Molecular Brain Research, 9 (1991) 157-160 Elsevier
157
BRESM 80069
Effects of nerve crush and transection on mRNA levels for nerve growth factor receptor in the rat facial motoneurons Takanori Saika ~, Emiko Senba 2, Koichi Noguchi 2, Makoto Sato 2, Shigetaka Yoshida 3, Takeshi Kubo ~, Toru Matsunaga 1 and Masaya Tohyama 2 ~Department of Otolaryngology, 2Department of Anatomy (II) and 3Department of Anesthesiology, Osaka University Medical School, Osaka (Japan) (Accepted 24 July 1990) Key words: Nerve growth factor receptor; Motoneuron; Facial nerve; Axotomy; In situ hybridization
We found that the level of nerve growth factor receptor (NGF-R) mRNA in facial motoneurons was increased after both facial nerve crushing and transection by means of in situ hybridization histochemistry. The increased level of NGF-R mRNA was maintained for at least 8 weeks after facial nerve transection, while facial nerve crushing caused only a transient increase. Thus, expression of NGF-R mRNA paralleled the axonal regeneration process. In addition, the increase of NGF-R mRNA with crushing was more pronounced than with transection from the 3rd to the 14th day after the insult. Nerve growth factor (NGF) is a t r o p h i c agent which promotes the survival and cellular metabolism of both sympathetic neurons and neural crest-derived sensory neurons. N G F also exerts similar effects on some populations of cholinergic neurons in the CNS. It is generally accepted though that N G F does not have any neurotrophic effect on motoneurons 4,11,1s, and that adult motoneurons express neither NGF receptor (NGF-R) m R N A nor NGF-R protein 9"14,2° under normal conditions. However, it has been reported that both NGF-R m R N A and N G F - R protein are expressed during development by spinal and brainstem motoneurons 2,3,6,21. Moreover, in the adult rat it has been shown that NGF-R m R N A and N G F - R protein are expressed by motoneurons under some experimental conditions. For example, colchicine treatment results in the visualization of NGFR-like immunoreactivity in brainstem motoneurons 12, and NGF-R m R N A is expressed by spinal motoneurons after the production of sciatic nerve crush lesions 3. A dramatic increase of N G F and NGF-R in the distal nerve segment after axotomy of the sciatic nerve has also been demonstrated 7"s'15'16. These findings suggest that NGF may play an important role in the regenerative process of motoneurons. To examine this possibility, we compared the changes of the synthesis of NGF-R in facial motoneurons subjected to crushing and transection, using in situ hybridization histochemistry. Thirty-five adult male Wistar rats weighing about 150
g were used. Under pentobarbital anesthesia (50 mg/kg, i.p.), the left facial nerve was exposed. At the point just distal to the posterior auricular branch, which diverges from the facial nerve as it leaves the stylomastoid foramen, the main trunk as either crushed for 30 s with thin forceps or was transected with a pair of scissors. After transection, about 5 mm of the distal nerve segment was removed. Following a postoperative interval of 1, 3, 7, 14, 21, 28, 42, or 56 days, the animals were deeply anesthetized with pentobarbital and killed. The brains were immediately excised and frozen with crushed dry ice. Then 20-/~m frozen sections were cut on a cryostat, mounted on gelatin-coated slides, and subjected to in situ hybridization histochemistry. These sections were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 5 min, followed by incubation in l x Denhardt's solution for 1 h and dehydration in a series of graded ethanols. An oligonucleotide probe complementary to bases 201-242 of the rat NGF-R eDNA 13 was synthesized with an Applied Biosystems 381A D N A synthesizer. The probes were labelled with [a -35 S]dATP using terminal deoxynucleotidyl transferase, giving a specific activity of 1.0-1.5 x 109 dpm//~g. Hybridization was performed at 42 °C with 50% formamide, 4× SSC, l x Denhardt's solution, 10% dextran sulfate, 10 ktg/ml of tRNA, 1% sarcosyl, 0.05 M dithiothreitol (DTI'), and 1.5 x 106 dpm/ml of the probe. Sections were then washed 4 times (for 15 min each time) at 55 °C in 1 x SSC and dehydrated with graded ethanols.
