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Effect of adrenaline synthesis inhibition on brain noradrenaline neurons in locus coeruleus
Brain Research, 223 (1981) 49-58
49
Elsevier/North-HollandBiomedicalPress
EFFECT OF ADRENALINE SYNTHESIS INHIBITION ON BRAIN NORADRENALINE NEURONS IN LOCUS COERULEUS
G. ENGBERG, M. ELAM and T. H. SVENSSON Department of Pharmacology, University of Gi~teborg, Box 33031, S-400 33 G~teborg (Sweden)
(Accepted February 26th, 1981) Key words: adrenalinesynthesisinhibition-- noradrenalineneurons -- locus coeruleus-- phenyl-
ethanolamine-N-methyltransferase
SUMMARY The functional significance of the morphologically identified adrenaline (A)mediated input to the noradrenergic nucleus locus coeruleus (LC) was pharmacologically analyzed. By means of single unit recording techniques the LC neurons in the rat brain were studied following administration of SK&F 64139 and DCMB, drugs which are both potent inhibitors of the A-forming enzyme phenylethanolamine-Nmethyltransferase (PNMT). SK&F 64139 (2-200 mg/kg i.v.) caused an immediate, dose-dependent and long-lasting increase in firing rate of the LC neurons. The dose-response curve for the LC inhibitory effect of the a2-receptor agonist clonidine was shifted in parallel to the right by pretreatment with SK&F 64139. All the above mentioned effects of SK&F 64139 were mimicked by SK&F 72223, a structurally analogous compound which is reported to lack PNMT inhibitory activity. Consequently, the activation of LC neurons by SK&F 64139 is probably not related to its capacity to inhibit the synthesis of A but rather to some other action of the drug, such as an a2-receptor blocking effect. In contrast to SK&F 64139, the other PNMT inhibitor tested, DCMB (1-60 mg/kg), produced no significant activation of the LC neurons and but little clonidine antagonistic action. Thus, judging from these experiments, DCMB is devoid of significant az-receptor blocking properties. At the time for maximal brain A depletion after DCMB administration (4-6 h) the average firing rate of randomly encountered LC neurons was unaltered when compared with controls. In contrast, pretreatment with the tyrosine hydroxylase inhibitor a-methyl-ptyrosine methylester, which causes depletion also of brain noradrenaline, significantly increased LC neuronal firing rates. These data indicate that if there exists a physiologically relevant A-mediated input to the LC, this is not of critical importance for the tonic activity of the LC neurons.
0006-8993/81/00004)000/$ 2.50 © Elsevier/North-HollandBiomedicalPress
50 INTRODUCTION Previous biochemical studies have confirmed the existence of adrenaline (A) in mammalian brain 11. More recently, immunohistochemical methods have allowed mapping of both cell bodies and nerve terminals containing the A-forming enzyme phenylethanolamine-N-methyltransferase (PNMT) in the central nervous system12,13. The identified two groups of reticular nerve cell bodies in the medulla oblongata send their axons terminating into visceral nuclei of the brain stem and spinal cord as well as to nuclei in the hypothalamus and a major noradrenergic nucleus: the locus coeruleus (LC) in the pons. These LC noradrenergic neurons, which innervate almost the entire central nervous systeml~, 16,2a, are thought to be involved in various, e.g. behavioural and cardiovascular functions2,20, 30. Consequently, morphological evidence per se suggests the possibility that the LC noradrenergic neurons might be regulated inter alia by A afferents. This notion is consonant with the electrophysiological finding that various adrenergic agonists such as clonidine, an as-receptor agonist, noradrenaline (NA) as well as A and the fl-receptor agonist isoproterenol cause inhibition of firing of the LC noradrenergic neurons when applied microiontophoretically4,6, 24. Further studies utilizing various adrenergic antagonists revealed that the receptors involved in this inhibition could be characterized as a2-receptors 4,23. Thus, as-receptor antagonists such as yohimbine or piperoxane cause accelerated firing of the LC neurons both when applied systemically and microiontophoretically. Indeed, clonidine and piperoxane have been hypothesized to act as agonist and antagonist, respectively, at A synapses in brain 3. Thus, the above findings could be compatible with an A-mediated tonic, inhibitory influence on the LC NA system. However, recently an NA-mediated axon collateral inhibitory system in the LC has been demonstrated, which specifically was blocked by microiontophoretically applied piperoxane 1. Consequently, the effects of the a2-receptor active agents on LC neurons cannot either prove or disprove a putative functional significance of the A nerve terminals in the LC. In the absence of 'A receptor' specific agonists or antagonists, inhibitors of A synthesis, i.e. PNMT inhibitors, might prove interesting tools to elucidate the role of the A input to the LC. Therefore, it is significant that treatment with the PNMT inhibitor SK&F 64139 (7,8-dichloro-l,2,3,4-tetrahydroisoquinoline hydrochloride) 17 elicited an enhanced lowering of brain NA levels in stressed rats, particularly pronounced in the LC innervated frontal cortex~L This phenomenon was explained by a decreased inhibitory A-mediated influence on the LC, caused by the PNMT inhibitor. However, another explanation could be provided by an a~-receptor blocking effect of SK&F 64139, which should also cause increased firing rate of LC neurons, and recently we have presented some experimental evidence to support this view2L In the present paper we have studied systematically the role of the A input to the LC utilizing single cell recording techniques. Thus, the effects of SK &F 64139 on LC neurons were compared with those of SK&F 72223 (5,8-dimethoxy-l,2,3,4-tetrahydroisoquinoline hydrochloride) 19, a structural analogue to this drug without PNMT inhibitory effect and DCMB (2,3-dichloro-a-methylbenzylamine)7, another PNMT inhibitor.
