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Reserpine-induced changes in dopamine-β-hydroxylase activity in the terminal projections of the nucleus locus coeruleus
Neuroscience Letters, 2 (1976) 279--283
279
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
RESERPINE-INDUCED CHANGES IN DOPAMINE-~-HYDROXYLASE ACTIVITY IN THE TERMINAL PROJECTIONS OF THE NUCLEUS LOCUS COERULEUS
S.M. BROOKE and H.C. FIBIGER
Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C., V6T I W5 (Canada) (Received April 13th, 1976) (Accepted May 18th, 1976)
SUMMARY
Rats received daily subcutaneous injections of reserpine (2.5 mg/kg) for 3 days. Dopamine-~-hydroxylase (DBH) activity was measured in the dorsal pons (which contained the locus coeruleus), cerebellum, hippocampus and frontal cortex 1, 2 and 4 weeks after the last injection. In accordance with previous reports, reserpine significantly increased DBH activity in dorsal pons 1 and 2 weeks after reserpine. In the other brain regions, which contain noradrenergic terminals of the locus coeruleus, reserpine either produced no change in DBH activity (cerebellum) or resulted in significant decreases in enzyme activity 1--4 weeks after the last injection (hippocampus and frontal cortex). Induction of DBH in the locus coeruleus is not, therefore, subsequently reflected in an overall increase in enzyme activity in the terminals arising from this nucleus; on the contrary, reserpine produces long-term decreases in DBH activity in these regions.
Increases in the activities of the enzymes tyrosine hydroxylase (TH) and dopamine-~-hydroxylase (DBH) in the cell bodies of noradrenergic neurons after reserpine administration have been demonstrated in both the central [1,2,12,13,17] and peripheral [9,11,14] nervous systems. Moreover, these increases have been shown to be the result of synthesis and enhanced accumulation of these enzymes rather than reflecting changes in the kinetics of preexisting enzyme molecules [6,9,11,13,16]. In the peripheral sympathetic nervous system the induction of TH and DBH takes place in both the cell bodies (e.g., ganglia) and nerve terminals (e.g., heart) but there is a delay between the induction in the perikarya and the appearance of increased activity in the terminals [9,14]. It has, therefore, been postulated that the increased TH and DBH activities appearing in peripheral noradrenergic terminals after reserpine are due, at least in part, to increased anterograde axonal transport of these proteins.
280 in the central nervous system, the induction of TH and DBH after reserpine treatment has been conclusively demonstrated in the cell bodies of the noradrenergic neurons in the locus coeruleus {LC) [12]. There has been some indication of increases in TH activity in the hypothalamus, which contains both noradrenergic axons en passage and nerve terminals, but these changes have been small [12,13]. Recently, however, Black [1], using a sensitive TH assay, demonstrated that compared to the LC, reserpine produced a delayed increase in activity of this enzyme in frontal cortex and cerebellum by 190 and 170% respectively. Insofar as the cerebellum and frontal cortex contain noradrenergic terminals which originate in the LC [ 7,15], it was suggested that the increased TH activity in these structures was mediated by axoplasmic transport [ 1 ]. In the present experiments we u n d e r t o o k to determine if similar increases in the activity of DBH occurs in the nerve terminals of LC neurons. Male Wistar rats weighed 350--400 g at the start of the experiment and were housed in individual cages with free access to food and water. Animals were injected subcutaneously with 2.5 mg/kg of reserpine (Serpasil) for three consecutive days and then sacrificed at 1, 2 and 4 weeks after the last injection. To study earlier effects of reserpine, animals were injected intraperitoneally with 10 mg/kg and sacrificed 24 h later. At appropriate intervals, animals were sacrificed by cervical fracture. The brains were removed and the cerebellum, hippocampus and frontal cortex were dissected out on ice. Tissue samples containing locus coeruleus were dissected from frozen sections of the pons. The samples containing locus coeruleus weighed approximately 8 mg. The various brain regions were homogenized in cold 0.25 M sucrose. For DBH, brain tissue was homogenized in cold 0.005 M Tris HC1 buffer {0.15% Triton X) and enzyme activity was determined according to the method of Coyle and Axelrod [3] as previously described [2]. To determine the effect of reserpine on noradrenaline levels, animals were injected with reserpine (2.5 mg/kg/day for 3 days) and sacrificed 4 weeks later. Noradrenaline levels in the brain stem, cortex, hippocampus and cerebellum were measured according to the method of McGeer and McGeer [8]. As shown in Fig. 1, we were readily able to demonstrate increases in the activity of DBH in the tissue sample containing locus coeruleus for 2 weeks after the injection of reserpine. Although this sample contained other dorsal pontine nuclei in addition to the locus coeruleus, it has been demonstrated that the induced enzyme activity is confined to the locus coeruleus [12,13]. After 4 weeks, DBH activity had returned to within normal values. Reserpine (10 mg/kg) produced a small but significant decrease (80% of control) in DBH activity in the locus coeruleus 24 h after the injection. The control value for cerebellar DBH activity was 48.55 + 1.51 nmoles octopamine/g/h. The experimental values were within 12% of the control values at all times examined. The results for hippocampus and frontal cortex are shown in Fig. 1. In the hippocampus, DBH activity was significantly decreased at 1 and 2 weeks after the reserpine injections {50--60% of control values). By the 4th
