322
Brain Research, 549 (1991) 322-326 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONL~ 0006899391246591
BRES 24659
Is nerve growth factor required for the survival of retinal ganglion ceils during development? Z. Henderson Department of Physiology, University of Leeds, Leeds ( U. K. )
(Accepted 5 February 1991) Key words: Nerve growth factor; Retina; Cell death
Staining for nerve growth factor receptor was observed in the ferret's retinal ganglion cell layer, optic nerve and tract, and in the lateral geniculate nucleus and superficial layers of the superior coUiculus in the prenatal period, but had disappeared by birth. Thus the incidence of this transient staining does not correspond with the ganglion cell death that is known to occur in the ferret retina during the first postnatal week. During the development of most regions of the vertebrate central nervous system there is an overproduction of neurons followed by a phase of naturally occuring cell death when a high proportion of the neurons are eliminated. It has been shown that the axons of the neurons reach their target field before the onset of cell death, and so one of the postulated mechanisms for cell death is that the axonal terminals compete with each other for trophic factors that are in short supply in the target area 3. The ganglion cell layer of the mammalian retina has provided a useful model for the study of the mechanisms of neuronal cell death and it has been shown that cell generation gives rise to retinas that initially have 1.5 to 4 times the adult number of ganglion cells and that the surplus is eliminated by spontaneous cell death 6. The search is on, therefore, for the identities of the trophic factors that influence the survival of mammalian retinal ganglion cells and one candidate is nerve growth factor (NGF), the established trophic factor for peripheral neurons derived from the neural crest 1°. Thus there is evidence in the rodent that N G F supports the survival of adult retinal ganglion cells after section of the optic nerve 2, and that high levels of NGF and NGF receptor immunoreactivity are found in the retinogeniculate system 1'4'5'u at about the time of maximum cell death in the retinal ganglion cell layer 8. The presence of the N G F receptor is a prerequisite for a tissue to be sensitive to NGF 1° and the question is whether transient immunoreactivity for the N G F receptor is found in the visual systems of other species during development, which would strengthen the case for NGF being involved in
target-controlled cell death of mammalian retinal ganglion cells. I sought to answer this question in the ferret, a carnivore species for which there is data available concerning the course of cell death in the retinal ganglion cell layer 6. The study was made on tissue available from brains of prenatal ferrets from timed pregnancies (gestation period 42 days) at E (embryonic day) 28, E31, E35 and E39, and brains of postnatal ferrets at ages P (postnatal day) 1, P4, P7, P l l , P16, P21, P29, P37, P46, P56, P67, and adulthood. The brains had been fixed by perfusion with 2% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (pH 7.4), and coronal sections were cut at 50/~m on a freezing microtome. The sections were stained for N G F receptor using an immunocytochemical method, and adjacent sections were stained for Nissl substance. During the incubations for the immunocytochemistry the sections were kept in continuous agitation on a shaker and the incubation steps were carried out at room temperature unless mentioned otherwise. In between the antibody steps the sections were rinsed several times with wash solution consisting of 0.1% Triton X-100 in phosphate buffered saline (pH 7.4), also the diluent for the antibody solutions. The antibody procedure was carried out using the avidin-biotinhorseradish peroxidase kit supplied by Vector Labs. The sections first went into 2% horse serum for 1 h, and were then incubated in 1/600 anti-NGF receptor (Amersham International) overnight at 4 °C. The primary antibody was omitted in control incubations. After incubation in primary antibody the sections went into 1/50 biotinylated
Correspondence: Z. Henderson, Department of Physiology, The Worsley Medical and Dental Building, The University, Leeds LS2 9NQ, U.K.
323
Fig. 1. Presence of NGF receptor immunoreactivity within the optic system of the E28 ferret. Staining for NGF receptor is visible in the retinal ganglion cell layer (arrowed), within soma in the retinal ganglion cell layer (framed) of an eye (E) that has been cut tangentially, and in the optic nerve (ON) and optic chiasm (OC). Staining for NGF receptor is found also in other structures not related to the visual system. Calibration bar = 0.2 mm for (A), 0.5 mm for (B), 100/~m for (C) and 50/~m for (D).