51 In addition, the effect of A synthesis inhibition on the LC noradrenergic neurons was compared with the effect of inhibition also of the NA synthesis by a tyrosine hydroxylase inhibitor. The results will be discussed not only with respect to the functional significance of the A nerve terminals in the LC, but also with regard to various pharmacological effects of the drugs, e.g. on blood pressure, and their possible relationship to A systems in the brain. MATERIALSAND METHODS
Single unit recording The electrophysiological experiments were performed essentially as has been described previously27 on male, Sprague-Dawley rats. Briefly, the animals were anesthetized with chloral hydrate (400 mg/kg i.p.) and mounted in a stereotaxic apparatus. Additional injections were given as needed. A burr hole was drilled with its centre at 1.1 mm lateral and 1.1 mm posterior to the lambda. For recording a micropipette with a tip diameter of approximately 1/~m filled with 2 M NaC1 saturated with fast-green (impedance in vitro 3-6 Mf~, measured at 135 Hz) was lowered with a hydraulic microdrive into the brain. The electrode potentials were passed through a high input-impedance amplifier and filters. Each spike was discriminated and fed into an integrator being reset every 10 s and finally displayed on an oscilloscope, an audiomonitor and an oscillographic recorder. The body temperature of the animals was kept at 36-37 °C by means of a heating pad. Drugs were injected through a tail vein.
Identification of cells The neurophysiological characteristics of the cells were identical to those previously described for NA neurons of the rat LCI,10,14. No cells outside LC were included in this study. The final recording site of an experiment was marked by iontophoretic ejection of fast green. The rats were then perfused through the heart with 10 ~o formaldehyde solution and serial 50-/~m frozen sections of the brain were cut, mounted and stained with cresylviolet and counterstained with neutral red and the position of the dye was verified under the microscope. RESULTS Administration of SK&F 64139 consistently produced increased firing rate (up to 150 ~o) of the NA neurons in the LC (Fig. 1A). The effect was instantaneous in onset and dose-dependent within a broad dose range (2-200 mg/kg, Fig. 2A). Since there exists some indication that systemic administration of SK&F 64139 lowers blood pressure, at least in spontaneously hypertensive rats ~, arterial blood pressure was recorded via a tail artery in a few rats simultaneously with the electrophysiological recording from the LC after intravenous administration of the drug. Although a shortlasting hypotensive effect (5-10 min) was seen, this did not correlate with the long-
52
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Fig. 1. Effects of administration of SK&F 64139, SK&F 72223 and DCMB on the spontaneous firing rate of single N A neurons in the LC. A: Stimulation by SK&F 64139 (10+ 10 + 10 mg/kg) given at arrows. The effect is instantaneous in onset and dose-dependent. After this treatment it took totally 175/~g/kg of clonidine to silence the cell. Normally, about 10/~g/kg of clonidine is required to cause complete cessation of LC neuronal activity z4. The inhibitory action of the large dose of clonidine is readily reversed by an additional dose of SK&F 64139 (10 mg/kg). B: Instantaneous and dosedependent stimulation by SK&F 72223 ( 4 ÷ 8 mg/kg). This treatment necessitated 1.1 mg/kg of clonidine to silence the cell. C: Lack of stimulation by DCMB, total dose 58 mg/kg. After this treatment a rather low dose of clonidine (s¢¢ above) was sufficient to inhibit the cell (18/~g/kg).
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Fig. 2. Dose-response curves for the effects of SK&F 64139 (A), SK&F 72223 (B) and DCMB (C), respectively, on NA neurons in the LC from totally 24 rats. Each point represents the response of one single neuron in the LC. Lines are calculated regression lines. The correlation coefficients were: A, 0.87, P < 0.001 ; B, 0.67, P < 0.001 ; C, 0.21, N.S. lasting L C activating action o f S K & F 64139 (data not shown). After pretreatment with S K & F 64139, the inhibitory effect on L C neurons exerted by the a2-receptor agonist clonidine, an effect which is also obtained when microiontophoretically applied onto L C neurons 24, was markedly antagonized (Fig. 1A) and, in fact, the dose-response curve for the a2-agonist was shifted in parallel to the right after pretreatment with the P N M T inhibitor (Fig. 3A).