281
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120
.~ 1 0 0
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80 60 40 i
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i
1
7
14
DAYS A F T E R
2I
8
RESERPINE
Fig. 1. Activity o f dopamine-~-hydroxylase in dorsal pons (o o ), h i p p o c a m p u s (= = ) and frontal c o r t e x (c- . . . . . o ) expressed as a per c e n t of control values at various intervals after reserpine administration (for injection schedule see text). Control Vma x values in nmoles o c t o p a m i n e / g / h were: dorsal pons 3312 -+ 120 (21 determinations); h i p p o c a m p u s 59.3 -+ 2.3 (21 determinations); and frontal c o r t e x 72.4 -+ 2.8 (21 determinations). Each p o i n t represents the m e a n (± S.E.M.) o f 4--12 determinations. *Significantly different f r o m control value, P < 0.01.
week, these values had returned to normal. In the frontal cortex the enzyme activity followed a different pattern. There was no change in DBH until the 4th week after reserpine when the activity decreased significantly to a b o u t 60% of control values. The control values for noradrenaline content in ~g/g in the various areas of brain examined were: brain stem, 0.46 -+ 0.04; cerebellum, 0.15 + 0.02; hippocampus, 0.21 -+ 0.02; and cortex, 0.34 + 0.03. Four weeks after reserpine treatment the experimental values were 32, 46, 19 and 26% of control values, respectively. In confirmation of earlier work reserpine produced large and significant increases in the activity of DBH in the locus coeruleus [2,12,13]. However, in contrast to peripheral noradrenergic neurons, we failed to observe increases in the activity of the enzyme in the terminals of these central neurons {cerebellum, hippocampus, and cortex). Either there was no change in activity (cerebellum), or, as indicated in Fig. 1, significant decreases in DBH activity occurred at various intervals after reserpine. The present data suggest therefore that differences may exist between central and peripheral noradrenergic neurons in their response to reserpine. Several factors may account for these apparent differences. One possibility is that the increased DBH formed in the locus coeruleus after reserpine is not transported to the terminal areas. Alter-
282
natively, the time sampling utilized in the present experiments may have missed transient increases in enzyme activity in the terminals. Another possibility is that reserpine may indeed increase the a m o u n t of axonally transported DBH, but at the same time increases the turnover rate of this protein in central noradrenergic terminals. The net effect of these two processes would be to maintain a constant level of enzyme activity in the axon terminals. Garcia et al. [4] have, in fact, recently obtained evidence suggesting that reserpine can increase the a m o u n t of axonally transported DBH in cat hypogastric nerve. This hypothesis may also provide an explanation for the decreased enzyme activity observed in hippocampus and frontal cortex 1--4 weeks after reserpine. These decreases could occur if the increased enzyme turnover in the noradrenergic terminals persisted beyond the period of increased axonal transport of the enzyme. The fact that the noradrenaline content of the hippocampus, cortex and cerebellum had returned i:o less than 50% of control values 4 weeks after the reserpine injections indicates that the recovery process in the terminals was far from complete at this time, despite the fact that DBH activity had returned to normal values in the locus coeruleus. With regard to the transient decreased enzyme activity in hippocampus and cortex, it is perhaps relevant that Haggendal and Dahlstrom [ 5] have demonstrated that although there is a gradual return of noradrenaline in heart and salivary glands during the first 4 weeks after reserpine, during the 5th week there is a transient decrease before the values return to normal levels in 6--7 weeks. These workers attributed the decrease to transient changes in the rate of axonal transport of catecholamine storage granules. In summary, it is apparent that the effects of reserpine on DBH activity in the terminal regions of the locus coeruleus projections are long-lasting, complex and vary from one region to another. Unlike peripheral noradrenergic neurons, induction of this enzyme in the locus coeruleus is not subsequently reflected in an overall increase in enzyme activity in the terminals arising from this nucleus. The nature of the long latency reserpine-induced decreases in DBH and the variable effects of reserpine on different structures containing locus coeruleus efferents are under further investigation. ACKNOWLEDGEMENTS
Supported by the Medical Research Council. H.C. Fibiger is an M.R.C. Scholar. REFERENCES 1 Black, I.B., Increased tyrosine hydroxylase activity in frontal cortex and cerebellum after reserpine, Brain Res., 95 (1975) 170--176. 2 Brooke, S.M. and Fibiger, H.C., Differential rates of increase in pontine tyrosine hydroxylase and dopamine-~-hydroxylase activities after reserpine, Brain Res., 85 (1975) 532--534.
