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Increased nerve growth factor receptor mRNA in contused rat spinal cord
238
Neuroscience Letters, 118 (1990) 238-240 Elsevier Scientific Publishers Ireland Ltd.
NSL 07226
Increased nerve growth factor receptor mRNA in contused rat spinal cord N i c o l e t t a Brunello*, M i c h a e l R e y n o l d s , J e a n R. W r a t h a l l a n d I t a l o M o c c h e t t i Department of Anatomy and Cell Biology, Georgetown University, School of Medicine, Washington,DC 20007, (U.S.A.)
(Received 18 May 1990; Revisedversion received 18 June 1990; Accepted 26 June 1990) Key words: Nervegrowth factor receptor mRNA; Spinal cord; Contusive injury; Adult rat
Peripheral nerve injury induces the expression of nerve growth factor receptor (NGFR). To determine whether a similar induction results from injury of the spinal cord, NGFR mRNA content was determined using Northern blot hybridization analysis of total RNA from spinal cords of rats contused in the mid-thoracic region. By four days after contusion NGFR mRNA was significantlyincreased in the thoracic segmentsthat included the injury site. The induction was maximal at 7 days, about 5- to 7-fold the level of uninjured controls, and remained 4 times higher than controls at 14 and 28 days after injury. These results suggest that axotomy in the CNS may also trigger the molecular mechanism(s)leading to up-regnlation of NGFR expression.
Nerve growth factor ( N G F ) exerts its neurotrophic activity after binding to the N G F receptor ( N G F R ) and being internalized and retrogradely transported to the neuronal cell body as N G F / N G F R complex. Therefore, biosynthesis and availability of N G F R play a crucial role in determining N G F biological activity. Sciatic nerve transection has been shown to induce N G F R synthesis both in Schwann cells [7, 11] and in spinal cord motoneurons [5] suggesting that axonal damage can induce the expression of N G F R as a mechanism to facilitate regeneration. To test whether a similar induction could occur after a CNS injury, N G F R m R N A was determined by Northern blot hybridization analysis in rat spinal cord after a standardized contusive injury in the mid-thoracic region. Mild contusive injury at the T8 vertebral level was produced using a weight drop device [13]. Female SpragueDawley rats (200-220 g) wre anesthetized with chloral hydrate, a laminectomy performed, and a 10 g weight was dropped 2.5 cm onto exposed dura. Rats were sacrificed by decapitation 1, 4, 7, 14 and 28 days after injury. Spinal cord was quickly removed and dissected into cervical, thoracic and lumbar sections. The thoracic section was divided at the injury site into two 15 m m segments, rostral and caudal to the contusion site. Tissues were immediately frozen on dry ice and kept at - 7 0 ° C until
*Permanent address: Center of Neuropharmacology,Institute of Pharmacological Sciences, University of Milan, Italy. Correspondence." I. Mocchetti, Department of Anatomy and Cell Biology, School of Medicine, 3900 Reservoir Rd. N.W., Washington, DC 20007, U.S.A. 0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
analysis. Total R N A was extracted according to the guanidine/cesium chloride method [3]. N G F R m R N A was determined by Northern blot analysis using a 600 base [32p]-cRNA probe (pNico) encoding rat N G F R m R N A (see legend of Fig. 1). In control spinal cord, N G F R m R N A was barely detectable. However, 7 days after contusive injury, a 5 to 7-fold increase in N G F R m R N A was observed in the rostral and caudal thoracic segments that include the injury site (Fig. 1). This increase was not due to a general induction of R N A synthesis since the content of the R N A encoding the structural protein cyclophilin [4] did not change (Fig. 1). Since the surgical procedure to expose the spinal cord dura requires laminectomy, which could potentially injure the dorsal roots, laminectomy per se could be responsible for the induction of N G F R m R N A . This possibility was ruled out by the evidence that N G F R m R N A in the thoracic segment of the spinal cord from laminectomized rats did not differ from that in normal control spinal cord (Fig. 1). Therefore, the increased content of N G F R m R N A results from injury of the spinal cord itself. Furthermore, since N G F R m R N A content was not changed in the cervical and lumbar spinal cord (Fig. 2), the induction o f N G F R gene appears to occur only at, or adjacent to, the contusion site. To gain insight into the possible mechanisms of induction, we analyzed the time course of N G F R m R N A increase in cervical, thoracic and lumbar segments after contusion. In cervical and lumbar segments of the spinal cord, N G F R m R N A expression was not increased at any time (data not shown) supporting the hypothesis that N G F R gene is only induced at the injury site and/or
