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Altered serotonin and norepinephrine metabolism in rat dorsal raphe nucleus after drug-induced hypertension
Life Sciences, Vol. 34, pp. 1581-1589 Printed in the U.S.A.
ALTERED
SEROTONIN AND NOREPINEPHRINE METABOLISM RAT DORSAL RAPHE NUCLEUS AFTER DRUG-INDUCED HYPERTENSION
Pergamon Press
IN
Hirotoshi Echizen and Curt R. Freed I University of Colorado School of Medicine Departments of Medicine and Pharmacology, Box C237 b.200 East Ninth Avenue Denver, Colorado 80262 (Received in final form February 17, 1984) Summary The e f f e c t of drug-induced hypertension on neurotransmitter release f r o m dorsal raphe nucleus was studied by in vivo electrochemical electrodes in urethane anesthetized male Sprague-Dawley rats. Carbon paste electrodes were stereotaxically placed into dorsal raphe nucleus and neurot r a n s m i t t e r release was estimated electrochemically. Blood pressure was recorded f r o m a femoral a r t e r i a l catheter. Voltammograms taken f r o m dorsal raphe nucleus showed two distinct peaks corresponding to norepinephrine and 5-hydroxyindole acetic acid (5-HIAA). A f t e r basal blood pressure and neurotransmitter release were monitored for 30 min, blood pressure was raised 50 mmHg by continuous intravenous infusion of I_phenylephrine hydrochloride. Drug infusion was discontinued a f t e r 50 min, but blood pressure and neurotransmitter release were measured for an additional 2 hr. Results showed that the 5-HIAA response increased i m m e d i a t e l y a f t e r the i n i t i a t i o n of hypertension and remained elevated. By contrast, norepinephrine release i n i t i a l l y decreased, then returned to the basal level and then rose in parallel with 5-HIAA to a level above baseline as drug-induced hypertension was discontinued. The same experimental protocol was used to study the electrochemical response to drug-induced hypotension. Blood pressure was lowered 20 mmHg by intravenous infusion of sodium nitroprusside dihydrate. During hypotension, no changes were seen in either t r a n s m i t t e r response. However, as reflex hypertension appeared following discontinuation of the sodium nitroprusside infusion, the 5-HIAA response increased and the norepinephrine response decreased. These results show that drug-induced and reflex hypertension reduce norepinephrine release and increase serotonin turnover in dorsal raphe nucleus in anesthetized normotensive rats. These reciprocal changes appear to be a part of the neural response to hypertension. Central norepinephrine and serotonin are believed to have an i m p o r t a n t role in regulating blood pressure. Since Dahlstrom and Fuxe (i) revealed that serotonergic neurons were located in brain stem nuclei known to be cardioregulatory centers, pharmacological and electrophysiological studies have shown a relationship between neuronal activity and blood pressure changes. These data have been recently reviewed (2). The dorsal raphe nucleus is an important locus of serotonergic neurons and also receives dense noradrenergic innervation (3). W e therefore felt that this nucleus would be a useful site for studying changes in noradrenergic and serotonergic neurotransmitter activity that might occur in association with changes in blood pressure.