283
3 Coyle, J. and Axelrod, J., Dopamine-#-hydroxylase in rat brain: developmental characteristics,J. Neurochem., 19 (1972) 449--459. 4 Garcia, A.G., Kirkepar, S.M., Prat, J.C. and Wakade, A.R., Metabolic and ionic requirements for the axoplasmic transport of dopamine-#-hydroxylase, J. Physiol. (Lond.), 241 (1974) 809--821. 5 Haggendal, J. and DahlstrSm, A., The recovery of noradrenaline in adrenergic nerve terminals of the rat after reserpine treatment, J. Pharm. Pharmacol., 23 (1971) 81--89. 6 Joh, T.H., Gehman, C. and Reis, D.J., Immunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicitedby reserpine, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2767--2771. 7 Kobayashi, R.M., Palkovits, M., Jacobowitz, D.M. and Kopin, I.J.,Biochemical mapping of the noradrenergic projection from the locus coeruleus, Neurology (Minneap.), 25 (1975) 223--233. 8 McGeer, E.G. and McGeer, P.L.. Catecholamine content of the spinal cord, Canad. J. Biochem., 40 (1962) 1141--1] 51. 9 Molinoff, P.B., Brimijoin, S. and Axelrod, J., Induction of dopamine-#-hydroxylase and tyrosine hydroxylase in rat hearts and sympathetic ganglia, J. Pharmacol. exp. Ther., 182 (1972) 116--129. 10 Molinoff, P.B., Brimijoin, S., Weinshilboum, R. and Axelrod, J., Neurally mediated increase in dopamine-#-hydroxylase activity, Proc. nat. Acad. Sci. (Wash.), 66 (1970) 453--458. 11 Mueller, R.A., Thoenen, H. and Axelrod, J., Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. exp. Ther., 169 (1969) 74--79. 12 Reis, D.J., Joh, T.H. and Ross, R.A., Effects of reserpine on activities and amounts of tyrosine hydroxylase and dopamine-#-hydroxylase in catecholamine neuronal systems in rat brain, J. Pharmacol. exp. Ther., 193 (1975) 775--784. 13 Reis, D.J., Joh, T.H., Ross, R.A. and Pickel, V.M., Reserpine selectively increases tyrosine hydroxylase and dopamine-#-hydroxylase enzyme protein in central noradrenergic neurons. Brain Res., 81 (1974) 380--386. 14 Thoenen, H., Mueller, R.A. and Axelrod, J., Phase difference in the induction of tyrosine hydroxylase in cell b o d y and nerve terminals of sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 65 (1970) 58--62. 15 Ungerstedt, U., Stereotaxic mapping of monamine pathways in the rat brain, Acta physiol, scand., 95, Suppl. 367, (1971) 1--48. 16 Zigmond, R.E. and MacKay, A.V.P., Dissociation of stimulatory and synthetic phases in the induction of tyrosine hydroxylase, Nature (Lond.), 247 (1974) 112--113. 17 Zigmond, R.E., Schon, F. and Iversen, L.L., Increased tyrosine hydroxylase activity in the locus coeruleus in rat brain stem after reserpine treatment and cold stress, Brain Res., 70 (1974) 547--552.