239
"*-'NGFR
"*-'C yc 1
2
3
4
5
to g4J
6
Fig. 1. Northern blot analysis of RNA in rat spinal cord. Total RNA was prepared from the thoracic region of the spinal cord from control (1,2), laminectomized (3,4) and injured rats, 7 days (5,6), after contusion. Tissues were homogenized in 5M guanidine isothiocyanate and total RNA was isolated by the cesium chloride method (3). Total RNA was electrophoresed in 1.1% agarose gel containing 6% formaldehyde and transferred to a nylon membrane (9). Prehybridization of blots was carded out at 65°C in 50% formamide, 12.5 mM Tris (pH7.4), 5 x Denhardt's, 0.5 x SSC, 25 mM EDTA, 0.1% SDS, 100/tg/ml ssDNA. Hybridization was carried out at 65°C in fresh prehybridization solution containing 1 x Denhardt's and 32p-labeled rat NGFR cRNA probe (pNico). This clone is a derivative of p5B plasmid (1) (a generous gift from Dr. M. Chao) lacking the 1000 bp BamHI-HindlII fragment. The in vitro transcription assay was performed according to the described procedure (8) to yield [a2P]-cRNA at a specific activity of 89 x 108 cpm//tg of RNA. The washing procedure was carded out in 0.1% SSC, 0.1% SDS at 70°C. Blots were then exposed to Kodak XOMAT film with intensifying screen at -70°C. After exposure, the radioactivity was removed by washing the blots at 75°C in 5 mM TrisHCI (pH 7.4), 0.2 mM EDTA, 50 mM sodium pyrophosphate and 1X Denhardt's (9). The blots were then rehybridized with 32P-nick-translated plB15 cDNA probe, encoding the mRNA for the structural protein cyclophilin (4), washed and exposed to a new X-ray film. This procedure enables us to correct for the relative amount of specific mRNA detected in the blot and to normalize for experimental artifacts (1O).
in a d j a c e n t spinal c o r d tissue. I n the t h o r a c i c segments N G F R m R N A increased b y 3 to 4-fold as early as 4 d a y s after c o n t u s i o n a n d it was still significantly higher (4fold) t h a n the c o n t r o l 14 a n d 28 d a y s after the injury (Fig. 3). T h e slow onset o f increase a n d the long lasting c h a n g e in N G F R m R N A suggest t h a t the increase o f N G F R m R N A m i g h t be due to increased t r a n s c r i p t i o n r a t h e r t h a n R N A stabilization. H o w e v e r , w i t h o u t the direct m e a s u r e m e n t o f the f o r m e r o u r d a t a d o n o t allow us to d i s c r i m i n a t e b e t w e e n these possibilities. T h e increased expression o f N G F R m R N A c o u l d be triggered b y increased levels o f the N G F protein. Infusion o f N G F has been s h o w n to induce N G F R expression in the b r a i n (2, 6). W e have recently f o u n d greatly increased levels o f spinal c o r d N G F after contusive injury (Bakhit, A r m a n i n i , W o n g , Bennett a n d W r a t h a l l , s u b m i t t e d ) . Thus, N G F itself c o u l d be one o f the m e c h a nisms r e s p o n s i b l e for the i n d u c t i o n o f its o w n receptor. T h e cellular l o c a l i z a t i o n o f this i n d u c t i o n is c u r r e n t l y u n k n o w n . A s injury to the sciatic nerve induces expression o f N G F R in a x o t o m i z e d spinal m o t o n e u r o n s , these cells are likely c a n d i d a t e s to r e s p o n d to the a x o t o m y t h a t
I trl
C
T IcJ
L
Fig. 2. Induction of NGFR mRNA in contused spinal cord. NGFR mRNA content was detected by Northern blot analysis (see legend of Fig. 1) in different sections of the spinal cord in control and contused rats: (C) cervical, (Tr) thoracic rostral to the injury site, (Tc) thoracic caudal to the injury site, (L) lumbar. The amount of NGFR mRNA detected on the blots is expressed in arbitrary units defined as the peak densitometric area of the NGFR mRNA hybridization band divided by the peak densitometric area of the cyclophilin mRNA hybridization band. The value of NGFR mRNA from control cervical was set equal to 1, and values for other samples were calculated relative to it. Values are presented as the mean + S.D. of 3 independent experiments.