Correspondence: T. Saika, Department of Anatomy (II), Osaka University Medical School, 4-3-57, Nakanoshima, Kitaku, Osaka 530, Japan. 0169-328X/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
158 Then the slides were dipped in Ilford K-5 diluted 1:1 with distilled water. After exposure for 1 month, the slides were developed, fixed, and counterstained with 1% thionin. For the quantitative analysis of labelled neurons, two representative sections were chosen from each animal. The number of labelled neurons was counted under a dark-field microscope. Neurons labelled by twice as many grains as background level were considered to be positive. The total number of motoneurons was determined from bright-field observation of thionin-stained sections. The neurons in the medial subnucleus were excluded from counting, because they innervate the posterior auricular branch which was left intact. No NGF-R m R N A was detected in the contralateral unoperated facial motoneurons at any time in rats subjected to either procedure. On the operated side, a few neurons with NGF-R m R N A could already be identified in the facial motor nucleus 1 day after both crushing and transection of the facial nerve. Fig. 1 shows the time-course of postoperative changes of NGF-R m R N A expression by facial motoneurons after crushing of the nerve. As shown in this figure, the number of
labelled cells and the signal intensity increased after the insult and reached its maximum from the 3rd (Fig. 1-3D) to the 7th day (Fig. 1-1W) after operation. From that time on, the decrease was rapid and no specific labelling was seen by 3 weeks after the procedure (Fig. 1-2W, Fig. 1-3W). The changes of NGF-R m R N A levels in the facial motoneurons after nerve transection differed from those seen after nerve crushing. On day 1, the number of labelled cells and the intensity of labelling increased in the facial motoneurons after transection, as it had after nerve crushing. The m R N A level reached its peak from the 3rd (Fig. 2-3D) to the 7th day after the procedure, although the signal intensity was weaker than after nerve crushing and the number of labelled neurons was only one-third of that after nerve crushing (Fig. 3). A conspicuous difference was noted in NGF-R m R N A levels in the facial motor nucleus between the rats with nerve transection and nerve crushing after day 7. In the case of nerve transection, the number of labelled neurons and the signal intensity reached a plateau and then showed no marked changes. In contrast, after nerve crushing, the m R N A level decreased rapidly and expres-
Fig. 1. Dark-field photomicrographs showing facial motoneurons on the operated side expressing NGF-R mRNA hybridization signals 3 days (3D), 1 week (lW), 2 weeks (2W), and 3 weeks (3W) after facial nerve crushing. Bar = I ram.
159
Fig. 2. Dark-field photomicrographs showing facial motoneurons on the operated side expressing NGF-R mRNA hybridization signals 1 day (1D), 3 days (3D), 6 weeks (6W), and 8 weeks (8W) after facial nerve transection. Bar = 1 mm.
sion had totally disappeared by the 3rd week after injury. After nerve crush injury, the function of the facial nerves seemed to be restored within 2 weeks, because recovery of movement of the whiskers occured at this
a
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2oi t 1
3
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21
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Fig. 3. Time-course of changes in the percentage of neurons expressing NGF-R mRNA following facial nerve crushing ((D) and transection (0). The percentages of positive neurons are expressed as the mean + S.D. of 3 rats.
time, whereas no functional recovery was observed following nerve transection. We demonstrated that facial nerve crushing caused a transient increase of NGF-R m R N A in the facial motor nucleus, while facial nerve transection produced a long-term increase in this nucleus. Thus, expression of NGF-R m R N A appears to closely parallel the regenerative process of motoneuron axons. Accordingly, the present findings provide a strong basis at the molecular level for the assumption that N G F - R has a trophic or axonal guidance function for regenerating motoneurons as has already been suggested by several studies 1'3'5'1°A7. It is known that axotomy of the adult rat sciatic nerve induces expression of both N G F and N G F - R in Schwann cells located distal to the injury, and this induction appears to be inversely regulated by axonal contact 7'8'15'16. A persistent increase of N G F m R N A has also been found after transection of the facial nerve 7. The present study demonstrated that N G F - R synthesis in the injured facial motoneurons was in line with the timecourse of nerve regeneration. This suggests that the persistent increase of N G F - R synthesis seen in axotomized cell bodies was due to the increased levels of N G F internalized by the peripheral processes and retrogradely
160 transported to the cell bodies. O n e of the most conspicuous findings in the present study was that the increase of N G F - R m R N A was more p r o n o u n c e d after crush injury than after nerve transec-
after nerve transection than crush injury. (2) In the case of crush injury, the continuity of the e n d o n e u r i u m is preserved, while transection allows the free flow of axoptasm from the cut end, Therefore, after a crush
tion. A t least 3 explanations for this can be postulated: (1) Crush injury may stimulate Schwann cells to produce
injury much more of the N G F produced by Schwann cells in the distal segment or by target muscles may have been
more N G F than does nerve transection. However, this hypothesis seems to be unlikely, because synthesis of both N G F s and N G F - R a'15 in Schwann cells of the distal
transection. (3) Transection may have caused a more marked perikaryal reaction than did crush injury, which
nerve segment has been shown to be more prominent
resulted in the production of less N G F - R .