279
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
RESERPINE-INDUCED CHANGES IN DOPAMINE-~-HYDROXYLASE ACTIVITY IN THE TERMINAL PROJECTIONS OF THE NUCLEUS LOCUS COERULEUS
S.M. BROOKE and H.C. FIBIGER
Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C., V6T I W5 (Canada) (Received April 13th, 1976) (Accepted May 18th, 1976)
SUMMARY
Rats received daily subcutaneous injections of reserpine (2.5 mg/kg) for 3 days. Dopamine-~-hydroxylase (DBH) activity was measured in the dorsal pons (which contained the locus coeruleus), cerebellum, hippocampus and frontal cortex 1, 2 and 4 weeks after the last injection. In accordance with previous reports, reserpine significantly increased DBH activity in dorsal pons 1 and 2 weeks after reserpine. In the other brain regions, which contain noradrenergic terminals of the locus coeruleus, reserpine either produced no change in DBH activity (cerebellum) or resulted in significant decreases in enzyme activity 1--4 weeks after the last injection (hippocampus and frontal cortex). Induction of DBH in the locus coeruleus is not, therefore, subsequently reflected in an overall increase in enzyme activity in the terminals arising from this nucleus; on the contrary, reserpine produces long-term decreases in DBH activity in these regions.
Increases in the activities of the enzymes tyrosine hydroxylase (TH) and dopamine-~-hydroxylase (DBH) in the cell bodies of noradrenergic neurons after reserpine administration have been demonstrated in both the central [1,2,12,13,17] and peripheral [9,11,14] nervous systems. Moreover, these increases have been shown to be the result of synthesis and enhanced accumulation of these enzymes rather than reflecting changes in the kinetics of preexisting enzyme molecules [6,9,11,13,16]. In the peripheral sympathetic nervous system the induction of TH and DBH takes place in both the cell bodies (e.g., ganglia) and nerve terminals (e.g., heart) but there is a delay between the induction in the perikarya and the appearance of increased activity in the terminals [9,14]. It has, therefore, been postulated that the increased TH and DBH activities appearing in peripheral noradrenergic terminals after reserpine are due, at least in part, to increased anterograde axonal transport of these proteins.
280 in the central nervous system, the induction of TH and DBH after reserpine treatment has been conclusively demonstrated in the cell bodies of the noradrenergic neurons in the locus coeruleus {LC) [12]. There has been some indication of increases in TH activity in the hypothalamus, which contains both noradrenergic axons en passage and nerve terminals, but these changes have been small [12,13]. Recently, however, Black [1], using a sensitive TH assay, demonstrated that compared to the LC, reserpine produced a delayed increase in activity of this enzyme in frontal cortex and cerebellum by 190 and 170% respectively. Insofar as the cerebellum and frontal cortex contain noradrenergic terminals which originate in the LC [ 7,15], it was suggested that the increased TH activity in these structures was mediated by axoplasmic transport [ 1 ]. In the present experiments we u n d e r t o o k to determine if similar increases in the activity of DBH occurs in the nerve terminals of LC neurons. Male Wistar rats weighed 350--400 g at the start of the experiment and were housed in individual cages with free access to food and water. Animals were injected subcutaneously with 2.5 mg/kg of reserpine (Serpasil) for three consecutive days and then sacrificed at 1, 2 and 4 weeks after the last injection. To study earlier effects of reserpine, animals were injected intraperitoneally with 10 mg/kg and sacrificed 24 h later. At appropriate intervals, animals were sacrificed by cervical fracture. The brains were removed and the cerebellum, hippocampus and frontal cortex were dissected out on ice. Tissue samples containing locus coeruleus were dissected from frozen sections of the pons. The samples containing locus coeruleus weighed approximately 8 mg. The various brain regions were homogenized in cold 0.25 M sucrose. For DBH, brain tissue was homogenized in cold 0.005 M Tris HC1 buffer {0.15% Triton X) and enzyme activity was determined according to the method of Coyle and Axelrod [3] as previously described [2]. To determine the effect of reserpine on noradrenaline levels, animals were injected with reserpine (2.5 mg/kg/day for 3 days) and sacrificed 4 weeks later. Noradrenaline levels in the brain stem, cortex, hippocampus and cerebellum were measured according to the method of McGeer and McGeer [8]. As shown in Fig. 1, we were readily able to demonstrate increases in the activity of DBH in the tissue sample containing locus coeruleus for 2 weeks after the injection of reserpine. Although this sample contained other dorsal pontine nuclei in addition to the locus coeruleus, it has been demonstrated that the induced enzyme activity is confined to the locus coeruleus [12,13]. After 4 weeks, DBH activity had returned to within normal values. Reserpine (10 mg/kg) produced a small but significant decrease (80% of control) in DBH activity in the locus coeruleus 24 h after the injection. The control value for cerebellar DBH activity was 48.55 + 1.51 nmoles octopamine/g/h. The experimental values were within 12% of the control values at all times examined. The results for hippocampus and frontal cortex are shown in Fig. 1. In the hippocampus, DBH activity was significantly decreased at 1 and 2 weeks after the reserpine injections {50--60% of control values). By the 4th
