Nrurophan~xzcoloy~. 1976, IS,509S510. Pergamon Press. Printed in Gt. Brmin
INDUCTION OF TYROSINE-3-MONOOXYGENASE IN SUPERIOR CERVICAL GANGLIA AFTER AXOTOMY OF POSTGANGLIONIC NERVES I. HANBAUER
Section on Biochemical Pharmacology,
National Heart and Lung Institute, Bethesda, MD 20014
(Accepted
9 February
1976)
Summary-Interruption of the major postganglionic nerve fibres did not prevent the induction of tyrosine-3-monooxygenase elicited by trans-synaptic or direct stimulation of postsynaptic receptors in superior cervical ganglia. The results suggest that the induction of tyrosine-3-monooxygenase does not depend on an intact retrograde transport system for trophic substances.
Axotomy of autonomic nerves always leads to the degeneration of the axon distal to the cut (EULER and PURKHOLD, 1951). This indicates that trophism of axonal terminals crucially depends on their connection to cell bodies. This is not surprising because little or no protein synthesis occurs in axons and the turnover of functional and structural proteins in nerve terminals is assured by axoplasmic transport (BARONDES, 1969). In considering the regulation of neuronal trophism, the question arises whether terminal axons can influence the metabolic regulation of the amount of functional or structural proteins which are synthesized in cell bodies. It is known that during development retrograde transport of trophic substances taken up by nerve endings regulates gene expression of constitutional proteins in the perikaryon of sympathetic neurones (HENDRY, 1975; IVERSEN, ST~CKEL and THOENEN, 1975). If axotomy is performed before a critical period in neuronal maturation the death of cell bodies ensues (HENDRY, 1975). Evidently, during the period of neuronal maturation trophism of noradrenergic neurones critically depends on the retrograde transport of nerve growth factor (HENDRY, ST~~CKEL,THOENEN and IVERSEN, 1974). Since this retrograde transport of nerve growth factor proceeds throughout life, it was of interest to evaluate whether it participates in the increase in tyrosine-3monooxygenase activity elicited by trans-synaptic or other exogenous stimuli. The present experiments examine the effects of prior axotomy on the induction of tyrosine-3monooxygenase, elicited by hormonal or transmitter stimulation of specific receptors. The results show that after neuronal maturation the induction of this cell specific functional protein is independent from the presence of intact postsynaptic axons. MATERIALS AND
METHODS
Male Sprague-Dawley rats (12&150 g body weight, Zivic Miller, Allison Park, PA) were kept 4-6 days 509
in our animal quarters under constant light-dark cycles (14 hr light) and were fed Purina chow ad lib. Under methohexital anaesthesia (0.139 pmol/kg, i.v.), the bilateral axotomy of the major superior cervical nerve trunks was performed five days prior to application of stimuli known to induce tyrosine-3monooxygenase in superior cervical ganglia (HANBAUERand COSTA, 1976). The stimuli used were: nicotine salycilate (50 pmol/kg, s.c.), reserpine (12.3 pmol/kg, s.c.), dexamethasone (3.28 pmol/kg, i.p.) or epinephrine together with phenoxybenzamine (120 pmol/kg, S.C. and 32 ,umol/kg, i.p., respectively). The rats were killed 48 hr after each injection and each ganglion was homogenized in 0.5 ml of 0.06 M potassium phosphate buffer, pH 6.2. Two aliquots of 100 ,ul were assayed for tyrosine-3-monooxygenase activity using 3,5-[3HJ-L-tyrosine as substrate (Amersham/Searle) and 6-methyl-3,5,6,7_tetrahydropterine (Calbiochem, San Diego, CA) as cofactor (HANBAUER, KOPIN, GUIDOTTI and COSTA, 1975). The sources of the chemicals used were: nicotine salycilate, K & K Laboratories, Plainview, NY; reserpine (Serpasil) Ciba-Geigy, Summit, NJ; dexamethasone (Decadrone) Merck, Sharp & Dohme. Westpoint, PA; (-)-epinephrine bitartrate, Calbiochem, San Diego, CA; phenoxybenzamine (Dibenzyhne) Smith, Kline & French Laboratories, Philadelphia, PA; methohexital sodium (Brevital-Sodium) Eli-Lilly & Co., Indianapolis, IN.