1 Chen, Y.S., Wang-Bennett, L.T. and Coker, N.J., Facial nerve regeneration in the silicone chamber: the influence of nerve growth factor, Exp. Neurol., 103 (1989) 52-60. 2 Eckenstein, E, Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord, Brain Res., 446 (1988) 149-154. 3 Ernfors, P., Henschen, A., Olson, L. and Persson, H., Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons, Neuron, 2 (1989) 1605-1613. 4 Gunderson, R.W. and Park, K.H.C., The effects of conditioned media on spinal neuritis: substrate-associated changes in neurite direction and adherence, Dev. Biol., 104 (1984) 18-27. 5 Heaton, M.B., Influence of nerve growth factor on chick trigeminal motor nucleus explants, J. Comp. Neurol., 265 (1987) 362-366. 6 Heuer, J.G., Fatemie-Nainie, S., Wheeler, E.E and Bothwell, M., Structure and developmental expression of the chicken NGF receptor, Dev. Biol., 137 (1990) 287-304. 7 Heumattn, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection, J. Cell Biol., 104 (1987) 1623-1631. 8 Heumann, R., Lindhotm, D., Bandtlow, C., Meyer, M., Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H., Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regeneration: role of macrophages, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8735-8739. 9 Koh, S., Oyler, G.A. and Higgins, G.A., Localization of nerve growth factor receptor messenger RNA and protein in the adult rat brain, Exp. Neurol., 106 (1989) 209-221. 10 Levi-Montalcini, R. and Aloe, L., Differentiating effects of murine nerve growth factor in the peripheral and central nervous systems of Xenopus laevis tadpoles, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 7111-7115.
11 Miyata, Y., Kashihara, Y., Homma, S. and Kuno, M., Effects of nerve growth factor on the survival and synaptic function of Ia sensory neurons axotomized in neonatal rats, J. Neurosci., 6 (1986) 2012-2018. 12 Pioro, E.P. and Cuello, A.C., Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system - II. Brainstem, cerebellum and spinal cord, Neuroscience, 34 (1990) 89-110. 13 Radeke, M.J., Misko, T.P., Hsu, C., Herzenberg, L.A. and Shooter, E.M., Gene transfer and molecular cloning of the rat nerve growth factor receptor, Nature, 325 (1987) 593-597. 14 Richardson, P.M., Verge-Issa, V.M.K. and Riopelle, R.J., Distribution of neuronal receptors for nerve growth factor in the rat, J. Neurosci., 6 (1986) 2312-2321. 15 Taniuchi, M., Clark, H.B. and Johnson, E.M. Jr., Induction of nerve growth factor receptor in Schwann cells after axotomy, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4094--4098. 16 Taniuehi, M., Clark, H.B., Schweitzer, J,B. and Johnson, E.M. Jr., Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastructural location, supression by axonal contact, and binding properties, J. Neurosci., 8 (1988) 664-681. 17 Wayne, D.B. and Heaton, M.B., The response of cultured trigeminal and spinal cord motoneurons to nerve growth factor, Dev. Biol., 138 (1990) 473-483. 18 Yan, Q., Snider, W.D., Pinzone, J.J. and Johnson, E.M. Jr., Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats: assessment of potential neurotrophic effects, Neuron, 1 (1988) 335-343. 19 Yan, Q. and Johnson, E.M. Jr., An immunohistochemical study of nerve growth factor receptor in developing rats, J. Neurosci., 8 (1988) 3481-3498. 20 Yip, H.K. and Johnson, E.M. Jr., Nerve growth factor receptors in rat spinal cord: an autoradiographic and immunohistochemical study, Neuroscience, 22 (1987) 267-279.
