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Nerve growth factor-mediated increase of the substance P content of chick embryo dorsal root ganglia
198
Brain Research, 170 (1979) 198-202, © Elsevier/North-Holland Biomedical Press
Nerve growth factor-mediated increase of the substance P content of chick embryo dorsal root ganglia
J. P. SCHWARTZ and E. COSTA Laboratory of PreclinicalPharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted March 8th, 1979)
The presence of substance P (SP) in the dorsal root ganglia (DRG) and the spinal cord of several species has been demonstrated by immunohistochemistryl, 5, by radioimmunoassayS, 14 and by bioassay 14. Current evidence suggests that SP functions as a putative neurotransmitter in sensory nerves afferent to the spinal cord 5,7,s,12. Immunohistochemical results have suggested that D R G contain SP cell bodies4, 5. Ligation o f the dorsal root between the ganglia and the spinal cord caused the appearance of more clearly identifiable SP immunofluorescent cell bodies in DRG. Such SP-positive cell bodies account for approximately 25 ~ of the cells present in the DRG. Moreover, such a ligation decreases the spinal cord content of SP and causes a corresponding increase of the SP content on the side of the ligation which is proximal to the D R G cell bodie sS,14. From day 8 to day 10 of their development, cells of D R G of chick embryo require nerve growth factor (NGF) 9. The loss of sensitivity to the biological effects of N G F after day 10 is apparently due to a disappearance of N G F receptors 2,3. During the sensitive stage, N G F stimulates overall protein synthesis, thereby increasing cell and D R G sizes, and stimulates neurite growth. In addition, the synthesis or activity of specific proteins and enzymes is affected by N G F 9,11,15. We have measured the SP (SP-like immunoreactive material) content of D R G dissected from chick embryos of different ages. Furthermore, we have examined the effects of in vitro culture on the SP content of D R G ; the addition of N G F to the culture medium increases the content of SP in the DRG. D R G were dissected using a stereomicroscope from chick embryos (Truslow Farms, Chesterville, Md.) of 9-18 days of age. Approximately 200-250 ganglia were dissected within one hour, pooled, and then aliquoted 10-15 per culture dish to form each sample. For the in vitro incubations, D R G were maintained, 10-50 per sample, in 30 mm petri dishes containing 1 ml of Ham's F-10 medium supplemented with 20 heat-inactivated fetal calf serum, penicillin (50 units/ml) and streptomycin (50/~g/ml). Incubation was carried out at 37 °C in a humidified atmosphere of 95 ~ air-5 ~ CO2. When present, fl-NGF prepared according to Smith et al. 13 was added at a concentration of 2 biological units/ml. SP-like immunoreactivity, hereafter referred to as SP, was measured by radioimmunoassay as previously described 6. Protein was assayed
199
I0
1A.
8
-;c o
2
AGE OF EMBRYO{OAYSI o,.