results f r o m spinal c o r d c o n t u s i o n . H o w e v e r , N G F R c o u l d also be i n d u c e d in o t h e r n e u r o n s o r n o n n e u r o n a l cells including those a s s o c i a t e d with b l o o d vessels a n d meninges. T h e l a t t e r cells have been s h o w n to t r a n s i e n t l y express the r e c e p t o r d u r i n g d e v e l o p m e n t [12]. I n situ h y b r i d i z a t i o n studies will e n a b l e us to d i s c r i m i n a t e a m o n g these possibilities b y d e t e r m i n i n g the cellular l o c a l i z a t i o n o f N G F R expression. In conclusion, o u r d a t a suggest t h a t a x o t o m y in the C N S m a y trigger the u p - r e g u l a t i o n o f N G F R expression. T h e m o l e c u l a r m e c h a n i s m ( s ) l e a d i n g to this upr e g u l a t i o n m a y be similar to those in p e r i p h e r a l nerve o r m a y involve a d d i t i o n a l factors such as the i n f l a m m a t o r y a n d C N S glial responses to injury. F u r t h e r studies c o m -
6OO 500 ~ 400 o
300
1:3
200 100 0
4
7
14
28
Days a f t e r i n j u r y
Fig. 3. Time course of NGFR mRNA expression in injured spinal cord. NGFR mRNA content was determined by Northern blot analysis in the thoracic segments of the spinal cord of rats injured at T8 and sacrificed at the indicated time. Data are expressed as percentage of laminectomized rats considered as control. Values are the mean + S.D. of 4 independent determinations considering (n = 2) the segments rostral and caudal to the injury site.
240 p a r i n g the effects o f spinal cord a n d peripheral nerve injury are needed to address these issues. The a u t h o r s are grateful to Drs. M. C h a o a n d R. Miller for the precious gift o f c D N A probes. This work was supported in part by N a t i o n a l Institute o f Health B R S G R R 05360 to I.M. a n d in part by G e n e n t e c h Inc., S. Francisco (CA) to J.W. 1 Buck, C.R., Martinez, H.J., Chao, M.V. and Black, I.B., Differential expression of the nerve growth factor receptor gene in multiple brain areas. Dev. Brain Res. 44 (1988) 259-268. 2 Cavicchioli, L., Flanigan, T.P., Vantini, G., Fusco, R., Polato, P., Toffano, G., Walsh, F.S. and Leon, A., NGF amplifiesexpression of NGF receptor mRNA in forebrain cholinergic neurons of rats, Eur. J. Neurosci., 1 (1989) 258-262. 3 Chirgwin, S.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologicallyactive ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 15 (1979) 5294-5299. 4 Danielson, P.E., Forss-Petter, S., Brown, M.A., Calavetta, L., Douglas, J., Milner, R.J. and Sutcliffe, G.J., plB15: a cDNA clone of the rat mRNA encoding cyclophilin, DNA, 4 (1988) 261-267. 5 Ernfors, P. Henschon, 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. 6 Gage, F.H., Batchelor, P., Chen, K.S., Chin, D., Higgins, G.A., Koh, S., Deputy, S., Rosenberg, M.B., Fisher, W. and Bj6rklund, A. NGF receptor reexpression and NGF-mediated cholinergic neuronal hypertrophy in the damaged adult neostriatum, Neuron, 2(1989) 1177 1184.