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Fig. 3. Dose-response curves for the inhibitory effect of clonidine on single cells in the LC of control rats (open symbols; n = 6) and rats pretreated (5 min, solid symbols) with (A) SK&F 64139 (30 mg/kg; n = 6) P < 0.01, (B) SK&F 72223 (3 mg/kg ;) P < 0.01 and (C) D C M B (40 mg/kg; n = 10) P < 0.01. Each point represents the response of one single cell in the LC, Lines have been plotted using linear regression analysis. Statistics: Regression lines for the individual animals were calculated; the intercepts of the control group were compared with the intercepts of the various treatment groups using Mann-Whitney's U-test.
55
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Fig. 4. Average spontaneous firing rate of randomly encountered NA neurons throughout the LC of control rats (n = 4) compared with rats pretreated with SK&F 64139 (35 mg/kg, 3-5 h; n = 4), SK&F 72223 (3 mg/kg, 4--6h; n = 4), DCMB (40 mg/kg, 4-6 h; n = 6) and a-MT (250 mg/kg, 5-6 h; n = 6). Shown are the means ± S.E.M. The number of cells recorded from are given in brackets in each column. *** P < 0.001. Also S K & F 72223, the structurally analogous compound without P N M T blocking properties, caused a dose-dependent excitation of all LC units tested (Figs. IB and 2B). In analogy with the results with S K & F 64139, pretreatment with S K & F 72223 antagonized the clonidine-induced depression of LC units (Fig. 1 B ) a n d caused a parallel shift to the right of the dose-response curve for clonidine. The other P N M T inhibitor tested, DCMB, produced no activation of the N A neurons in the LC (Fig. 1C), although at high doses, a slight stimulation of the LC neurons could possibly be detected (Fig. 2C). At low doses of D C M B no significant clonidine antagonistic action could be obtained and, in fact, a dose of 40 mg/kg i.v. of the P N M T inhibitor was necessary to obtain a slight parallel shift to the right of the dose-response curve for the a2-agonist (Fig. 3C). The activation of N A neurons in the LC by S K & F 64139 or S K & F 72223 remained approximately at the same level for several hours. Thus, 4-6 h after drug administration the average firing rate of neurons, randomly encountered throughout the LC, was significantly increased compared to that of the controls (Fig. 4). DCMB, however, did not cause any increased activity of the LC neuron also at this interval (4-6 h). In contrast, pretreatment with the tyrosine hydroxylase inhibitor a-methyl-ptyrosine methylester (a-MT), which inhibits also the synthesis of NA, causeda marked activation of the N A cells in the LC (Fig. 4). DISCUSSION
From the present data one can conclude that the activation of brain N A neurons in the LC by S K & F 64139 cannot solely be ascribed to inhibition of A synthesis for several reasons. Thus, the effect is instantaneous in onset, i.e. it occurs within seconds after intravenous administration of the drug. Clearly even if there exists a very small,
56 functionally important pool of newly synthetized A with a rapid turnover, the immediate effect by SK&F 64139 on the NA neurons must, in all probability, involve some other action than enzyme inhibition. Furthermore, the stimulatory effect on brain NA neurons in the LC is dose-dependent up to about 200 mg/kg, whereas brain P N M T activity is almost completely inhibited already after 10 mg/kg TM. Moreover, the LC activation was essentially of the same order of magnitude when studied immediately after injection of SK&F 64139 and when estimated 3-4 h later. Thus, the stimulation of the NA neurons does not correlate either with the time for maximal P N M T inhibition or that for maximal brain A depletion 22. It does not either correlate with the cardiovascular effects of the drug (c.f. above), which is important considering the documental peripheral, e.g. vagal inputs to the LC 5,26,2s. The rapid onset of the LC stimulatory effect by SK&F 64139 would rather suggest a receptor-mediated phenomenon to be involved. This is also indicated by the parallel shift to the right of the dose-response curve for the LC inhibitory effect of the a2-receptor agonist clonidine, without significant attenuation of its maximum response. The finding provides classical pharmacological evidence for an a2-receptor blocking effect of SK&F 64139. The question then arises whether A synthesis inhibition at all contributes to the stimulation of brain NA neurons in the LC by S K & F 64139. Such a contribution seems, in fact, distinctly unlikely for several reasons. Thus, the structurally analogous compound SK&F 72223, which does not possess P N M T inhibitory effect19, but in all probability exerts an az-receptor antagonistic action as is suggested by our experiments, also caused an immediate, dose-dependent stimulatory effect on the activity of the NA neurons in the LC. Moreover, the other P N M T inhibitor used, DCMB, did