lB. 6.0 4.0 2.01
HRS OF INCUBATION
Fig. 1. Substance P content of chick embryo dorsal root ganglia. A: ganglia were dissected from embryos aged 9, 12, 15 or 18 days and SP measured. The results are presented as ng SP/mg protein. The corresponding concentrations, in pg SP/ganglion are: 9-day, 39.5 ; 12-day,76.0; 15-day, 103.3 ; 18-day, 115.7. B: Ganglia from 9-day chick embryoswere incubated in the absence (--O--) or presence (--O--) of cycloheximide(25/~g/ml) for the indicated times. Substance P was assayed in 2 N acetic acid extracts of the ganglia. Results are presented as mean -I- S.E.M. (n = 5 in Fig. 1A; n = 4 in Fig. 1B). The n refers to the number of culture dishes per point, each of which contained 10-15 ganglia. using the Lowry method 10 and bovine serum albumin as the standard. Tissue culture reagents were obtained from Grand Island Biological; cycloheximide from Sigma; substance P from Boehringer-Mannheim. The content of SP in the D R G of chick embryos, whether expressed as pg SP/ ganglion or ng SP/mg protein, increases with age (9-18 days) of the embryo (Fig. 1A). There are comparable amounts of SP in various regions of the spinal cord of chick embryo (cervical - - 2.4 ng/mg; thoracic - - 4.7; lumbar - - 3.5). Since D R G contain the cell bodies of SP neurons, they offer an opportunity to study the synthesis and turnover of SP. Incubation of D R G from 9-day-old chick embryos in growth medium containing fetal calf serum led to a 2-3-fold increase of the SP content by 20 h (Fig. 1B). Inclusion of cycloheximide (25/~g/ml), a blocker of protein synthesis, prevented this increase. This dose of cycloheximide had no effect on the total protein content of the cultured DRG. During incubations which lasted up to 24 h, there was no evidence of SP secretion from the ganglia. Incubation of D R G taken from 9-day-old chick embryos with fl-NGF led to an increase in the content of SP (Table I). During incubations as short as 4 h, N G F stimulated SP accumulation by about 60 ~ . As shown previously (Fig. IB), the incubation of D R G in media devoid of fl-NGF led
200 TABLE 1 Effect o f nerve growth factor on substance P content o f dorsal root ganglia ]rom 9- and 15-day chick embryos
Ganglia were dissected and incubated as described in the methods. Ten to fifteen ganglia were pooled in one culture dish for each sample: there were 4 samples for any given treatment (n = 4). Substance P was measured in 2 N acetic acid extracts of the DRG. The results are presented as mean ~ S.E.M. The experiment was repeated on 3 separate occasions, using 3 lots of embryos. Treatment
Substance P (ng/mg protein) 9-Day DRG
Control NGF (2 BU/ml)
15-Day DRG
4h
24h
4h
24h
3.38 ± 0.21 5.33 ± 0.26
12.7 ± 1.8 19.3 ± 0.7
6.22 £ 1.06 7.92 ± 0.54
16.2 i 3.2 12.8 :i:: 1.2
to an increase o f 2.7-fold in SP c o n t e n t f r o m 4 to 24 h: the presence o f N G F for 24 h caused a further 5 0 ~ increase. A l t h o u g h D R G from 15-day-old e m b r y o s showed a c o m p a r a b l e increase in SP c o n t e n t (l.6-fold) d u r i n g a 20 h i n c u b a t i o n period, N G F failed to cause the further s t i m u l a t i o n seen in D R G f r o m 9-day-old chick e m b r y o s (Table I). These results fit with the k n o w n loss o f N G F receptors a n d the decrease o f sensitivity for N G F in D R G o f o l d e r embryos2,~, 9. T h e increase in SP c o n t e n t o f D R G f r o m 9-day-old e m b r y o s resulting f r o m i n c u b a t i o n o f the ganglia with N G F can also be p r e v e n t e d by inclusion o f cycloheximide in the i n c u b a t i o n m e d i u m (Table II). I n this experiment, N G F caused a 60 ~ increase in the D R G c o n t e n t o f SP; this increase was c o m p l e t e l y b l o c k e d b y cycloheximide. C y c l o h e x i m i d e alone had no effect on the SP c o n t e n t o f the ganglia. N G F h a d no significant effect on the p r o t e i n c o n t e n t o f the ganglia d u r i n g either a 4 or a 24 h incubation. In p r e l i m i n a r y gel filtration e x p e r i m e n t s with a Sephadex G-25 c o l u m n , we have o b t a i n e d evidence for the existence o f two m o l e c u l a r forms o f SP in D R G (Fig. 2). One
TABLE II Effect o f cycloheximide on N G F stimulation o f substance P content o f 9-day chick embryo dorsal root ganglia
200-300 ganglia from 9-day chick embryos were incubated in groups of 50 per culture dish for each treatment. N G F was added at 2 biological units/ml and cycloheximide at 25/~g/ml. Incubations were carried out for 4 h. The results are presented for 2 experiments, in which different lots of embryos were used. Treatment
ng SP/mg protein Expt. 1
Control 3.33 NGF 5.19 NGF + Cycloheximide 3.88
Expt. 2
3.02 5.45 3.09
201
8tA
NGF-TREATED
"6
A
P =a.