RESULTS AND
DISCUSSION
The data reported in Table 1 show that seven days after interruption of the major postganglionic branches, which has been shown to affect at least four fifths of the neurones in the ganglion (PURVES, 1975). there was no change in tyrosine-3-monooxygenase activity in the ganglion. This finding is in agreement with a previous report by HENDRY (1975) but is at
510
1. HANBAUER
Table I. Induction of tyrosine-3-monooxygenase
in intact and in bilaterally axotomized superior cervical ganglia of rats
Drugs (~~moI~kg) Saline Nicotine (SO s.c.) Reserpine (12.3s.c.] Dexamethasone (3.28 i.p.) Epinephrine (120 s.c.) + Pllenoxybenzamine
Tyrosine-3-monooxygenase Intact
(32 i.p.)
4.5 5.6 6.5 6.8 6.1
(nmol DOPA~ganglion per hr) Bilateral axafomy
ri: 0.38 + 0.15 +- 0.57** + 0.48* + 0.43**
4.3 6.3 6.1 6.4 6.0
+ ) ) * *
0.16 0.29* 0.27* 0.40s 0.48*
Five days after axotomy rats were injected with the various drugs and were killed 48 hr thereafter. Results are the mean k S.E.M. of 6-8 samples. * P > 0.01 : **P > 0.02.
variance with another report showing that tyrosine-3monooxygcnase activity in locus coeruleus is decreased following axotomy (REIS,Ross and JOH, 1974). The reasons for this discrepancy in the response to axotomy of noradrenergic neurones in superior cervical ganglia and central nervous system is not elucidated by the present experiments. The data reported in Table I also show that reserpine, dexamethasone, nicotine or epinephrine increased tyrosine-3-monooxygenase activity seven days after axotomy. Since none of the various stimuli which have been shown to elicit tyrosine-3-monooxygenase induction in ganglia (HANBAUERand COSTA. 1976) require the presence of axonterminals. it can be inferred that the retrograde transport of nerve growth factor, which evidently has physiological significance during neuronal maturation, has no functionaJ role in the mediation of changes in gene expression elicited by various stimuli. If the interpret~~tion of the results presented in this report are correct, it can be inferred that in sympathetic ganglia of adult rats, trophic factors (including nerve growth factor) which reach the cell body by retrograde transport, have an insignificant role in the trans-synaptic regulation of gene expression of functional proteins. The present results suggest that if nerve growth factor plays a role in gene expression, it is probably carried to its receptor sites in noradrenergic cell bodies either via circulation or communication with the supporting cells.
REFERENCES
BARONDES, S. H. (1969). Axoplasmic transport. In: Hun& book of Neurochemistry, Vol. 2, (LAJTIIA, A., Ed.), pp. 435-445, Plenum Press, New York. EULER,U. S. VON and PURKI~OLD,A. (1951). Effect of sympathetic denervation on the noradrenaline and adrenaline content of spleen, kidney and salivary glands in the sheep. Acta physiol. stand. 24: 214-717. HANBAUER,I. and COSTA,I?. (1976). Induction of tyrosine hydroxylase in the superior cervical ganglion of rats: opposite influence of nuscarinic and nicotinic receptor agonists. Neuropharmacoloyy 15: G-90. HANBAUER,I.. KOPIN, I. J.. GLWIOTTI. A. and COSTA.E. (1975). Induction of tyrosine hydroxylase elicited by beta-adrenergic receptor agonists in normal and decentralized sympathetic ganglion: role of cyclic AMP. Yr. Phannac.
e-up. Ther. 193: 95-104.
HENDRY.I. A. (1975). The effects of axotomy on the development of the rat superior cervical ganglion. Bruin Rcs. 90: 235.-244. HENDRY, I. A., ST&XEI., K., TI-IOENEN. I-i. and IWRSEP~, L. L. (1974). The retrograde axonal transport of nerve growth factor. Bruit? Res. 68: IOi-121. IVERSEN,L. L., ST&XX, K. and ‘T’HOENEN,H. (1975). Autoradiograpbic studies of the retrograde axonal transport of nerve growth factor in mouse sympathetic neurones. Brain Rex 88: 37-43. PURVES,D. (1975). Functional and structural changes in mammalian sympathetic neurons following interruption of their axons. .I. Physiol.. Lond. 252: 429-463. REIS,D. J., Ross, R. A. and JOH, T. H. (1974): Some aspects of the reaction of central and peripheral noradrenergic neurons to injury. In: Dynamics of’ Degeneration and Growrh in Neurons (FLJXE.K., OLSON’, L. and ZOTTERMAN, Y., Eds.), pp. 109-125. Pergamon Press. Oxford.