retrogradely transported to the cell body than after
157
BRESM 80069
Effects of nerve crush and transection on mRNA levels for nerve growth factor receptor in the rat facial motoneurons Takanori Saika ~, Emiko Senba 2, Koichi Noguchi 2, Makoto Sato 2, Shigetaka Yoshida 3, Takeshi Kubo ~, Toru Matsunaga 1 and Masaya Tohyama 2 ~Department of Otolaryngology, 2Department of Anatomy (II) and 3Department of Anesthesiology, Osaka University Medical School, Osaka (Japan) (Accepted 24 July 1990) Key words: Nerve growth factor receptor; Motoneuron; Facial nerve; Axotomy; In situ hybridization
We found that the level of nerve growth factor receptor (NGF-R) mRNA in facial motoneurons was increased after both facial nerve crushing and transection by means of in situ hybridization histochemistry. The increased level of NGF-R mRNA was maintained for at least 8 weeks after facial nerve transection, while facial nerve crushing caused only a transient increase. Thus, expression of NGF-R mRNA paralleled the axonal regeneration process. In addition, the increase of NGF-R mRNA with crushing was more pronounced than with transection from the 3rd to the 14th day after the insult. Nerve growth factor (NGF) is a t r o p h i c agent which promotes the survival and cellular metabolism of both sympathetic neurons and neural crest-derived sensory neurons. N G F also exerts similar effects on some populations of cholinergic neurons in the CNS. It is generally accepted though that N G F does not have any neurotrophic effect on motoneurons 4,11,1s, and that adult motoneurons express neither NGF receptor (NGF-R) m R N A nor NGF-R protein 9"14,2° under normal conditions. However, it has been reported that both NGF-R m R N A and N G F - R protein are expressed during development by spinal and brainstem motoneurons 2,3,6,21. Moreover, in the adult rat it has been shown that NGF-R m R N A and N G F - R protein are expressed by motoneurons under some experimental conditions. For example, colchicine treatment results in the visualization of NGFR-like immunoreactivity in brainstem motoneurons 12, and NGF-R m R N A is expressed by spinal motoneurons after the production of sciatic nerve crush lesions 3. A dramatic increase of N G F and NGF-R in the distal nerve segment after axotomy of the sciatic nerve has also been demonstrated 7"s'15'16. These findings suggest that NGF may play an important role in the regenerative process of motoneurons. To examine this possibility, we compared the changes of the synthesis of NGF-R in facial motoneurons subjected to crushing and transection, using in situ hybridization histochemistry. Thirty-five adult male Wistar rats weighing about 150
g were used. Under pentobarbital anesthesia (50 mg/kg, i.p.), the left facial nerve was exposed. At the point just distal to the posterior auricular branch, which diverges from the facial nerve as it leaves the stylomastoid foramen, the main trunk as either crushed for 30 s with thin forceps or was transected with a pair of scissors. After transection, about 5 mm of the distal nerve segment was removed. Following a postoperative interval of 1, 3, 7, 14, 21, 28, 42, or 56 days, the animals were deeply anesthetized with pentobarbital and killed. The brains were immediately excised and frozen with crushed dry ice. Then 20-/~m frozen sections were cut on a cryostat, mounted on gelatin-coated slides, and subjected to in situ hybridization histochemistry. These sections were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 5 min, followed by incubation in l x Denhardt's solution for 1 h and dehydration in a series of graded ethanols. An oligonucleotide probe complementary to bases 201-242 of the rat NGF-R eDNA 13 was synthesized with an Applied Biosystems 381A D N A synthesizer. The probes were labelled with [a -35 S]dATP using terminal deoxynucleotidyl transferase, giving a specific activity of 1.0-1.5 x 109 dpm//~g. Hybridization was performed at 42 °C with 50% formamide, 4× SSC, l x Denhardt's solution, 10% dextran sulfate, 10 ktg/ml of tRNA, 1% sarcosyl, 0.05 M dithiothreitol (DTI'), and 1.5 x 106 dpm/ml of the probe. Sections were then washed 4 times (for 15 min each time) at 55 °C in 1 x SSC and dehydrated with graded ethanols.