not cause significant stimulation of the NA neurons in the LC. This was the case also at the time for maximal brain A depletion after DCMB administration (4-6 h, Fig. 4) 7. Finally, judging from our experiments DCMB seems to possess but little, if any, a2-receptor blocking effect in contrast to SK&F 72223. Consequently, the stimulation of brain NA neurons in the LC by SK&F 64139 cannot be taken as evidence for an inhibitory, A-mediated control of these neurons, although such a regulatory mechanism may yet exist. Clearly, the absence of activation of LC neurons even at the time for maximal A depletion in brain after DCMB administration, if anything argues against a significant regulatory influence on the LC by A afferents. This is so, particularly in consideration of the fact that synthesis inhibition also involving NA systems by means of a-MT pretreatment actually did cause activation of the NA neurons in the LC. Thus, the present data taken together support the previously shown tonic inhibitory control of the LC neurons by noradrenergic nerve terminals, but leaves no support to the notion of a similar Amediated mechanism. Previously, the P N M T inhibitor SK&F 64139 has been used as a tool to disclose the significance of brain A systems for e.g. cardiovascular control. For example, intracerebrally administered SK&F 64139 has been found to elicit a hypertensive response in the rat a. This almost instantaneous effect, also obtained with SK&F 72223, which lacks PN MT inhibitory action, could presumably be related to an a2-receptor blocking
57 effect of the drugs. This interpretation is supported by the finding that intracerebral administration of other az-receptor blocking agents such as yohimbine also causes a hypertensive response 8,9. In any case, the present data make the interpretation of the blood pressure effects of the SK&F substances very difficult with respect to central A systems. Generally, our results imply that great caution must be exercised when using SK&F 64139 to elucidate the functional significance of brain A systems. ACKNOWLEDGEMENTS
The excellent technical assistance of Mrs. Birgitta Andersson is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (project No. 4747), ' T o r s t e n och R a g n a r S6derbergs Stiftelse', ' M a g n u s Bergvalls Stiftelse', 'Vilhelm och M a r t i n a L u n d g r e n s Vetenskaps fond', ' F o n d e n f6r N e u r o b i o l o g i s k F o r s k n i n g ' a n d the Medical Faculty, University of G6teborg.
REFERENCES 1 Aghajanian, G. K., Cedarbaum, J. M. and Wang, R. Y., Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons, Brain Research, 136 (1977) 570-577. 2 Amaral, D. G. and Sinnamon, H. M., The locus coeruleus: neurobiology of a central noradrenergic nucleus, Progr. Neurobiol., 9 (1977) 147-196. 3 Bolme, P. H., Corrodi, H., Fuxe, K., H6kfelt, T., Lidbrink, P. and Goldstein, M., Possible involvement of central adrenaline neurons in vasomotor and respiratory control. Studies with clonidine and its interaction with piperoxane and yohimbine, Europ. J. Pharmacol., 28 (1974) 89-94. 4 Cedarbaum, J. M. and Aghajanian, G. K., Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and activation by the a-antagonist piperoxane, Brain Research, 112 (1976) 413-419. 5 Cedarbaum, J. M. and Aghajanian, G. K., Activation of locus coeruleus neurons by peripheral stimuli: Modulation by a collateral inhibitory mechanism, Life Sci., 23 (1978) 1383-1392. 6 Cedarbaum, J. M. and Aghajanian, G. K., Catecholamine receptors on the locus coeruleus neurons: pharmacological characterization, Europ. J. PharmacoL, 44 (1977) 375-385. 7 Fuller, R. W. and Perry, K. W., Lowering of epinephrine concentration in rat brain by 2,3dichloro-a-methylbenzylamine, an inhibitor of norepinephrine N-methyltransferase, Biochem. Pharmacol., 26 (1977) 2087-2090. 8 Fuxe, K., Bolme, P., Johnsson, G., Agnati, L. F., Goldstein, M., H6kfelt, T., Schwarcz, R. and Engel, J., On the cardiovascular role of noradrenaline, adrenaline and peptide containing neuron systems in the brain. In P. Meyer and H. Schmitt (Eds.), Nervous System and Hypertension, Flammarion M6decine-Science, Paris, 1979, pp. 1-17. 9 Gomes, C., Trolin, G., Henning, M. and Persson, B., Pre- and postsynaptic a-adrenoceptor antagonists: differentiated cardiovascular effects in the rat, Clin. exp. Hypertension, 2 (1980) 273-296. 10 Graham, A. W. and Aghajanian, G. K., Effects of amphetamine on single cell activity in a catecholamine nucleus, the locus coeruleus, Nature (Lond.), 234 (1971) 100-102. 11 Holzbauer, M. and Sharman, D. F., The distribution of catecholamines in vertebrates. In H. Blaschko and E. Muscholl (Eds.), Catecholamines, Springer, Berlin, 1972, pp. 110-185. 12 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Evidence for adrenaline neurons in the rat brain, Acta physiol, scand., 89 (1973) 286-288. 13 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235-251.
