganglia for each of the three groups in the study. The Student's t-test was used to determine statistical differences with a level of confidence set at P ~
261
BRESD 51533
Distribution and relative density of p75 nerve growth factor receptors in the rat spinal cord as a function of age and treatment with antibodies to nerve growth factor B e t h a n y A. Urschel a a n d Claire E. H u l s e b o s c h b a Department of Neuroscience, McMaster University, Hamilton, Ont., L8N 3Z5 (Canada) and h Department of Anatomy and Neurosciences and the Marine Biomedical Institute, Unicersity of Texas Medical Branch, Gah'eston, TX 77550-2772 (USA) (Accepted 30 June 1992)
Key words: Primary afferent; Sprouting; Dorsal horn
It has been postulated that nerve growth factor (NGF) binding to the low-affinity fast-dissociating NGF receptor (p75 NGFR) on Schwann cells and growing neurites is involved with the molecular feedback necessary for continued neurite extension during development and regeneration. Since central projections of somatosensory fibers sprout into the ,~pinal cord after daily neonatal injections of antibodies to NGF (ANTI-NGF) for a one month period, it is of interest to determine if the distribution of p75 NGFR correlates with the occurrence of sprouting. Spinal cords from three groups of rats: untreated, preimmune sera treated and ANTI-NGF treated were examined on postnatal days (PD) 0, 14 and 30. The p75 NGFR distribution was determined using the monoclonal antibody 192 with standard immunohistochemical techniques and the optical density of the immunoreaction product was quantified using an Amersham image analysis system. The 192 immunoreaction product was localized to laminae l-IV, the dorsal columns, the dorsolateral funiculus, Lissauer's tract (LT) and the ventral horn on PD 0; to laminae 1-111and medial IV and LT on PD 14; and laminae !-!! and LT on PD 30. The untreated and preimmune sera treated groups show no difference in distribution. In the ANTI-NGF treated group, the 192 immunoreaction product was localized to laminae I-V and LT on PD 14 and to laminae I-!ii and medial IV and LT on PD 30. Similarly, the optical density of the ANTI-NGF treated group was significantly greater than same aged untreated and preimumne sera treated groups, but was not statistically different from these two groups examined 14 days earlier. Thus, ANTI-NGF treatment interferes with the postnatal downregulation of p75 NGFR in the dorsal horn and may provide for continued neurite growth.
INTRODUCTION Nerve growth factor (NGF) is one of a family of proteins known collectively as neurotrophins. Included in this family of related factors is brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and possibly other neurotrophins such as NT4 and N T 5 4't5'21'32'38°45'46'58. However, NGF is the most well characterized of the neurotrophins in terms of neurotrophic (neuron sun, ival) activities and neurotropic (neurite elongation) activities both in the peripheral n e r v o u s s y s t e m 13't4'42'66 and in the central nervous system 2°m. Neurons that are known to be responsive to NGF are sympathetic postganglionic neurons 13'1s'3°'39-42 a subpopulation of dorsal root ganglion neurons 13'25'30'39'41'42'54'75 and some cholinergic neurons in the C N S 36'59'7l.
The trophic and tropic effects of NGF are thought to be mediated through a process of NGF binding to a NGF receptor complex 34'75-77. The current thinking is that the NGF receptor complex is composed of two proteins: a transmembrane glycoprotein with low affinity for binding NGF, p75 NGFR 74, and a transmembrane tyrosine kinase protein, the gene product of the trk-1 proto-oncogene (p140)which, in combination with p75, is thought to confer high-affinity binding for N G F 17'33'35. It is postulated that the internalization of NGF and thus, NGF-induced biological activity, is associated with the high-affinity combined receptor complex of p75 and p140. Another speculated role of the low affinity p75 NGFR on glial cells is to provide a scaffold for neurotropic binding which provides for a continuous supply of neurotropic factors for neurite growth during development and regeneration ~9'31'76.For
Correspondence: C.E. Hulsebosch, Department of Anatomy and Neurosciences, M.B.I., University of Texas Medical Branch, 200 University Blvd., Galveston, TX 77550-2772, USA.
262 example, elongating neurites grow over the NGF-laden substrate provided by the NGF bound to the p75 NGFR on Schwann cells. The high-affinity combined complex of p75 and p140 NGFR on the neurites provides for transfer of the NGF from the p75 low affinity NGFR on the Schwann cells, to the combined NGFR complex on the neurite where either NGF is then internalized and retrogradely transported to the soma ~9 or tyrosine kinase activity is initiated ~7'67 and subsequent molecular events ensure continued elongation of the neurite. in addition to developmental and regenerative neurite growth, another paradigm in which postnatal sensory neurite growth is observed is the neonatal administration of antibodies to NGF (ANTI-NGF), which produces intraspinal sprouting and synaptogenesis of primary afferents in adult rats 22'25-27.Therefore, it was of interest to determine if the p75 NGFR changed in distribution or density which would suggest involvement in this phenomenon. To this end, we used the monoclonal antibody 192, which has been shown to recognize the rat low-affinity receptors in vitro 7, to determine the laminar distribution and relative density of the p75 NGFR in the rat spinal cord at various ages and with NGF suppression. This work was presented in preliminary form elsewhere ~'H. MATERIALS AND METHODS Pregnant Sprague-Dawl~y rats were obtained from llarlan Sprague-Dawley. All procedures involving rats were performed in compliance with USDA Animal Welfare Act and amendments, the revised Guide for the Care and Use of Laboratory Animals DHEW (NIH) and were approved by the UTMB Animal Care and Use Committee. Three groups of rats were examined: (I) rats treated with antibodies to NGF (ANTI-NGF), (2) rats treated with preimmune sera from the same rabbits used to generate the ANTI-NGF (similar injection doses and schedules to the rats treated with ANTINGF, PRP-IMM) and (3) untreated littermates (UNTR). Neonatal rats were given daily subcutaneous injections of ANTI-NGF (3 p.l of undiluted rabbit serum per gram b.wt.) or similar doses of preimmune sera near the dorsal fat pad for a period of ! month beginning on postnatal day (PD) 0 (day of birth). The ANTI-NGF was raised in rabbits which had received subcutaneous inoculations of purified /3-NGF (10-20 ng/ml for I biological unit/ml) isolated from the 7S NGF purified from mouse submaxillary glands according to the methods described in Varon ct al. 7°. The classic bioassays with extirpated embryonic chick sensory ganglia11'12'4°'41 and morphological differentiation of cultured human neuroblastoma cells ~''m were used and compared to NGF preparations whose specific biological activity was 10-20 ng/ml (I biological unit/ml) as described elsewhere'~k By both assays, the biological activity of the NGF and the ANTI-NGF used in this study was found to remain stable during the course of the experiments. An antiserum dilution of 1:1,500 blocked the effects of exogenously applied NGF at concentrations containing 1 biological unit/ml 4:' and by both assays the blocking activity of the ANTI-NGF was stable during the course of the experiments. The ANTI-NGF injections were doses of undiluted rabbit antiserum prepared against the purified/3-NGF subunit. To determine in vivo injection levels, it was determined empirically that increases in antibody concentration in the rabbit sera by ultrafiltra-
tion 2s greatly affected mortality. Consequently, the use of undiluted antisera in these experiments was deemed sufficient to block endogenous levels of NGF, which is known to be in the nanogram range. As an internal assay for the ANTI-NGF activity in vivo, wet weights were taken of the superior cervical ganglia for each of the three groups in the study. The Student's t-test was used to determine statistical differences with a level of confidence set at P ~