Correspondence: T. Saika, Department of Anatomy (II), Osaka University Medical School, 4-3-57, Nakanoshima, Kitaku, Osaka 530, Japan. 0169-328X/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
158 Then the slides were dipped in Ilford K-5 diluted 1:1 with distilled water. After exposure for 1 month, the slides were developed, fixed, and counterstained with 1% thionin. For the quantitative analysis of labelled neurons, two representative sections were chosen from each animal. The number of labelled neurons was counted under a dark-field microscope. Neurons labelled by twice as many grains as background level were considered to be positive. The total number of motoneurons was determined from bright-field observation of thionin-stained sections. The neurons in the medial subnucleus were excluded from counting, because they innervate the posterior auricular branch which was left intact. No NGF-R m R N A was detected in the contralateral unoperated facial motoneurons at any time in rats subjected to either procedure. On the operated side, a few neurons with NGF-R m R N A could already be identified in the facial motor nucleus 1 day after both crushing and transection of the facial nerve. Fig. 1 shows the time-course of postoperative changes of NGF-R m R N A expression by facial motoneurons after crushing of the nerve. As shown in this figure, the number of
labelled cells and the signal intensity increased after the insult and reached its maximum from the 3rd (Fig. 1-3D) to the 7th day (Fig. 1-1W) after operation. From that time on, the decrease was rapid and no specific labelling was seen by 3 weeks after the procedure (Fig. 1-2W, Fig. 1-3W). The changes of NGF-R m R N A levels in the facial motoneurons after nerve transection differed from those seen after nerve crushing. On day 1, the number of labelled cells and the intensity of labelling increased in the facial motoneurons after transection, as it had after nerve crushing. The m R N A level reached its peak from the 3rd (Fig. 2-3D) to the 7th day after the procedure, although the signal intensity was weaker than after nerve crushing and the number of labelled neurons was only one-third of that after nerve crushing (Fig. 3). A conspicuous difference was noted in NGF-R m R N A levels in the facial motor nucleus between the rats with nerve transection and nerve crushing after day 7. In the case of nerve transection, the number of labelled neurons and the signal intensity reached a plateau and then showed no marked changes. In contrast, after nerve crushing, the m R N A level decreased rapidly and expres-
Fig. 1. Dark-field photomicrographs showing facial motoneurons on the operated side expressing NGF-R mRNA hybridization signals 3 days (3D), 1 week (lW), 2 weeks (2W), and 3 weeks (3W) after facial nerve crushing. Bar = I ram.
159
Fig. 2. Dark-field photomicrographs showing facial motoneurons on the operated side expressing NGF-R mRNA hybridization signals 1 day (1D), 3 days (3D), 6 weeks (6W), and 8 weeks (8W) after facial nerve transection. Bar = 1 mm.
sion had totally disappeared by the 3rd week after injury. After nerve crush injury, the function of the facial nerves seemed to be restored within 2 weeks, because recovery of movement of the whiskers occured at this
a
8
ii
ii
m
lg
#
2oi t 1
3
7
t4
21
28
d a y s a f t e r i:~rm:edure
Fig. 3. Time-course of changes in the percentage of neurons expressing NGF-R mRNA following facial nerve crushing ((D) and transection (0). The percentages of positive neurons are expressed as the mean + S.D. of 3 rats.
time, whereas no functional recovery was observed following nerve transection. We demonstrated that facial nerve crushing caused a transient increase of NGF-R m R N A in the facial motor nucleus, while facial nerve transection produced a long-term increase in this nucleus. Thus, expression of NGF-R m R N A appears to closely parallel the regenerative process of motoneuron axons. Accordingly, the present findings provide a strong basis at the molecular level for the assumption that N G F - R has a trophic or axonal guidance function for regenerating motoneurons as has already been suggested by several studies 1'3'5'1°A7. It is known that axotomy of the adult rat sciatic nerve induces expression of both N G F and N G F - R in Schwann cells located distal to the injury, and this induction appears to be inversely regulated by axonal contact 7'8'15'16. A persistent increase of N G F m R N A has also been found after transection of the facial nerve 7. The present study demonstrated that N G F - R synthesis in the injured facial motoneurons was in line with the timecourse of nerve regeneration. This suggests that the persistent increase of N G F - R synthesis seen in axotomized cell bodies was due to the increased levels of N G F internalized by the peripheral processes and retrogradely
160 transported to the cell bodies. O n e of the most conspicuous findings in the present study was that the increase of N G F - R m R N A was more p r o n o u n c e d after crush injury than after nerve transec-
after nerve transection than crush injury. (2) In the case of crush injury, the continuity of the e n d o n e u r i u m is preserved, while transection allows the free flow of axoptasm from the cut end, Therefore, after a crush
tion. A t least 3 explanations for this can be postulated: (1) Crush injury may stimulate Schwann cells to produce
injury much more of the N G F produced by Schwann cells in the distal segment or by target muscles may have been
more N G F than does nerve transection. However, this hypothesis seems to be unlikely, because synthesis of both N G F s and N G F - R a'15 in Schwann cells of the distal
transection. (3) Transection may have caused a more marked perikaryal reaction than did crush injury, which
nerve segment has been shown to be more prominent
resulted in the production of less N G F - R .