4~
325 ,6Fig. 2. Presence of NGR receptor immunoreactivity within the optic system of the E28 ferret; the section in (B) has been stained for Nissl substance. Staining for NGF receptor is visible in fibrous structures (arrowed) in the lateral geniculate nucleus (LG) and superficial layers of the superior coUieulus (SC). Staining for NGF-receptor is found also in other structures not related to the visual system. Calibration bar = 0.5 mm for (A, B and D) and 0.2 mm for (C).
anti-mouse IgG for 1 h, and then into a 1/25 dilution of the avidin-biotin-horseradish peroxidase complex for 1 h. For added sensitivity the sections were incubated in 1/100 mouse peroxidase anti-peroxidase, which should in theory bind to vacant sites on the secondary antibody thus increasing the number of horseradish peroxidase units associated with the antibody-antigen complexes. In practice this step was found to increase the intensity of the specific staining slightly without compromising the background. The sections were reacted for horseradish peroxidase activity for 30 min in 0.1% diaminobenzidine and 0.004% H 2 0 2 in 0.1 M phosphate buffer (pH 7.4), dehydrated and mounted under coverslips in DPX. Transient staining for N G F receptor is observed in many parts of the prenatal ferret brain, as is evident in the prenatal rodent brain 4,n. In the visual system of the developing ferret brain, NGF receptor immunoreactivity is found within cell bodies in the retinal ganglion cell layer, in the optic nerve, optic chiasm and optic tract (Fig. 1), and in fibrous structures in the lateral geniculate nucleus and superficial layers of the superior colliculus (Fig. 2). This staining for NGF receptor is visible at E28, becomes faint by E39 and has disappeared at P2. Thus in the ferret, the incidence of transient staining for N G F receptor in the visual system, which is prevalent prenatally, does not coincide with the major incidence of cell death in the retinal ganglion cell layer which has been shown in a previous study to occur postnatally 6. The ganglion cell numbers in the developing ferret retina peak at twice their normal value at P3 and fall to adult levels by P6, and this is accompanied by an adundance of degenerating profiles in the ganglion cell layer in the immediate postnatal period. In the developing rat visual system, the retinal ganglion cell layer and the optic nerve are positive for N G F receptor from El6, the staining decreases at P6 and has gone by P10 (ref. 11). Unlike in the ferret, this staining for N G F receptor coincides quite well with the event of cell death in the rat retinal ganglion
cell layer. At birth in the rat there are twice as many retinal ganglion cells as there are in the adult and the excess ganglion cells are lost over the first ten postnatal days 8. The differences between the rat and ferret may be explained, however, by the fact that brain development progresses much more rapidly in rodents than in carnivores, and therefore unrelated developmental events are more likely to overlap in the rodent. The evidence in the ferret suggests that N G F is unlikely to be involved in target-controlled cell death in the retinal ganglion cell layer. This does not rule out other functions for N G F in the developing visual system which may not strictly be within the confines of the role of N G F as a true neurotrophic factor. For example, axotomy of adult rat sciatic nerve induces Schwann cells distal to the lesion to express NGF receptor during regeneration of the nerve 9, and intense staining for N G F receptor is found transiently in the nerves of cranial nerve nuclei 4"11. From this evidence it has been proposed that Schwann or glial cell NGF receptors serve to concentrate N G F molecules upon their surface, over which regeneration or growing axons travel 9. Indeed, in the study on the ferret it was not possible at light microscopy level to determine whether the transient staining for N G F receptor in the optic nerve and tract is localised in axons or glia. Alternatively, N G F may influence the survival and maintenance of only a small proportion of retinal ganglion cells, since it has been found that some optic related structures continue to express NGF-receptor into adulthood, e.g. the suprachiasmatic nucleus 12. A more likely candidate for a targetderived trophic factor for the majority of the retinal ganglion cell population is the less well characterised 'brain-derived neurotrophic factor', which has recently been shown to support the survival of rat perinatal retinal ganglion cells in culture 7.