58 14 Korf, J., Bunney, B. S. and Aghajanian, G. K., Noradrenergic neurons: morphine inhibition of spontaneous activity, Europ. J. Pharmacol., 25 (1974) 165-179. 15 Lindvall, O. and Bj6rklund, A., The organization of the ascending catecholamine neuron system in the rat brain as revealed by the glyoxylic acid fluorescence method, Aetaphysiol. scand. Suppl. 412 (1974) 1--48. 16 Nygren, L. and Olsson, L., A new major projection from locus coeruleus: the main source of noradrenergic nerve terminals in the ventral and dorsal columns of the spinal cord, Brain Research, 132 (1977) 85-93. 17 Pendleton, R. G., Kaiser, C. and Gessner, G., Studies on adrenal phenylethanolamine-N-methyltransferase (PNMT) with SK&F 64139, a selective inhibitor, J. Pharmacol. exp. Ther., 197 (1976) 623-632. 18 Pendleton, R. G., Khalsa, J., Gessner, G. and Sawyer, J., Studies on the characterization and inhibition of rat brain phenylethanolamine-N-transferase, Naunyn-Schmiedeberg's Arch. Pharmacol., 299 (1977) 219-224. 19 Pendleton, R. G., McCafferty, J. P. and Roesler, J. M., The effects of PNMT inhibitors upon cardiovascular changes induced by hemorrhage in the rat, Europ. J. Pharmacol., 66 (1980) 1-10. 20 Redmond, D. E. Jr., Huang, Y. H., Snyder, D. R. and Maas, J. W., Behavioural effects of stimulation of the nucleus locus coeruleus in the stump-tailed monkey, Macaca arctoides, Brain Research, 116 (1976) 502-510. 21 Sauter, A. M., Baba, Y., Stone, E. A. and Goldstein, M., Effects of stress and of phenylethanolamine-N-methyl-transferase inhibition on central norepinephrine and epinephrine levels, Brain Research, 144 (1978) 415-419. 22 Sauter, A. M., Lew, J. Y., Baba, Y. and Goldstein, M., Effect of phenylethanolamine-N-methyltransferase and dopamine-fl-hydroxylase inhibition on epinephrine levels in the brain, Life Sci., 21 (1977) 261-266. 23 Svensson, T. H., Central a-adrenoceptors and affective symptoms and disorder. In E. Lindenlaub and S. Garattini (Eds.), Depressive Disorders, 13th Symposium Medicum Hoechst, Rome, F. K. Schattauer, Stuttgart, 1978, pp. 245-254. 24 Svensson, T. H., Bunney, B. S. and Aghajanian, G. K., Inhibition of both noradrenergic and serotonergic neurons in brain by the a-adrenergic agonist clonidine, Brain Research, 92 (1975) 291-306. 25 Svensson, T. H. and Engberg, G., Putative regulation of brain norepinephrine: electrophysiological studies. In K. Fuxe, M. Goldstein, B. H/Skfelt and T. HSkfelt (Eds.), Central Adrenaline Neurons, Pergamon Oxford, 1980, pp. 105-111. 26 Svensson, T. H. and Thor6n, P., Brain noradrenergic neurons in the locus coeruleus: inhibition by blood volume through vagal afferents, Brain Research, 172 (1979) 174-178. 27 Svensson, T. H. and Usdin, T., Feedback inhibition of brain noradrenaline neurons by tricyclic antidepressants: a-receptor mediation, Science, 202 (1978) 1089-1091. 28 Takigawa, M. and Mogenson, G. J., A study of inputs to antidromically identified neurons of the locus coeruleus, Brain Research, 135 (1977) 217-230. 29 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand. Suppl. 367 (1971) 1-48. 30 Ward, D. G. and Gunn, C. G., Locus coeruleus complex: differential modulation of depressor mechanism, Brain Research, 107 (1976) 407-411.