B
CONTROL
z 0
4
• @
•
2 0 "~------7"=
10
20 FRACTION
I -30
Fig. 2. Sephadex G-25 column chromatography of extracts of ganglia incubated with or without nerve growth factor. For each sample 800--1000gangliafrom 9-day chick embryos were pooled and incubated, 50 ganglia per culture dish for 4 h in either control medium (Fig. 2B) or medium containing 2 BU/ml of fl-NGF (Fig. 2A). The ganglia were extracted with 2 N acetic acid, the extracts were centrifuged at 5000g × 10 min and the supernatants lyophilized. The residues were dissolvedin 0.1 N acetic acid and applied to a Sephadex G-25 column (0.9 × 55 cm) equilibrated in 0.1 N acetic acid. The flow rate was 1 ml/5 min. One ml fractions were collected and aliquots assayed for substance P. [125I]SubstanceP eluted at fraction 24 when applied separately: the void volume was at fraction 14. The control sample (Fig. 2B) contained 4.5 mg protein and the NGF-treated sample (Fig. 2A) 3.5 mg protein.
elutes in the same position as authentic SP; the other, which represents a small percentage of the total immunoactivity, has a molecular weight larger than that of SP. This larger mol.wt, peak, which is destroyed by incubation with trypsin, is increased following N G F treatment of D R G from 9-day-old embryos (Fig. 2A). Sufficient evidence supports the possibility that substance P is a putative neurotransmitter but little is known about the regulation of its biosynthesis and biological inactivation. We have begun an investigation of these problems by choosing as a model the chick embryo D R G which are known to contain SP cell bodies4,5,14. The D R G can be cultured in vitro, thereby permitting manipulation of the growth medium. Furthermore, they are responsive to N G F , a well-known physiological regulator, during a certain stage of development and thus the effect of such a physiological regulator on SP biosynthesis and content can be studied in these ganglia. There is some variability in the levels of SP in the ganglia from experiment to experiment, which we believe reflects the difficulty of the dissection, the variable size of the ganglia, and the possibility that ganglia may contain different numbers of neu-
202 t o n s o r m o r e specifically, SP-containing neurons. However, within a given e x p e r i m e n t , a consistent SP c o n t e n t was found. Using chick e m b r y o D R G we have f o u n d SP to be p r e s e n t a n d to increase in c o n c e n t r a t i o n with increasing age o f the e m b r y o . T w o o b s e r v a t i o n s suggest that in D R G the synthesis o f SP follows one o f the classic schemes for the regulation o f p r o t e i n synthesis. F i r s t , the SP c o n t e n t o f D R G increases d u r i n g a n in vitro i n c u b a t i o n o f these ganglia in g r o w t h m e d i u m : this increase is b l o c k e d by cycloheximide. Secondly, inc u b a t i o n o f D R G t a k e n f r o m 9-day-old chick e m b r y o s with N G F also results in an increased SP c o n t e n t : this increase is also b l o c k e d by cycloheximide. F u r t h e r m o r e , our p r e l i m i n a r y experiments using gel filtration suggest t h a t there are at least 2 m o l e c u l a r forms o f i m m u n o r e a c t i v e SP present in D R G a n d t h a t i n c u b a t i o n with N G F causes an increase in the larger mol.wt, form. O u r results thus suggest t h a t chick e m b r y o d o r s a l r o o t ganglia m a y provide a g o o d m o d e l system for studying the biosynthesis a n d met a b o l i s m o f SP. 1 Chan-Palay, V. and Palay, S. L., Ultrastructural identification of substance P cells and their processes in rat sensory ganglia and their terminals in the spinal cord by immunocytochemistry, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4050-4054. 2 Frazier, W. A., Boyd, L. F. and Bradshaw, R. A., Properties of the specific binding of 125I-nerve growth factor to responsive peripheral neurons, J. bioL Chem., 249 (1974) 5513-5519. 3 Herrup, K. and Shooter, E. M., Properties of the fl-nerve growth factor receptor in development, J. CellBioL, 67 (1975) 118-125. 4 Hi~kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Substance P: localization in the central nervous system and in some primary sensory neurons, Science, 190 (1975) 889-890. 