INDUCTION OF TYROSINE-3-MONOOXYGENASE IN SUPERIOR CERVICAL GANGLIA AFTER AXOTOMY OF POSTGANGLIONIC NERVES I. HANBAUER
Section on Biochemical Pharmacology,
National Heart and Lung Institute, Bethesda, MD 20014
(Accepted
9 February
1976)
Summary-Interruption of the major postganglionic nerve fibres did not prevent the induction of tyrosine-3-monooxygenase elicited by trans-synaptic or direct stimulation of postsynaptic receptors in superior cervical ganglia. The results suggest that the induction of tyrosine-3-monooxygenase does not depend on an intact retrograde transport system for trophic substances.
Axotomy of autonomic nerves always leads to the degeneration of the axon distal to the cut (EULER and PURKHOLD, 1951). This indicates that trophism of axonal terminals crucially depends on their connection to cell bodies. This is not surprising because little or no protein synthesis occurs in axons and the turnover of functional and structural proteins in nerve terminals is assured by axoplasmic transport (BARONDES, 1969). In considering the regulation of neuronal trophism, the question arises whether terminal axons can influence the metabolic regulation of the amount of functional or structural proteins which are synthesized in cell bodies. It is known that during development retrograde transport of trophic substances taken up by nerve endings regulates gene expression of constitutional proteins in the perikaryon of sympathetic neurones (HENDRY, 1975; IVERSEN, ST~CKEL and THOENEN, 1975). If axotomy is performed before a critical period in neuronal maturation the death of cell bodies ensues (HENDRY, 1975). Evidently, during the period of neuronal maturation trophism of noradrenergic neurones critically depends on the retrograde transport of nerve growth factor (HENDRY, ST~~CKEL,THOENEN and IVERSEN, 1974). Since this retrograde transport of nerve growth factor proceeds throughout life, it was of interest to evaluate whether it participates in the increase in tyrosine-3monooxygenase activity elicited by trans-synaptic or other exogenous stimuli. The present experiments examine the effects of prior axotomy on the induction of tyrosine-3monooxygenase, elicited by hormonal or transmitter stimulation of specific receptors. The results show that after neuronal maturation the induction of this cell specific functional protein is independent from the presence of intact postsynaptic axons. MATERIALS AND
METHODS
Male Sprague-Dawley rats (12&150 g body weight, Zivic Miller, Allison Park, PA) were kept 4-6 days 509
in our animal quarters under constant light-dark cycles (14 hr light) and were fed Purina chow ad lib. Under methohexital anaesthesia (0.139 pmol/kg, i.v.), the bilateral axotomy of the major superior cervical nerve trunks was performed five days prior to application of stimuli known to induce tyrosine-3monooxygenase in superior cervical ganglia (HANBAUERand COSTA, 1976). The stimuli used were: nicotine salycilate (50 pmol/kg, s.c.), reserpine (12.3 pmol/kg, s.c.), dexamethasone (3.28 pmol/kg, i.p.) or epinephrine together with phenoxybenzamine (120 pmol/kg, S.C. and 32 ,umol/kg, i.p., respectively). The rats were killed 48 hr after each injection and each ganglion was homogenized in 0.5 ml of 0.06 M potassium phosphate buffer, pH 6.2. Two aliquots of 100 ,ul were assayed for tyrosine-3-monooxygenase activity using 3,5-[3HJ-L-tyrosine as substrate (Amersham/Searle) and 6-methyl-3,5,6,7_tetrahydropterine (Calbiochem, San Diego, CA) as cofactor (HANBAUER, KOPIN, GUIDOTTI and COSTA, 1975). The sources of the chemicals used were: nicotine salycilate, K & K Laboratories, Plainview, NY; reserpine (Serpasil) Ciba-Geigy, Summit, NJ; dexamethasone (Decadrone) Merck, Sharp & Dohme. Westpoint, PA; (-)-epinephrine bitartrate, Calbiochem, San Diego, CA; phenoxybenzamine (Dibenzyhne) Smith, Kline & French Laboratories, Philadelphia, PA; methohexital sodium (Brevital-Sodium) Eli-Lilly & Co., Indianapolis, IN.