49
Elsevier/North-HollandBiomedicalPress
EFFECT OF ADRENALINE SYNTHESIS INHIBITION ON BRAIN NORADRENALINE NEURONS IN LOCUS COERULEUS
G. ENGBERG, M. ELAM and T. H. SVENSSON Department of Pharmacology, University of Gi~teborg, Box 33031, S-400 33 G~teborg (Sweden)
(Accepted February 26th, 1981) Key words: adrenalinesynthesisinhibition-- noradrenalineneurons -- locus coeruleus-- phenyl-
ethanolamine-N-methyltransferase
SUMMARY The functional significance of the morphologically identified adrenaline (A)mediated input to the noradrenergic nucleus locus coeruleus (LC) was pharmacologically analyzed. By means of single unit recording techniques the LC neurons in the rat brain were studied following administration of SK&F 64139 and DCMB, drugs which are both potent inhibitors of the A-forming enzyme phenylethanolamine-Nmethyltransferase (PNMT). SK&F 64139 (2-200 mg/kg i.v.) caused an immediate, dose-dependent and long-lasting increase in firing rate of the LC neurons. The dose-response curve for the LC inhibitory effect of the a2-receptor agonist clonidine was shifted in parallel to the right by pretreatment with SK&F 64139. All the above mentioned effects of SK&F 64139 were mimicked by SK&F 72223, a structurally analogous compound which is reported to lack PNMT inhibitory activity. Consequently, the activation of LC neurons by SK&F 64139 is probably not related to its capacity to inhibit the synthesis of A but rather to some other action of the drug, such as an a2-receptor blocking effect. In contrast to SK&F 64139, the other PNMT inhibitor tested, DCMB (1-60 mg/kg), produced no significant activation of the LC neurons and but little clonidine antagonistic action. Thus, judging from these experiments, DCMB is devoid of significant az-receptor blocking properties. At the time for maximal brain A depletion after DCMB administration (4-6 h) the average firing rate of randomly encountered LC neurons was unaltered when compared with controls. In contrast, pretreatment with the tyrosine hydroxylase inhibitor a-methyl-ptyrosine methylester, which causes depletion also of brain noradrenaline, significantly increased LC neuronal firing rates. These data indicate that if there exists a physiologically relevant A-mediated input to the LC, this is not of critical importance for the tonic activity of the LC neurons.
0006-8993/81/00004)000/$ 2.50 © Elsevier/North-HollandBiomedicalPress
50 INTRODUCTION Previous biochemical studies have confirmed the existence of adrenaline (A) in mammalian brain 11. More recently, immunohistochemical methods have allowed mapping of both cell bodies and nerve terminals containing the A-forming enzyme phenylethanolamine-N-methyltransferase (PNMT) in the central nervous system12,13. The identified two groups of reticular nerve cell bodies in the medulla oblongata send their axons terminating into visceral nuclei of the brain stem and spinal cord as well as to nuclei in the hypothalamus and a major noradrenergic nucleus: the locus coeruleus (LC) in the pons. These LC noradrenergic neurons, which innervate almost the entire central nervous systeml~, 16,2a, are thought to be involved in various, e.g. behavioural and cardiovascular functions2,20, 30. Consequently, morphological evidence per se suggests the possibility that the LC noradrenergic neurons might be regulated inter alia by A afferents. This notion is consonant with the electrophysiological finding that various adrenergic agonists such as clonidine, an as-receptor agonist, noradrenaline (NA) as well as A and the fl-receptor agonist isoproterenol cause inhibition of firing of the LC noradrenergic neurons when applied microiontophoretically4,6, 24. Further studies utilizing various adrenergic antagonists revealed that the receptors involved in this inhibition could be characterized as a2-receptors 4,23. Thus, as-receptor antagonists such as yohimbine or piperoxane cause accelerated firing of the LC neurons both when applied systemically and microiontophoretically. Indeed, clonidine and piperoxane have been hypothesized to act as agonist and antagonist, respectively, at A synapses in brain 3. Thus, the above findings could be compatible with an A-mediated tonic, inhibitory influence on the LC NA system. However, recently an NA-mediated axon collateral inhibitory system in the LC has been demonstrated, which specifically was blocked by microiontophoretically applied piperoxane 1. Consequently, the effects of the a2-receptor active agents on LC neurons cannot either prove or disprove a putative functional significance of the A nerve terminals in the LC. In the absence of 'A receptor' specific agonists or antagonists, inhibitors of A synthesis, i.e. PNMT inhibitors, might prove interesting tools to elucidate the role of the A input to the LC. Therefore, it is significant that treatment with the PNMT inhibitor SK&F 64139 (7,8-dichloro-l,2,3,4-tetrahydroisoquinoline hydrochloride) 17 elicited an enhanced lowering of brain NA levels in stressed rats, particularly pronounced in the LC innervated frontal cortex~L This phenomenon was explained by a decreased inhibitory A-mediated influence on the LC, caused by the PNMT inhibitor. However, another explanation could be provided by an a~-receptor blocking effect of SK&F 64139, which should also cause increased firing rate of LC neurons, and recently we have presented some experimental evidence to support this view2L In the present paper we have studied systematically the role of the A input to the LC utilizing single cell recording techniques. Thus, the effects of SK &F 64139 on LC neurons were compared with those of SK&F 72223 (5,8-dimethoxy-l,2,3,4-tetrahydroisoquinoline hydrochloride) 19, a structural analogue to this drug without PNMT inhibitory effect and DCMB (2,3-dichloro-a-methylbenzylamine)7, another PNMT inhibitor.