5 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 6 Hong, J. S., Costa, E. and Yang, H.-Y. T., Effects of habenular lesions on the substance P content of various brain regions, Brain Research, 118 (1976) 523-525. 7 Iversen, L. L., Jessell, T. M. and Kanazawa, I., Release and metabolism of substance P in rat hypothalamus, Nature (Lond.), 264 (1976) 81-83. 8 Jessell, T. M. and Iversen, L. L., Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature (Lond.), 268 (1977) 549-551. 9 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 MacDonnell, P. C., Nagaiah, K., Lakshmanan, J. and Guroff, G., Nerve growth factor increases activity of ornithine decarboxylase in superior cervical ganglia of young rats, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4681-4684. 12 Otsuka, M., Konishi, S. and Takahashi, T., Hypothalamic substance P as a candidate for transmitter of primary afferent neurons, Fed. Proc., 34 (1975) 1922-1928. 13 Smith, A. P., Varon, S. and Shooter, E. M., Multiple forms of the nerve growth factor protein and its subunits, Biochemistry, 7 (1968) 3259-3268. 14 Takahashi, T. and Otsuka, M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section, Brain Research, 87 (1975) 1-11. 15 Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. and Kettler, R., Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-fl-hydroxylase in the rat superior cervical ganglia, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 1598-1602.
Brain Research, 170 (1979) 198-202, © Elsevier/North-Holland Biomedical Press
Nerve growth factor-mediated increase of the substance P content of chick embryo dorsal root ganglia
J. P. SCHWARTZ and E. COSTA Laboratory of PreclinicalPharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted March 8th, 1979)
The presence of substance P (SP) in the dorsal root ganglia (DRG) and the spinal cord of several species has been demonstrated by immunohistochemistryl, 5, by radioimmunoassayS, 14 and by bioassay 14. Current evidence suggests that SP functions as a putative neurotransmitter in sensory nerves afferent to the spinal cord 5,7,s,12. Immunohistochemical results have suggested that D R G contain SP cell bodies4, 5. Ligation o f the dorsal root between the ganglia and the spinal cord caused the appearance of more clearly identifiable SP immunofluorescent cell bodies in DRG. Such SP-positive cell bodies account for approximately 25 ~ of the cells present in the DRG. Moreover, such a ligation decreases the spinal cord content of SP and causes a corresponding increase of the SP content on the side of the ligation which is proximal to the D R G cell bodie sS,14. From day 8 to day 10 of their development, cells of D R G of chick embryo require nerve growth factor (NGF) 9. The loss of sensitivity to the biological effects of N G F after day 10 is apparently due to a disappearance of N G F receptors 2,3. During the sensitive stage, N G F stimulates overall protein synthesis, thereby increasing cell and D R G sizes, and stimulates neurite growth. In addition, the synthesis or activity of specific proteins and enzymes is affected by N G F 9,11,15. We have measured the SP (SP-like immunoreactive material) content of D R G dissected from chick embryos of different ages. Furthermore, we have examined the effects of in vitro culture on the SP content of D R G ; the addition of N G F to the culture medium increases the content of SP in the DRG. D R G were dissected using a stereomicroscope from chick embryos (Truslow Farms, Chesterville, Md.) of 9-18 days of age. Approximately 200-250 ganglia were dissected within one hour, pooled, and then aliquoted 10-15 per culture dish to form each sample. For the in vitro incubations, D R G were maintained, 10-50 per sample, in 30 mm petri dishes containing 1 ml of Ham's F-10 medium supplemented with 20 heat-inactivated fetal calf serum, penicillin (50 units/ml) and streptomycin (50/~g/ml). Incubation was carried out at 37 °C in a humidified atmosphere of 95 ~ air-5 ~ CO2. When present, fl-NGF prepared according to Smith et al. 13 was added at a concentration of 2 biological units/ml. SP-like immunoreactivity, hereafter referred to as SP, was measured by radioimmunoassay as previously described 6. Protein was assayed
199
I0
1A.