1 Ayer-Lelievre, L.A., Ebendal, T., Olson, S. and Sieger, A., Localization of nerve growth factor-like immunoreactivity in rat nervous tissue, Med. Biol., 61 (1983) 293-304. 2 Carmignoto, G., Maffei, L., Candeo, P., Canella, R. and Comelli, C., Effect of NGF on survival of rat retinal ganglion cells following optic nerve section, J. Neurosci., 9 (1989) 1263-1273. 3 Cowan, W.M., Fawcett, J.W., O'Leary, D.D.M. and Stanfield, B.B., Regressive events in neurogenesis, Science, 225 (1984) 1258-1265.
4 Eckenstein, E, Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord, Brain Research, 446 (1988) 149-154. 5 Finn, P.J., Ferguson, I.A., Wilson, P.A., Vahaviolos, J. and Rush, R.A., Immunohistochemical evidence for the distribution of nerve growth factor in the embryonic mouse, J. Neurocytol., 16 (1987) 639-647. 6 Henderson, Z., Finlay, B.L. and Wikler, K.C., Development of ganglion cell topography in ferret retina, J. Neurosci., 8 (1988) 1194-1205.
This research was supported by MRC Grant No. G8621410N. I am grateful to Ann Stamper and David Johanson for their technical assistance.
326 7 Johnson, J.E., Barde, ¥.A., Schwab, M. and Thoenen, H., Brain-derived neurotrophic factor supports the survival of cultured rat retinal ganglion cells, J. Neurosci., 6 (1986) 3031-3038. 8 Perry, V.H., Henderson, Z. and Linden, R., Postnatal changes in retinal ganglion cell and optic axon populations in the pigmented rat, J. Comp. Neurol., 219 (1983) 356-368. 9 Taniuchi, M., Clark, H.B. and Johnson, E.M., Induction of nerve growth factor receptor in Schwann cells after axotomy,
Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4094-4098. 10 Thoenen, H. and Barde, Y.A., Physiology of nerve growth factor, Physiol. Rev., 60 (1980) 1284-1335. 11 Yan, Q. and Johnson, E.M., An immunohistochemical study ol the nerve growth factor receptor in developing rats, J. Neurosci., 8 (1988) 3481-3498. 12 Yan, Q. and Johnson, E.M., Immunohistochemical localisation and biochemical characterization of NGF receptor in adult rat brain, J. Comp. NeuroL, 290 (1989) 585-598.
Brain Research, 549 (1991) 322-326 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONL~ 0006899391246591
BRES 24659
Is nerve growth factor required for the survival of retinal ganglion ceils during development? Z. Henderson Department of Physiology, University of Leeds, Leeds ( U. K. )
(Accepted 5 February 1991) Key words: Nerve growth factor; Retina; Cell death
Staining for nerve growth factor receptor was observed in the ferret's retinal ganglion cell layer, optic nerve and tract, and in the lateral geniculate nucleus and superficial layers of the superior coUiculus in the prenatal period, but had disappeared by birth. Thus the incidence of this transient staining does not correspond with the ganglion cell death that is known to occur in the ferret retina during the first postnatal week. During the development of most regions of the vertebrate central nervous system there is an overproduction of neurons followed by a phase of naturally occuring cell death when a high proportion of the neurons are eliminated. It has been shown that the axons of the neurons reach their target field before the onset of cell death, and so one of the postulated mechanisms for cell death is that the axonal terminals compete with each other for trophic factors that are in short supply in the target area 3. The ganglion cell layer of the mammalian retina has provided a useful model for the study of the mechanisms of neuronal cell death and it has been shown that cell generation gives rise to retinas that initially have 1.5 to 4 times the adult number of ganglion cells and that the surplus is eliminated by spontaneous cell death 6. The search is on, therefore, for the identities of the trophic factors that influence the survival of mammalian retinal ganglion cells and one candidate is nerve growth factor (NGF), the established trophic factor for peripheral neurons derived from the neural crest 1°. Thus there is evidence in the rodent that N G F supports the survival of adult retinal ganglion cells after section of the optic nerve 2, and that high levels of NGF and NGF receptor immunoreactivity are found in the retinogeniculate system 1'4'5'u at about the time of maximum cell death in the retinal ganglion cell layer 8. The presence of the N G F receptor is a prerequisite for a tissue to be sensitive to NGF 1° and the question is whether transient immunoreactivity for the N G F receptor is found in the visual systems of other species during development, which would strengthen the case for NGF being involved in