51 In addition, the effect of A synthesis inhibition on the LC noradrenergic neurons was compared with the effect of inhibition also of the NA synthesis by a tyrosine hydroxylase inhibitor. The results will be discussed not only with respect to the functional significance of the A nerve terminals in the LC, but also with regard to various pharmacological effects of the drugs, e.g. on blood pressure, and their possible relationship to A systems in the brain. MATERIALSAND METHODS
Single unit recording The electrophysiological experiments were performed essentially as has been described previously27 on male, Sprague-Dawley rats. Briefly, the animals were anesthetized with chloral hydrate (400 mg/kg i.p.) and mounted in a stereotaxic apparatus. Additional injections were given as needed. A burr hole was drilled with its centre at 1.1 mm lateral and 1.1 mm posterior to the lambda. For recording a micropipette with a tip diameter of approximately 1/~m filled with 2 M NaC1 saturated with fast-green (impedance in vitro 3-6 Mf~, measured at 135 Hz) was lowered with a hydraulic microdrive into the brain. The electrode potentials were passed through a high input-impedance amplifier and filters. Each spike was discriminated and fed into an integrator being reset every 10 s and finally displayed on an oscilloscope, an audiomonitor and an oscillographic recorder. The body temperature of the animals was kept at 36-37 °C by means of a heating pad. Drugs were injected through a tail vein.
Identification of cells The neurophysiological characteristics of the cells were identical to those previously described for NA neurons of the rat LCI,10,14. No cells outside LC were included in this study. The final recording site of an experiment was marked by iontophoretic ejection of fast green. The rats were then perfused through the heart with 10 ~o formaldehyde solution and serial 50-/~m frozen sections of the brain were cut, mounted and stained with cresylviolet and counterstained with neutral red and the position of the dye was verified under the microscope. RESULTS Administration of SK&F 64139 consistently produced increased firing rate (up to 150 ~o) of the NA neurons in the LC (Fig. 1A). The effect was instantaneous in onset and dose-dependent within a broad dose range (2-200 mg/kg, Fig. 2A). Since there exists some indication that systemic administration of SK&F 64139 lowers blood pressure, at least in spontaneously hypertensive rats ~, arterial blood pressure was recorded via a tail artery in a few rats simultaneously with the electrophysiological recording from the LC after intravenous administration of the drug. Although a shortlasting hypotensive effect (5-10 min) was seen, this did not correlate with the long-
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Fig. 3. Dose-response curves for the inhibitory effect of clonidine on single cells in the LC of control rats (open symbols; n = 6) and rats pretreated (5 min, solid symbols) with (A) SK&F 64139 (30 mg/kg; n = 6) P < 0.01, (B) SK&F 72223 (3 mg/kg ;) P < 0.01 and (C) D C M B (40 mg/kg; n = 10) P < 0.01. Each point represents the response of one single cell in the LC, Lines have been plotted using linear regression analysis. Statistics: Regression lines for the individual animals were calculated; the intercepts of the control group were compared with the intercepts of the various treatment groups using Mann-Whitney's U-test.