7 Heumann, R., Lindholm, 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. 8 Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T. Zinn, K. and Green, M.R., Efficientin vitro synthesis of biologically active RNA and RNA hybridization probes from plasmide containing a bacteriophage SP6 promoter, Nucleic Acid Res., 12 (1984) 54975520. 9 Mocchetti, I., De Bernardi, M.A., Szekely, A.M., Alho, H. Brooker, G. and Costa, E., Regulation of nerve growth factor biosynthesis by beta-adrenergic receptor activation in astrocytoma cells: a potential role of c-Fos protein, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 3891-3895. 10 Mocchetti, I., Naranjo, J.R. and Costa, E. Regulation of striatal enkephalin turnover in rats receivingantagonists of specific dopamine receptor subtypes, J. Pharmacol. Exp. Ther., 241 (1987) 11201124. 11 Taniuchi, M., Clark, H.B. and Johnson, E.M., Induction of nerve growth factor receptor in Schwann cell after axotomy, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4094-4098. 12 Taniuchi, M., Clark, H.B., Schweizer, J.B. and Johnson, E.M., Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties, J. Neurosci., 8 (1988) 664-681. 13 Wrathall, J.R., Pettegrew, R.K. and Harvey, F. Spinal cord contusion in the rat: production of graded, reproducible, injury groups, Exp. Neurol., 88 (1985) 108-122.
Neuroscience Letters, 118 (1990) 238-240 Elsevier Scientific Publishers Ireland Ltd.
NSL 07226
Increased nerve growth factor receptor mRNA in contused rat spinal cord N i c o l e t t a Brunello*, M i c h a e l R e y n o l d s , J e a n R. W r a t h a l l a n d I t a l o M o c c h e t t i Department of Anatomy and Cell Biology, Georgetown University, School of Medicine, Washington,DC 20007, (U.S.A.)
(Received 18 May 1990; Revisedversion received 18 June 1990; Accepted 26 June 1990) Key words: Nervegrowth factor receptor mRNA; Spinal cord; Contusive injury; Adult rat
Peripheral nerve injury induces the expression of nerve growth factor receptor (NGFR). To determine whether a similar induction results from injury of the spinal cord, NGFR mRNA content was determined using Northern blot hybridization analysis of total RNA from spinal cords of rats contused in the mid-thoracic region. By four days after contusion NGFR mRNA was significantlyincreased in the thoracic segmentsthat included the injury site. The induction was maximal at 7 days, about 5- to 7-fold the level of uninjured controls, and remained 4 times higher than controls at 14 and 28 days after injury. These results suggest that axotomy in the CNS may also trigger the molecular mechanism(s)leading to up-regnlation of NGFR expression.