8
-;c o
2
AGE OF EMBRYO{OAYSI o,.
lB. 6.0 4.0 2.01
HRS OF INCUBATION
Fig. 1. Substance P content of chick embryo dorsal root ganglia. A: ganglia were dissected from embryos aged 9, 12, 15 or 18 days and SP measured. The results are presented as ng SP/mg protein. The corresponding concentrations, in pg SP/ganglion are: 9-day, 39.5 ; 12-day,76.0; 15-day, 103.3 ; 18-day, 115.7. B: Ganglia from 9-day chick embryoswere incubated in the absence (--O--) or presence (--O--) of cycloheximide(25/~g/ml) for the indicated times. Substance P was assayed in 2 N acetic acid extracts of the ganglia. Results are presented as mean -I- S.E.M. (n = 5 in Fig. 1A; n = 4 in Fig. 1B). The n refers to the number of culture dishes per point, each of which contained 10-15 ganglia. using the Lowry method 10 and bovine serum albumin as the standard. Tissue culture reagents were obtained from Grand Island Biological; cycloheximide from Sigma; substance P from Boehringer-Mannheim. The content of SP in the D R G of chick embryos, whether expressed as pg SP/ ganglion or ng SP/mg protein, increases with age (9-18 days) of the embryo (Fig. 1A). There are comparable amounts of SP in various regions of the spinal cord of chick embryo (cervical - - 2.4 ng/mg; thoracic - - 4.7; lumbar - - 3.5). Since D R G contain the cell bodies of SP neurons, they offer an opportunity to study the synthesis and turnover of SP. Incubation of D R G from 9-day-old chick embryos in growth medium containing fetal calf serum led to a 2-3-fold increase of the SP content by 20 h (Fig. 1B). Inclusion of cycloheximide (25/~g/ml), a blocker of protein synthesis, prevented this increase. This dose of cycloheximide had no effect on the total protein content of the cultured DRG. During incubations which lasted up to 24 h, there was no evidence of SP secretion from the ganglia. Incubation of D R G taken from 9-day-old chick embryos with fl-NGF led to an increase in the content of SP (Table I). During incubations as short as 4 h, N G F stimulated SP accumulation by about 60 ~ . As shown previously (Fig. IB), the incubation of D R G in media devoid of fl-NGF led
200 TABLE 1 Effect o f nerve growth factor on substance P content o f dorsal root ganglia ]rom 9- and 15-day chick embryos
Ganglia were dissected and incubated as described in the methods. Ten to fifteen ganglia were pooled in one culture dish for each sample: there were 4 samples for any given treatment (n = 4). Substance P was measured in 2 N acetic acid extracts of the DRG. The results are presented as mean ~ S.E.M. The experiment was repeated on 3 separate occasions, using 3 lots of embryos. Treatment
Substance P (ng/mg protein) 9-Day DRG
Control NGF (2 BU/ml)
15-Day DRG
4h
24h
4h
24h
3.38 ± 0.21 5.33 ± 0.26
12.7 ± 1.8 19.3 ± 0.7
6.22 £ 1.06 7.92 ± 0.54
16.2 i 3.2 12.8 :i:: 1.2