1 Chen, Y.S., Wang-Bennett, L.T. and Coker, N.J., Facial nerve regeneration in the silicone chamber: the influence of nerve growth factor, Exp. Neurol., 103 (1989) 52-60. 2 Eckenstein, E, Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord, Brain Res., 446 (1988) 149-154. 3 Ernfors, P., Henschen, A., Olson, L. and Persson, H., Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons, Neuron, 2 (1989) 1605-1613. 4 Gunderson, R.W. and Park, K.H.C., The effects of conditioned media on spinal neuritis: substrate-associated changes in neurite direction and adherence, Dev. Biol., 104 (1984) 18-27. 5 Heaton, M.B., Influence of nerve growth factor on chick trigeminal motor nucleus explants, J. Comp. Neurol., 265 (1987) 362-366. 6 Heuer, J.G., Fatemie-Nainie, S., Wheeler, E.E and Bothwell, M., Structure and developmental expression of the chicken NGF receptor, Dev. Biol., 137 (1990) 287-304. 7 Heumattn, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection, J. Cell Biol., 104 (1987) 1623-1631. 8 Heumann, R., Lindhotm, D., Bandtlow, C., Meyer, M., Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H., Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regeneration: role of macrophages, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8735-8739. 9 Koh, S., Oyler, G.A. and Higgins, G.A., Localization of nerve growth factor receptor messenger RNA and protein in the adult rat brain, Exp. Neurol., 106 (1989) 209-221. 10 Levi-Montalcini, R. and Aloe, L., Differentiating effects of murine nerve growth factor in the peripheral and central nervous systems of Xenopus laevis tadpoles, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 7111-7115.
11 Miyata, Y., Kashihara, Y., Homma, S. and Kuno, M., Effects of nerve growth factor on the survival and synaptic function of Ia sensory neurons axotomized in neonatal rats, J. Neurosci., 6 (1986) 2012-2018. 12 Pioro, E.P. and Cuello, A.C., Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system - II. Brainstem, cerebellum and spinal cord, Neuroscience, 34 (1990) 89-110. 13 Radeke, M.J., Misko, T.P., Hsu, C., Herzenberg, L.A. and Shooter, E.M., Gene transfer and molecular cloning of the rat nerve growth factor receptor, Nature, 325 (1987) 593-597. 14 Richardson, P.M., Verge-Issa, V.M.K. and Riopelle, R.J., Distribution of neuronal receptors for nerve growth factor in the rat, J. Neurosci., 6 (1986) 2312-2321. 15 Taniuchi, M., Clark, H.B. and Johnson, E.M. Jr., Induction of nerve growth factor receptor in Schwann cells after axotomy, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4094--4098. 16 Taniuehi, M., Clark, H.B., Schweitzer, J,B. and Johnson, E.M. Jr., Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastructural location, supression by axonal contact, and binding properties, J. Neurosci., 8 (1988) 664-681. 17 Wayne, D.B. and Heaton, M.B., The response of cultured trigeminal and spinal cord motoneurons to nerve growth factor, Dev. Biol., 138 (1990) 473-483. 18 Yan, Q., Snider, W.D., Pinzone, J.J. and Johnson, E.M. Jr., Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats: assessment of potential neurotrophic effects, Neuron, 1 (1988) 335-343. 19 Yan, Q. and Johnson, E.M. Jr., An immunohistochemical study of nerve growth factor receptor in developing rats, J. Neurosci., 8 (1988) 3481-3498. 20 Yip, H.K. and Johnson, E.M. Jr., Nerve growth factor receptors in rat spinal cord: an autoradiographic and immunohistochemical study, Neuroscience, 22 (1987) 267-279.
retrogradely transported to the cell body than after