Nerve growth factor ( N G F ) exerts its neurotrophic activity after binding to the N G F receptor ( N G F R ) and being internalized and retrogradely transported to the neuronal cell body as N G F / N G F R complex. Therefore, biosynthesis and availability of N G F R play a crucial role in determining N G F biological activity. Sciatic nerve transection has been shown to induce N G F R synthesis both in Schwann cells [7, 11] and in spinal cord motoneurons [5] suggesting that axonal damage can induce the expression of N G F R as a mechanism to facilitate regeneration. To test whether a similar induction could occur after a CNS injury, N G F R m R N A was determined by Northern blot hybridization analysis in rat spinal cord after a standardized contusive injury in the mid-thoracic region. Mild contusive injury at the T8 vertebral level was produced using a weight drop device [13]. Female SpragueDawley rats (200-220 g) wre anesthetized with chloral hydrate, a laminectomy performed, and a 10 g weight was dropped 2.5 cm onto exposed dura. Rats were sacrificed by decapitation 1, 4, 7, 14 and 28 days after injury. Spinal cord was quickly removed and dissected into cervical, thoracic and lumbar sections. The thoracic section was divided at the injury site into two 15 m m segments, rostral and caudal to the contusion site. Tissues were immediately frozen on dry ice and kept at - 7 0 ° C until
*Permanent address: Center of Neuropharmacology,Institute of Pharmacological Sciences, University of Milan, Italy. Correspondence." I. Mocchetti, Department of Anatomy and Cell Biology, School of Medicine, 3900 Reservoir Rd. N.W., Washington, DC 20007, U.S.A. 0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
analysis. Total R N A was extracted according to the guanidine/cesium chloride method [3]. N G F R m R N A was determined by Northern blot analysis using a 600 base [32p]-cRNA probe (pNico) encoding rat N G F R m R N A (see legend of Fig. 1). In control spinal cord, N G F R m R N A was barely detectable. However, 7 days after contusive injury, a 5 to 7-fold increase in N G F R m R N A was observed in the rostral and caudal thoracic segments that include the injury site (Fig. 1). This increase was not due to a general induction of R N A synthesis since the content of the R N A encoding the structural protein cyclophilin [4] did not change (Fig. 1). Since the surgical procedure to expose the spinal cord dura requires laminectomy, which could potentially injure the dorsal roots, laminectomy per se could be responsible for the induction of N G F R m R N A . This possibility was ruled out by the evidence that N G F R m R N A in the thoracic segment of the spinal cord from laminectomized rats did not differ from that in normal control spinal cord (Fig. 1). Therefore, the increased content of N G F R m R N A results from injury of the spinal cord itself. Furthermore, since N G F R m R N A content was not changed in the cervical and lumbar spinal cord (Fig. 2), the induction o f N G F R gene appears to occur only at, or adjacent to, the contusion site. To gain insight into the possible mechanisms of induction, we analyzed the time course of N G F R m R N A increase in cervical, thoracic and lumbar segments after contusion. In cervical and lumbar segments of the spinal cord, N G F R m R N A expression was not increased at any time (data not shown) supporting the hypothesis that N G F R gene is only induced at the injury site and/or
239
"*-'NGFR
"*-'C yc 1
2
3
4
5
to g4J
6
Fig. 1. Northern blot analysis of RNA in rat spinal cord. Total RNA was prepared from the thoracic region of the spinal cord from control (1,2), laminectomized (3,4) and injured rats, 7 days (5,6), after contusion. Tissues were homogenized in 5M guanidine isothiocyanate and total RNA was isolated by the cesium chloride method (3). Total RNA was electrophoresed in 1.1% agarose gel containing 6% formaldehyde and transferred to a nylon membrane (9). Prehybridization of blots was carded out at 65°C in 50% formamide, 12.5 mM Tris (pH7.4), 5 x Denhardt's, 0.5 x SSC, 25 mM EDTA, 0.1% SDS, 100/tg/ml ssDNA. Hybridization was carried out at 65°C in fresh prehybridization solution containing 1 x Denhardt's and 32p-labeled rat NGFR cRNA probe (pNico). This clone is a derivative of p5B plasmid (1) (a generous gift from Dr. M. Chao) lacking the 1000 bp BamHI-HindlII fragment. The in vitro transcription assay was performed according to the described procedure (8) to yield [a2P]-cRNA at a specific activity of 89 x 108 cpm//tg of RNA. The washing procedure was carded out in 0.1% SSC, 0.1% SDS at 70°C. Blots were then exposed to Kodak XOMAT film with intensifying screen at -70°C. After exposure, the radioactivity was removed by washing the blots at 75°C in 5 mM TrisHCI (pH 7.4), 0.2 mM EDTA, 50 mM sodium pyrophosphate and 1X Denhardt's (9). The blots were then rehybridized with 32P-nick-translated plB15 cDNA probe, encoding the mRNA for the structural protein cyclophilin (4), washed and exposed to a new X-ray film. This procedure enables us to correct for the relative amount of specific mRNA detected in the blot and to normalize for experimental artifacts (1O).