to an increase o f 2.7-fold in SP c o n t e n t f r o m 4 to 24 h: the presence o f N G F for 24 h caused a further 5 0 ~ increase. A l t h o u g h D R G from 15-day-old e m b r y o s showed a c o m p a r a b l e increase in SP c o n t e n t (l.6-fold) d u r i n g a 20 h i n c u b a t i o n period, N G F failed to cause the further s t i m u l a t i o n seen in D R G f r o m 9-day-old chick e m b r y o s (Table I). These results fit with the k n o w n loss o f N G F receptors a n d the decrease o f sensitivity for N G F in D R G o f o l d e r embryos2,~, 9. T h e increase in SP c o n t e n t o f D R G f r o m 9-day-old e m b r y o s resulting f r o m i n c u b a t i o n o f the ganglia with N G F can also be p r e v e n t e d by inclusion o f cycloheximide in the i n c u b a t i o n m e d i u m (Table II). I n this experiment, N G F caused a 60 ~ increase in the D R G c o n t e n t o f SP; this increase was c o m p l e t e l y b l o c k e d b y cycloheximide. C y c l o h e x i m i d e alone had no effect on the SP c o n t e n t o f the ganglia. N G F h a d no significant effect on the p r o t e i n c o n t e n t o f the ganglia d u r i n g either a 4 or a 24 h incubation. In p r e l i m i n a r y gel filtration e x p e r i m e n t s with a Sephadex G-25 c o l u m n , we have o b t a i n e d evidence for the existence o f two m o l e c u l a r forms o f SP in D R G (Fig. 2). One
TABLE II Effect o f cycloheximide on N G F stimulation o f substance P content o f 9-day chick embryo dorsal root ganglia
200-300 ganglia from 9-day chick embryos were incubated in groups of 50 per culture dish for each treatment. N G F was added at 2 biological units/ml and cycloheximide at 25/~g/ml. Incubations were carried out for 4 h. The results are presented for 2 experiments, in which different lots of embryos were used. Treatment
ng SP/mg protein Expt. 1
Control 3.33 NGF 5.19 NGF + Cycloheximide 3.88
Expt. 2
3.02 5.45 3.09
201
8tA
NGF-TREATED
"6
A
P =a.
B
CONTROL
z 0
4
• @
•
2 0 "~------7"=
10
20 FRACTION
I -30
Fig. 2. Sephadex G-25 column chromatography of extracts of ganglia incubated with or without nerve growth factor. For each sample 800--1000gangliafrom 9-day chick embryos were pooled and incubated, 50 ganglia per culture dish for 4 h in either control medium (Fig. 2B) or medium containing 2 BU/ml of fl-NGF (Fig. 2A). The ganglia were extracted with 2 N acetic acid, the extracts were centrifuged at 5000g × 10 min and the supernatants lyophilized. The residues were dissolvedin 0.1 N acetic acid and applied to a Sephadex G-25 column (0.9 × 55 cm) equilibrated in 0.1 N acetic acid. The flow rate was 1 ml/5 min. One ml fractions were collected and aliquots assayed for substance P. [125I]SubstanceP eluted at fraction 24 when applied separately: the void volume was at fraction 14. The control sample (Fig. 2B) contained 4.5 mg protein and the NGF-treated sample (Fig. 2A) 3.5 mg protein.