RESULTS AND
DISCUSSION
The data reported in Table 1 show that seven days after interruption of the major postganglionic branches, which has been shown to affect at least four fifths of the neurones in the ganglion (PURVES, 1975). there was no change in tyrosine-3-monooxygenase activity in the ganglion. This finding is in agreement with a previous report by HENDRY (1975) but is at
510
1. HANBAUER
Table I. Induction of tyrosine-3-monooxygenase
in intact and in bilaterally axotomized superior cervical ganglia of rats
Drugs (~~moI~kg) Saline Nicotine (SO s.c.) Reserpine (12.3s.c.] Dexamethasone (3.28 i.p.) Epinephrine (120 s.c.) + Pllenoxybenzamine
Tyrosine-3-monooxygenase Intact
(32 i.p.)
4.5 5.6 6.5 6.8 6.1
(nmol DOPA~ganglion per hr) Bilateral axafomy
ri: 0.38 + 0.15 +- 0.57** + 0.48* + 0.43**
4.3 6.3 6.1 6.4 6.0
+ ) ) * *
0.16 0.29* 0.27* 0.40s 0.48*
Five days after axotomy rats were injected with the various drugs and were killed 48 hr thereafter. Results are the mean k S.E.M. of 6-8 samples. * P > 0.01 : **P > 0.02.
variance with another report showing that tyrosine-3monooxygcnase activity in locus coeruleus is decreased following axotomy (REIS,Ross and JOH, 1974). The reasons for this discrepancy in the response to axotomy of noradrenergic neurones in superior cervical ganglia and central nervous system is not elucidated by the present experiments. The data reported in Table I also show that reserpine, dexamethasone, nicotine or epinephrine increased tyrosine-3-monooxygenase activity seven days after axotomy. Since none of the various stimuli which have been shown to elicit tyrosine-3-monooxygenase induction in ganglia (HANBAUERand COSTA. 1976) require the presence of axonterminals. it can be inferred that the retrograde transport of nerve growth factor, which evidently has physiological significance during neuronal maturation, has no functionaJ role in the mediation of changes in gene expression elicited by various stimuli. If the interpret~~tion of the results presented in this report are correct, it can be inferred that in sympathetic ganglia of adult rats, trophic factors (including nerve growth factor) which reach the cell body by retrograde transport, have an insignificant role in the trans-synaptic regulation of gene expression of functional proteins. The present results suggest that if nerve growth factor plays a role in gene expression, it is probably carried to its receptor sites in noradrenergic cell bodies either via circulation or communication with the supporting cells.
REFERENCES
BARONDES, S. H. (1969). Axoplasmic transport. In: Hun& book of Neurochemistry, Vol. 2, (LAJTIIA, A., Ed.), pp. 435-445, Plenum Press, New York. EULER,U. S. VON and PURKI~OLD,A. (1951). Effect of sympathetic denervation on the noradrenaline and adrenaline content of spleen, kidney and salivary glands in the sheep. Acta physiol. stand. 24: 214-717. HANBAUER,I. and COSTA,I?. (1976). Induction of tyrosine hydroxylase in the superior cervical ganglion of rats: opposite influence of nuscarinic and nicotinic receptor agonists. Neuropharmacoloyy 15: G-90. HANBAUER,I.. KOPIN, I. J.. GLWIOTTI. A. and COSTA.E. (1975). Induction of tyrosine hydroxylase elicited by beta-adrenergic receptor agonists in normal and decentralized sympathetic ganglion: role of cyclic AMP. Yr. Phannac.
e-up. Ther. 193: 95-104.
HENDRY.I. A. (1975). The effects of axotomy on the development of the rat superior cervical ganglion. Bruin Rcs. 90: 235.-244. HENDRY, I. A., ST&XEI., K., TI-IOENEN. I-i. and IWRSEP~, L. L. (1974). The retrograde axonal transport of nerve growth factor. Bruit? Res. 68: IOi-121. IVERSEN,L. L., ST&XX, K. and ‘T’HOENEN,H. (1975). Autoradiograpbic studies of the retrograde axonal transport of nerve growth factor in mouse sympathetic neurones. Brain Rex 88: 37-43. PURVES,D. (1975). Functional and structural changes in mammalian sympathetic neurons following interruption of their axons. .I. Physiol.. Lond. 252: 429-463. REIS,D. J., Ross, R. A. and JOH, T. H. (1974): Some aspects of the reaction of central and peripheral noradrenergic neurons to injury. In: Dynamics of’ Degeneration and Growrh in Neurons (FLJXE.K., OLSON’, L. and ZOTTERMAN, Y., Eds.), pp. 109-125. Pergamon Press. Oxford.