in a d j a c e n t spinal c o r d tissue. I n the t h o r a c i c segments N G F R m R N A increased b y 3 to 4-fold as early as 4 d a y s after c o n t u s i o n a n d it was still significantly higher (4fold) t h a n the c o n t r o l 14 a n d 28 d a y s after the injury (Fig. 3). T h e slow onset o f increase a n d the long lasting c h a n g e in N G F R m R N A suggest t h a t the increase o f N G F R m R N A m i g h t be due to increased t r a n s c r i p t i o n r a t h e r t h a n R N A stabilization. H o w e v e r , w i t h o u t the direct m e a s u r e m e n t o f the f o r m e r o u r d a t a d o n o t allow us to d i s c r i m i n a t e b e t w e e n these possibilities. T h e increased expression o f N G F R m R N A c o u l d be triggered b y increased levels o f the N G F protein. Infusion o f N G F has been s h o w n to induce N G F R expression in the b r a i n (2, 6). W e have recently f o u n d greatly increased levels o f spinal c o r d N G F after contusive injury (Bakhit, A r m a n i n i , W o n g , Bennett a n d W r a t h a l l , s u b m i t t e d ) . Thus, N G F itself c o u l d be one o f the m e c h a nisms r e s p o n s i b l e for the i n d u c t i o n o f its o w n receptor. T h e cellular l o c a l i z a t i o n o f this i n d u c t i o n is c u r r e n t l y u n k n o w n . A s injury to the sciatic nerve induces expression o f N G F R in a x o t o m i z e d spinal m o t o n e u r o n s , these cells are likely c a n d i d a t e s to r e s p o n d to the a x o t o m y t h a t
I trl
C
T IcJ
L
Fig. 2. Induction of NGFR mRNA in contused spinal cord. NGFR mRNA content was detected by Northern blot analysis (see legend of Fig. 1) in different sections of the spinal cord in control and contused rats: (C) cervical, (Tr) thoracic rostral to the injury site, (Tc) thoracic caudal to the injury site, (L) lumbar. The amount of NGFR mRNA detected on the blots is expressed in arbitrary units defined as the peak densitometric area of the NGFR mRNA hybridization band divided by the peak densitometric area of the cyclophilin mRNA hybridization band. The value of NGFR mRNA from control cervical was set equal to 1, and values for other samples were calculated relative to it. Values are presented as the mean + S.D. of 3 independent experiments.
results f r o m spinal c o r d c o n t u s i o n . H o w e v e r , N G F R c o u l d also be i n d u c e d in o t h e r n e u r o n s o r n o n n e u r o n a l cells including those a s s o c i a t e d with b l o o d vessels a n d meninges. T h e l a t t e r cells have been s h o w n to t r a n s i e n t l y express the r e c e p t o r d u r i n g d e v e l o p m e n t [12]. I n situ h y b r i d i z a t i o n studies will e n a b l e us to d i s c r i m i n a t e a m o n g these possibilities b y d e t e r m i n i n g the cellular l o c a l i z a t i o n o f N G F R expression. In conclusion, o u r d a t a suggest t h a t a x o t o m y in the C N S m a y trigger the u p - r e g u l a t i o n o f N G F R expression. T h e m o l e c u l a r m e c h a n i s m ( s ) l e a d i n g to this upr e g u l a t i o n m a y be similar to those in p e r i p h e r a l nerve o r m a y involve a d d i t i o n a l factors such as the i n f l a m m a t o r y a n d C N S glial responses to injury. F u r t h e r studies c o m -
6OO 500 ~ 400 o
300
1:3
200 100 0
4
7
14
28
Days a f t e r i n j u r y
Fig. 3. Time course of NGFR mRNA expression in injured spinal cord. NGFR mRNA content was determined by Northern blot analysis in the thoracic segments of the spinal cord of rats injured at T8 and sacrificed at the indicated time. Data are expressed as percentage of laminectomized rats considered as control. Values are the mean + S.D. of 4 independent determinations considering (n = 2) the segments rostral and caudal to the injury site.