elutes in the same position as authentic SP; the other, which represents a small percentage of the total immunoactivity, has a molecular weight larger than that of SP. This larger mol.wt, peak, which is destroyed by incubation with trypsin, is increased following N G F treatment of D R G from 9-day-old embryos (Fig. 2A). Sufficient evidence supports the possibility that substance P is a putative neurotransmitter but little is known about the regulation of its biosynthesis and biological inactivation. We have begun an investigation of these problems by choosing as a model the chick embryo D R G which are known to contain SP cell bodies4,5,14. The D R G can be cultured in vitro, thereby permitting manipulation of the growth medium. Furthermore, they are responsive to N G F , a well-known physiological regulator, during a certain stage of development and thus the effect of such a physiological regulator on SP biosynthesis and content can be studied in these ganglia. There is some variability in the levels of SP in the ganglia from experiment to experiment, which we believe reflects the difficulty of the dissection, the variable size of the ganglia, and the possibility that ganglia may contain different numbers of neu-
202 t o n s o r m o r e specifically, SP-containing neurons. However, within a given e x p e r i m e n t , a consistent SP c o n t e n t was found. Using chick e m b r y o D R G we have f o u n d SP to be p r e s e n t a n d to increase in c o n c e n t r a t i o n with increasing age o f the e m b r y o . T w o o b s e r v a t i o n s suggest that in D R G the synthesis o f SP follows one o f the classic schemes for the regulation o f p r o t e i n synthesis. F i r s t , the SP c o n t e n t o f D R G increases d u r i n g a n in vitro i n c u b a t i o n o f these ganglia in g r o w t h m e d i u m : this increase is b l o c k e d by cycloheximide. Secondly, inc u b a t i o n o f D R G t a k e n f r o m 9-day-old chick e m b r y o s with N G F also results in an increased SP c o n t e n t : this increase is also b l o c k e d by cycloheximide. F u r t h e r m o r e , our p r e l i m i n a r y experiments using gel filtration suggest t h a t there are at least 2 m o l e c u l a r forms o f i m m u n o r e a c t i v e SP present in D R G a n d t h a t i n c u b a t i o n with N G F causes an increase in the larger mol.wt, form. O u r results thus suggest t h a t chick e m b r y o d o r s a l r o o t ganglia m a y provide a g o o d m o d e l system for studying the biosynthesis a n d met a b o l i s m o f SP. 1 Chan-Palay, V. and Palay, S. L., Ultrastructural identification of substance P cells and their processes in rat sensory ganglia and their terminals in the spinal cord by immunocytochemistry, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4050-4054. 2 Frazier, W. A., Boyd, L. F. and Bradshaw, R. A., Properties of the specific binding of 125I-nerve growth factor to responsive peripheral neurons, J. bioL Chem., 249 (1974) 5513-5519. 3 Herrup, K. and Shooter, E. M., Properties of the fl-nerve growth factor receptor in development, J. CellBioL, 67 (1975) 118-125. 4 Hi~kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Substance P: localization in the central nervous system and in some primary sensory neurons, Science, 190 (1975) 889-890. 5 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 6 Hong, J. S., Costa, E. and Yang, H.-Y. T., Effects of habenular lesions on the substance P content of various brain regions, Brain Research, 118 (1976) 523-525. 7 Iversen, L. L., Jessell, T. M. and Kanazawa, I., Release and metabolism of substance P in rat hypothalamus, Nature (Lond.), 264 (1976) 81-83. 8 Jessell, T. M. and Iversen, L. L., Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature (Lond.), 268 (1977) 549-551. 9 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 MacDonnell, P. C., Nagaiah, K., Lakshmanan, J. and Guroff, G., Nerve growth factor increases activity of ornithine decarboxylase in superior cervical ganglia of young rats, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4681-4684. 12 Otsuka, M., Konishi, S. and Takahashi, T., Hypothalamic substance P as a candidate for transmitter of primary afferent neurons, Fed. Proc., 34 (1975) 1922-1928. 13 Smith, A. P., Varon, S. and Shooter, E. M., Multiple forms of the nerve growth factor protein and its subunits, Biochemistry, 7 (1968) 3259-3268. 14 Takahashi, T. and Otsuka, M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section, Brain Research, 87 (1975) 1-11. 15 Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. and Kettler, R., Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-fl-hydroxylase in the rat superior cervical ganglia, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 1598-1602.