55
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Fig. 4. Average spontaneous firing rate of randomly encountered NA neurons throughout the LC of control rats (n = 4) compared with rats pretreated with SK&F 64139 (35 mg/kg, 3-5 h; n = 4), SK&F 72223 (3 mg/kg, 4--6h; n = 4), DCMB (40 mg/kg, 4-6 h; n = 6) and a-MT (250 mg/kg, 5-6 h; n = 6). Shown are the means ± S.E.M. The number of cells recorded from are given in brackets in each column. *** P < 0.001. Also S K & F 72223, the structurally analogous compound without P N M T blocking properties, caused a dose-dependent excitation of all LC units tested (Figs. IB and 2B). In analogy with the results with S K & F 64139, pretreatment with S K & F 72223 antagonized the clonidine-induced depression of LC units (Fig. 1 B ) a n d caused a parallel shift to the right of the dose-response curve for clonidine. The other P N M T inhibitor tested, DCMB, produced no activation of the N A neurons in the LC (Fig. 1C), although at high doses, a slight stimulation of the LC neurons could possibly be detected (Fig. 2C). At low doses of D C M B no significant clonidine antagonistic action could be obtained and, in fact, a dose of 40 mg/kg i.v. of the P N M T inhibitor was necessary to obtain a slight parallel shift to the right of the dose-response curve for the a2-agonist (Fig. 3C). The activation of N A neurons in the LC by S K & F 64139 or S K & F 72223 remained approximately at the same level for several hours. Thus, 4-6 h after drug administration the average firing rate of neurons, randomly encountered throughout the LC, was significantly increased compared to that of the controls (Fig. 4). DCMB, however, did not cause any increased activity of the LC neuron also at this interval (4-6 h). In contrast, pretreatment with the tyrosine hydroxylase inhibitor a-methyl-ptyrosine methylester (a-MT), which inhibits also the synthesis of NA, causeda marked activation of the N A cells in the LC (Fig. 4). DISCUSSION
From the present data one can conclude that the activation of brain N A neurons in the LC by S K & F 64139 cannot solely be ascribed to inhibition of A synthesis for several reasons. Thus, the effect is instantaneous in onset, i.e. it occurs within seconds after intravenous administration of the drug. Clearly even if there exists a very small,
56 functionally important pool of newly synthetized A with a rapid turnover, the immediate effect by SK&F 64139 on the NA neurons must, in all probability, involve some other action than enzyme inhibition. Furthermore, the stimulatory effect on brain NA neurons in the LC is dose-dependent up to about 200 mg/kg, whereas brain P N M T activity is almost completely inhibited already after 10 mg/kg TM. Moreover, the LC activation was essentially of the same order of magnitude when studied immediately after injection of SK&F 64139 and when estimated 3-4 h later. Thus, the stimulation of the NA neurons does not correlate either with the time for maximal P N M T inhibition or that for maximal brain A depletion 22. It does not either correlate with the cardiovascular effects of the drug (c.f. above), which is important considering the documental peripheral, e.g. vagal inputs to the LC 5,26,2s. The rapid onset of the LC stimulatory effect by SK&F 64139 would rather suggest a receptor-mediated phenomenon to be involved. This is also indicated by the parallel shift to the right of the dose-response curve for the LC inhibitory effect of the a2-receptor agonist clonidine, without significant attenuation of its maximum response. The finding provides classical pharmacological evidence for an a2-receptor blocking effect of SK&F 64139. The question then arises whether A synthesis inhibition at all contributes to the stimulation of brain NA neurons in the LC by S K & F 64139. Such a contribution seems, in fact, distinctly unlikely for several reasons. Thus, the structurally analogous compound SK&F 72223, which does not possess P N M T inhibitory effect19, but in all probability exerts an az-receptor antagonistic action as is suggested by our experiments, also caused an immediate, dose-dependent stimulatory effect on the activity of the NA neurons in the LC. Moreover, the other P N M T inhibitor used, DCMB, did not cause significant stimulation of the NA neurons in the LC. This was the case also at the time for maximal brain A depletion after DCMB administration (4-6 h, Fig. 4) 7. Finally, judging from our experiments DCMB seems to possess but little, if any, a2-receptor blocking effect in contrast to SK&F 72223. Consequently, the stimulation of brain NA neurons in the LC by SK&F 64139 cannot be taken as evidence for an inhibitory, A-mediated control of these neurons, although such a regulatory mechanism may yet exist. Clearly, the absence of activation of LC neurons even at the time for maximal A depletion in brain after DCMB administration, if anything argues against a significant regulatory influence on the LC by A afferents. This is so, particularly in consideration of the fact that synthesis inhibition also involving NA systems by means of a-MT pretreatment actually did cause activation of the NA neurons in the LC. Thus, the present data taken together support the previously shown tonic inhibitory control of the LC neurons by noradrenergic nerve terminals, but leaves no support to the notion of a similar Amediated mechanism. Previously, the P N M T inhibitor SK&F 64139 has been used as a tool to disclose the significance of brain A systems for e.g. cardiovascular control. For example, intracerebrally administered SK&F 64139 has been found to elicit a hypertensive response in the rat a. This almost instantaneous effect, also obtained with SK&F 72223, which lacks PN MT inhibitory action, could presumably be related to an a2-receptor blocking
57 effect of the drugs. This interpretation is supported by the finding that intracerebral administration of other az-receptor blocking agents such as yohimbine also causes a hypertensive response 8,9. In any case, the present data make the interpretation of the blood pressure effects of the SK&F substances very difficult with respect to central A systems. Generally, our results imply that great caution must be exercised when using SK&F 64139 to elucidate the functional significance of brain A systems. ACKNOWLEDGEMENTS
The excellent technical assistance of Mrs. Birgitta Andersson is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (project No. 4747), ' T o r s t e n och R a g n a r S6derbergs Stiftelse', ' M a g n u s Bergvalls Stiftelse', 'Vilhelm och M a r t i n a L u n d g r e n s Vetenskaps fond', ' F o n d e n f6r N e u r o b i o l o g i s k F o r s k n i n g ' a n d the Medical Faculty, University of G6teborg.
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