target-controlled cell death of mammalian retinal ganglion cells. I sought to answer this question in the ferret, a carnivore species for which there is data available concerning the course of cell death in the retinal ganglion cell layer 6. The study was made on tissue available from brains of prenatal ferrets from timed pregnancies (gestation period 42 days) at E (embryonic day) 28, E31, E35 and E39, and brains of postnatal ferrets at ages P (postnatal day) 1, P4, P7, P l l , P16, P21, P29, P37, P46, P56, P67, and adulthood. The brains had been fixed by perfusion with 2% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (pH 7.4), and coronal sections were cut at 50/~m on a freezing microtome. The sections were stained for N G F receptor using an immunocytochemical method, and adjacent sections were stained for Nissl substance. During the incubations for the immunocytochemistry the sections were kept in continuous agitation on a shaker and the incubation steps were carried out at room temperature unless mentioned otherwise. In between the antibody steps the sections were rinsed several times with wash solution consisting of 0.1% Triton X-100 in phosphate buffered saline (pH 7.4), also the diluent for the antibody solutions. The antibody procedure was carried out using the avidin-biotinhorseradish peroxidase kit supplied by Vector Labs. The sections first went into 2% horse serum for 1 h, and were then incubated in 1/600 anti-NGF receptor (Amersham International) overnight at 4 °C. The primary antibody was omitted in control incubations. After incubation in primary antibody the sections went into 1/50 biotinylated
Correspondence: Z. Henderson, Department of Physiology, The Worsley Medical and Dental Building, The University, Leeds LS2 9NQ, U.K.
323
Fig. 1. Presence of NGF receptor immunoreactivity within the optic system of the E28 ferret. Staining for NGF receptor is visible in the retinal ganglion cell layer (arrowed), within soma in the retinal ganglion cell layer (framed) of an eye (E) that has been cut tangentially, and in the optic nerve (ON) and optic chiasm (OC). Staining for NGF receptor is found also in other structures not related to the visual system. Calibration bar = 0.2 mm for (A), 0.5 mm for (B), 100/~m for (C) and 50/~m for (D).
4~
325 ,6Fig. 2. Presence of NGR receptor immunoreactivity within the optic system of the E28 ferret; the section in (B) has been stained for Nissl substance. Staining for NGF receptor is visible in fibrous structures (arrowed) in the lateral geniculate nucleus (LG) and superficial layers of the superior coUieulus (SC). Staining for NGF-receptor is found also in other structures not related to the visual system. Calibration bar = 0.5 mm for (A, B and D) and 0.2 mm for (C).
anti-mouse IgG for 1 h, and then into a 1/25 dilution of the avidin-biotin-horseradish peroxidase complex for 1 h. For added sensitivity the sections were incubated in 1/100 mouse peroxidase anti-peroxidase, which should in theory bind to vacant sites on the secondary antibody thus increasing the number of horseradish peroxidase units associated with the antibody-antigen complexes. In practice this step was found to increase the intensity of the specific staining slightly without compromising the background. The sections were reacted for horseradish peroxidase activity for 30 min in 0.1% diaminobenzidine and 0.004% H 2 0 2 in 0.1 M phosphate buffer (pH 7.4), dehydrated and mounted under coverslips in DPX. Transient staining for N G F receptor is observed in many parts of the prenatal ferret brain, as is evident in the prenatal rodent brain 4,n. In the visual system of the developing ferret brain, NGF receptor immunoreactivity is found within cell bodies in the retinal ganglion cell layer, in the optic nerve, optic chiasm and optic tract (Fig. 1), and in fibrous structures in the lateral geniculate nucleus and superficial layers of the superior colliculus (Fig. 2). This staining for NGF receptor is visible at E28, becomes faint by E39 and has disappeared at P2. Thus in the ferret, the incidence of transient staining for N G F receptor in the visual system, which is prevalent prenatally, does not coincide with the major incidence of cell death in the retinal ganglion cell layer which has been shown in a previous study to occur postnatally 6. The ganglion cell numbers in the developing ferret retina peak at twice their normal value at P3 and fall to adult levels by P6, and this is accompanied by an adundance of degenerating profiles in the ganglion cell layer in the immediate postnatal period. In the developing rat visual system, the retinal ganglion cell layer and the optic nerve are positive for N G F receptor from El6, the staining decreases at P6 and has gone by P10 (ref. 11). Unlike in the ferret, this staining for N G F receptor coincides quite well with the event of cell death in the rat retinal ganglion