281
200
'~ 1 8 0 0n.. ~" 1 6 0 Z 0
O
'=1("
C) 1 4 0
120
.~ 1 0 0
INIIll
IllllllltllllllllllllllLIIIIIIltl
I,-
80 60 40 i
i
i
1
7
14
DAYS A F T E R
2I
8
RESERPINE
Fig. 1. Activity o f dopamine-~-hydroxylase in dorsal pons (o o ), h i p p o c a m p u s (= = ) and frontal c o r t e x (c- . . . . . o ) expressed as a per c e n t of control values at various intervals after reserpine administration (for injection schedule see text). Control Vma x values in nmoles o c t o p a m i n e / g / h were: dorsal pons 3312 -+ 120 (21 determinations); h i p p o c a m p u s 59.3 -+ 2.3 (21 determinations); and frontal c o r t e x 72.4 -+ 2.8 (21 determinations). Each p o i n t represents the m e a n (± S.E.M.) o f 4--12 determinations. *Significantly different f r o m control value, P < 0.01.
week, these values had returned to normal. In the frontal cortex the enzyme activity followed a different pattern. There was no change in DBH until the 4th week after reserpine when the activity decreased significantly to a b o u t 60% of control values. The control values for noradrenaline content in ~g/g in the various areas of brain examined were: brain stem, 0.46 -+ 0.04; cerebellum, 0.15 + 0.02; hippocampus, 0.21 -+ 0.02; and cortex, 0.34 + 0.03. Four weeks after reserpine treatment the experimental values were 32, 46, 19 and 26% of control values, respectively. In confirmation of earlier work reserpine produced large and significant increases in the activity of DBH in the locus coeruleus [2,12,13]. However, in contrast to peripheral noradrenergic neurons, we failed to observe increases in the activity of the enzyme in the terminals of these central neurons {cerebellum, hippocampus, and cortex). Either there was no change in activity (cerebellum), or, as indicated in Fig. 1, significant decreases in DBH activity occurred at various intervals after reserpine. The present data suggest therefore that differences may exist between central and peripheral noradrenergic neurons in their response to reserpine. Several factors may account for these apparent differences. One possibility is that the increased DBH formed in the locus coeruleus after reserpine is not transported to the terminal areas. Alter-
282
natively, the time sampling utilized in the present experiments may have missed transient increases in enzyme activity in the terminals. Another possibility is that reserpine may indeed increase the a m o u n t of axonally transported DBH, but at the same time increases the turnover rate of this protein in central noradrenergic terminals. The net effect of these two processes would be to maintain a constant level of enzyme activity in the axon terminals. Garcia et al. [4] have, in fact, recently obtained evidence suggesting that reserpine can increase the a m o u n t of axonally transported DBH in cat hypogastric nerve. This hypothesis may also provide an explanation for the decreased enzyme activity observed in hippocampus and frontal cortex 1--4 weeks after reserpine. These decreases could occur if the increased enzyme turnover in the noradrenergic terminals persisted beyond the period of increased axonal transport of the enzyme. The fact that the noradrenaline content of the hippocampus, cortex and cerebellum had returned i:o less than 50% of control values 4 weeks after the reserpine injections indicates that the recovery process in the terminals was far from complete at this time, despite the fact that DBH activity had returned to normal values in the locus coeruleus. With regard to the transient decreased enzyme activity in hippocampus and cortex, it is perhaps relevant that Haggendal and Dahlstrom [ 5] have demonstrated that although there is a gradual return of noradrenaline in heart and salivary glands during the first 4 weeks after reserpine, during the 5th week there is a transient decrease before the values return to normal levels in 6--7 weeks. These workers attributed the decrease to transient changes in the rate of axonal transport of catecholamine storage granules. In summary, it is apparent that the effects of reserpine on DBH activity in the terminal regions of the locus coeruleus projections are long-lasting, complex and vary from one region to another. Unlike peripheral noradrenergic neurons, induction of this enzyme in the locus coeruleus is not subsequently reflected in an overall increase in enzyme activity in the terminals arising from this nucleus. The nature of the long latency reserpine-induced decreases in DBH and the variable effects of reserpine on different structures containing locus coeruleus efferents are under further investigation. ACKNOWLEDGEMENTS
Supported by the Medical Research Council. H.C. Fibiger is an M.R.C. Scholar. REFERENCES 1 Black, I.B., Increased tyrosine hydroxylase activity in frontal cortex and cerebellum after reserpine, Brain Res., 95 (1975) 170--176. 2 Brooke, S.M. and Fibiger, H.C., Differential rates of increase in pontine tyrosine hydroxylase and dopamine-~-hydroxylase activities after reserpine, Brain Res., 85 (1975) 532--534.