240 p a r i n g the effects o f spinal cord a n d peripheral nerve injury are needed to address these issues. The a u t h o r s are grateful to Drs. M. C h a o a n d R. Miller for the precious gift o f c D N A probes. This work was supported in part by N a t i o n a l Institute o f Health B R S G R R 05360 to I.M. a n d in part by G e n e n t e c h Inc., S. Francisco (CA) to J.W. 1 Buck, C.R., Martinez, H.J., Chao, M.V. and Black, I.B., Differential expression of the nerve growth factor receptor gene in multiple brain areas. Dev. Brain Res. 44 (1988) 259-268. 2 Cavicchioli, L., Flanigan, T.P., Vantini, G., Fusco, R., Polato, P., Toffano, G., Walsh, F.S. and Leon, A., NGF amplifiesexpression of NGF receptor mRNA in forebrain cholinergic neurons of rats, Eur. J. Neurosci., 1 (1989) 258-262. 3 Chirgwin, S.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologicallyactive ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 15 (1979) 5294-5299. 4 Danielson, P.E., Forss-Petter, S., Brown, M.A., Calavetta, L., Douglas, J., Milner, R.J. and Sutcliffe, G.J., plB15: a cDNA clone of the rat mRNA encoding cyclophilin, DNA, 4 (1988) 261-267. 5 Ernfors, P. Henschon, 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. 6 Gage, F.H., Batchelor, P., Chen, K.S., Chin, D., Higgins, G.A., Koh, S., Deputy, S., Rosenberg, M.B., Fisher, W. and Bj6rklund, A. NGF receptor reexpression and NGF-mediated cholinergic neuronal hypertrophy in the damaged adult neostriatum, Neuron, 2(1989) 1177 1184.
7 Heumann, R., Lindholm, 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. 8 Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T. Zinn, K. and Green, M.R., Efficientin vitro synthesis of biologically active RNA and RNA hybridization probes from plasmide containing a bacteriophage SP6 promoter, Nucleic Acid Res., 12 (1984) 54975520. 9 Mocchetti, I., De Bernardi, M.A., Szekely, A.M., Alho, H. Brooker, G. and Costa, E., Regulation of nerve growth factor biosynthesis by beta-adrenergic receptor activation in astrocytoma cells: a potential role of c-Fos protein, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 3891-3895. 10 Mocchetti, I., Naranjo, J.R. and Costa, E. Regulation of striatal enkephalin turnover in rats receivingantagonists of specific dopamine receptor subtypes, J. Pharmacol. Exp. Ther., 241 (1987) 11201124. 11 Taniuchi, M., Clark, H.B. and Johnson, E.M., Induction of nerve growth factor receptor in Schwann cell after axotomy, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4094-4098. 12 Taniuchi, M., Clark, H.B., Schweizer, J.B. and Johnson, E.M., Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties, J. Neurosci., 8 (1988) 664-681. 13 Wrathall, J.R., Pettegrew, R.K. and Harvey, F. Spinal cord contusion in the rat: production of graded, reproducible, injury groups, Exp. Neurol., 88 (1985) 108-122.