cell layer. At birth in the rat there are twice as many retinal ganglion cells as there are in the adult and the excess ganglion cells are lost over the first ten postnatal days 8. The differences between the rat and ferret may be explained, however, by the fact that brain development progresses much more rapidly in rodents than in carnivores, and therefore unrelated developmental events are more likely to overlap in the rodent. The evidence in the ferret suggests that N G F is unlikely to be involved in target-controlled cell death in the retinal ganglion cell layer. This does not rule out other functions for N G F in the developing visual system which may not strictly be within the confines of the role of N G F as a true neurotrophic factor. For example, axotomy of adult rat sciatic nerve induces Schwann cells distal to the lesion to express NGF receptor during regeneration of the nerve 9, and intense staining for N G F receptor is found transiently in the nerves of cranial nerve nuclei 4"11. From this evidence it has been proposed that Schwann or glial cell NGF receptors serve to concentrate N G F molecules upon their surface, over which regeneration or growing axons travel 9. Indeed, in the study on the ferret it was not possible at light microscopy level to determine whether the transient staining for N G F receptor in the optic nerve and tract is localised in axons or glia. Alternatively, N G F may influence the survival and maintenance of only a small proportion of retinal ganglion cells, since it has been found that some optic related structures continue to express NGF-receptor into adulthood, e.g. the suprachiasmatic nucleus 12. A more likely candidate for a targetderived trophic factor for the majority of the retinal ganglion cell population is the less well characterised 'brain-derived neurotrophic factor', which has recently been shown to support the survival of rat perinatal retinal ganglion cells in culture 7.
1 Ayer-Lelievre, L.A., Ebendal, T., Olson, S. and Sieger, A., Localization of nerve growth factor-like immunoreactivity in rat nervous tissue, Med. Biol., 61 (1983) 293-304. 2 Carmignoto, G., Maffei, L., Candeo, P., Canella, R. and Comelli, C., Effect of NGF on survival of rat retinal ganglion cells following optic nerve section, J. Neurosci., 9 (1989) 1263-1273. 3 Cowan, W.M., Fawcett, J.W., O'Leary, D.D.M. and Stanfield, B.B., Regressive events in neurogenesis, Science, 225 (1984) 1258-1265.
4 Eckenstein, E, Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord, Brain Research, 446 (1988) 149-154. 5 Finn, P.J., Ferguson, I.A., Wilson, P.A., Vahaviolos, J. and Rush, R.A., Immunohistochemical evidence for the distribution of nerve growth factor in the embryonic mouse, J. Neurocytol., 16 (1987) 639-647. 6 Henderson, Z., Finlay, B.L. and Wikler, K.C., Development of ganglion cell topography in ferret retina, J. Neurosci., 8 (1988) 1194-1205.
This research was supported by MRC Grant No. G8621410N. I am grateful to Ann Stamper and David Johanson for their technical assistance.
326 7 Johnson, J.E., Barde, ¥.A., Schwab, M. and Thoenen, H., Brain-derived neurotrophic factor supports the survival of cultured rat retinal ganglion cells, J. Neurosci., 6 (1986) 3031-3038. 8 Perry, V.H., Henderson, Z. and Linden, R., Postnatal changes in retinal ganglion cell and optic axon populations in the pigmented rat, J. Comp. Neurol., 219 (1983) 356-368. 9 Taniuchi, M., Clark, H.B. and Johnson, E.M., Induction of nerve growth factor receptor in Schwann cells after axotomy,
Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4094-4098. 10 Thoenen, H. and Barde, Y.A., Physiology of nerve growth factor, Physiol. Rev., 60 (1980) 1284-1335. 11 Yan, Q. and Johnson, E.M., An immunohistochemical study ol the nerve growth factor receptor in developing rats, J. Neurosci., 8 (1988) 3481-3498. 12 Yan, Q. and Johnson, E.M., Immunohistochemical localisation and biochemical characterization of NGF receptor in adult rat brain, J. Comp. NeuroL, 290 (1989) 585-598.