283
3 Coyle, J. and Axelrod, J., Dopamine-#-hydroxylase in rat brain: developmental characteristics,J. Neurochem., 19 (1972) 449--459. 4 Garcia, A.G., Kirkepar, S.M., Prat, J.C. and Wakade, A.R., Metabolic and ionic requirements for the axoplasmic transport of dopamine-#-hydroxylase, J. Physiol. (Lond.), 241 (1974) 809--821. 5 Haggendal, J. and DahlstrSm, A., The recovery of noradrenaline in adrenergic nerve terminals of the rat after reserpine treatment, J. Pharm. Pharmacol., 23 (1971) 81--89. 6 Joh, T.H., Gehman, C. and Reis, D.J., Immunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicitedby reserpine, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2767--2771. 7 Kobayashi, R.M., Palkovits, M., Jacobowitz, D.M. and Kopin, I.J.,Biochemical mapping of the noradrenergic projection from the locus coeruleus, Neurology (Minneap.), 25 (1975) 223--233. 8 McGeer, E.G. and McGeer, P.L.. Catecholamine content of the spinal cord, Canad. J. Biochem., 40 (1962) 1141--1] 51. 9 Molinoff, P.B., Brimijoin, S. and Axelrod, J., Induction of dopamine-#-hydroxylase and tyrosine hydroxylase in rat hearts and sympathetic ganglia, J. Pharmacol. exp. Ther., 182 (1972) 116--129. 10 Molinoff, P.B., Brimijoin, S., Weinshilboum, R. and Axelrod, J., Neurally mediated increase in dopamine-#-hydroxylase activity, Proc. nat. Acad. Sci. (Wash.), 66 (1970) 453--458. 11 Mueller, R.A., Thoenen, H. and Axelrod, J., Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. exp. Ther., 169 (1969) 74--79. 12 Reis, D.J., Joh, T.H. and Ross, R.A., Effects of reserpine on activities and amounts of tyrosine hydroxylase and dopamine-#-hydroxylase in catecholamine neuronal systems in rat brain, J. Pharmacol. exp. Ther., 193 (1975) 775--784. 13 Reis, D.J., Joh, T.H., Ross, R.A. and Pickel, V.M., Reserpine selectively increases tyrosine hydroxylase and dopamine-#-hydroxylase enzyme protein in central noradrenergic neurons. Brain Res., 81 (1974) 380--386. 14 Thoenen, H., Mueller, R.A. and Axelrod, J., Phase difference in the induction of tyrosine hydroxylase in cell b o d y and nerve terminals of sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 65 (1970) 58--62. 15 Ungerstedt, U., Stereotaxic mapping of monamine pathways in the rat brain, Acta physiol, scand., 95, Suppl. 367, (1971) 1--48. 16 Zigmond, R.E. and MacKay, A.V.P., Dissociation of stimulatory and synthetic phases in the induction of tyrosine hydroxylase, Nature (Lond.), 247 (1974) 112--113. 17 Zigmond, R.E., Schon, F. and Iversen, L.L., Increased tyrosine hydroxylase activity in the locus coeruleus in rat brain stem after reserpine treatment and cold stress, Brain Res., 70 (1974) 547--552.