1 Address reprint requests to Curt R. Freed, M.D. 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Until recently, the push-pull cannula was the only technique available for detecting neurotransmitter release in vivo. Using this method, Philippu et al. (4,5) revealed that anterior hypothalamus released norepinephrine in response to druginduced hypotension, while posterior hypothalamus released norepinephrine during druginduced hypertension. The recent development of Ln viv___£oelectrochemistry has made it feasible to measure extraneuronal catecholamines and indoleamines (6,7). We have previously reported that the in vivo electrochemical electrode measures 5-hydroxyindole acetic acid (5-HIAA) and norepinephrine in rat dorsal raphe nucleus (8). Since 5HIAA concentration is a direct index of serotonin turnover in dorsal raphe nucleus and since noradrenergic neurons are known to synapse on dorsal raphe nucleus, we decided to study both serotonin and norepinephrine release in this nucleus during drug-induced hypertension and hypotension. Methods Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA), weighing 350-450 g, were anesthetized with urethane (1.25 g/kg) and were implanted with polyethylene catheters (PE-50) in the femoral artery for blood pressure monitoring and in the femoral vein for drug infusion. Stereotaxie coordinates for electrode implantation in dorsal raphe nucleus were taken from the atlas of Pellegrino et al. (9). The upper incisor bar of the stereotaxic frame was raised 5 mm above the interaural line. Bregma was used as the rostral-cauda] zero point. Coordinates used were A, -6.5 ram; L, +1.22 ram; V, -5.87 mm from the surface of dura. Electrodes were lowered at an angle of 12 degrees from vertical to avoid the sagittal sinus. Histological verification was done to confirm electrode placement. Carbon paste electrodes were fabricated accoding to Kissinger et al. (6), using a teflon coated 32-gauge stainless steel wire ( l e i c o Industries, Inc., New York, NY) filled with carbon paste (CP-O, Bioanalytieal Systems, Inc., West Lafayette, IN). Ag/AgCI electrodes and 27-gauge stainless steel needles were used as reference and auxiliary electrodes, respectively. A DCV-5 cyclic voltammetry amplifier with semiderivative signal processing (Bioanalytical Systems, Inc.) was used to measure the electrochemical responses. The working electrodes were scanned at the rate of either 10 mV/sec or 5 mV/sec. The slower sweep enhanced resolution of electrochemical peaks. Electrodes were scanned from -0.2 V to +0.5 V every 5 rain. Peak height was measured from trough-to-trough baseline. We have shown in previous experiments that carbon paste electrodes implanted in dorsal raphe nucleus measure two peaks in the voltage range -0.2 to +0.5 V. Pharmacological studies have indicated that peak 1 is related to extracellular norepinephrine while peak 2 represents primarily 5-HIAA (8). Briefly, in in vitro experiments, buffer solutions containing indoles (5-HTP, serotonin, 5-HIAA) or catecholamines (dopamine and norepinephrine) were scanned over the range of -0.2 to +0.5 V and showed well defined peaks at +0.25 and +0.15 V, respectively. No other precursors or major metabolites showed distinct peaks in this potential range. Tested compounds included tryptophan, tyrosine, 3,4-dihydroxyphenyla]anine(E)OPA), 3,/4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxymandelie acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3-methoxytyramine, normetanephrine and ascorbic acid. Some of those compounds, most particularly DOPAC and ascorbic acid, did produce continuously increasing current over the voltage range scanned but no peaks were observed for these compounds. Since peaks were measured from trough-to-trough baseline, the increasing baseline current did not interfere with peak height measurement. In vivo experiments were also performed using various pharmacologic treatments. The peak 1 response decreased after tyrosine hydroxylase inhibition with alpha-methyl-p-tyrosine and after dopamine-beta-hydroxylase inhibition with fusaric acid. Monoamine oxidase inhibition with pargyline did not change the peak 1 response. The peak 2 response decreased after tryptophan hydroxylase inhibition with p-chlorphenylalanine and monoamine oxidase
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i n h i b i t i o n w i t h pargy]ine. A l l of these results suggest t h a t peak 1 p r i m a r i l y represents norepinephrine and peak 2 is p r i m a r i l y produced by 5 - H I A A . A f t e r i n i t i a l s t a b i l i z a t i o n of the e l e c t r o c h e m i c a l response and the blood pressure, 3 0 - m i n u t e recordings of basal e l e c t r o c h e m i c a l and blood pressure data were obtained. Blood pressure was then e i t h e r raised or lowered w i t h drugs, in one group of animals, blood pressure was increased 50 m m H g above baseline f o r 50 rain by infusion of L phenylephrine hydrochloride at a dose of 1-10 p g / m i n ( A l d r i c h C h e m i c a l Company, Inc., M i l w a u k e e , WI). In another group of animals blood pressure was lowered 20 mmHg below the basal level f o r 50 min by infusion of sodium nitroprusside d i h y d r a t e at a dose of 0.25-1.24 p g / m i n (Roche L a b o r a t o r i e s , Nutiey, N0). Because the urethane anesthet i z e d animals had low baseline blood pressures (75+5 m m H g ) , only a 20 m m H g reduction in pressure could be sustained. Both drugs were continuously infused using a p e r i s t a l t i c infusion pump (Gilson Medical E l e c t r o n i c s , Inc., M i d d i e t o n , W]) at a rate to m a i n t a i n the desired blood pressure. A f t e r 50 rain, drug infusions were discontinued, but blood pressure and n e u r o t r a n s m i t t e r release were m o n i t o r e d for an a d d i t i o n a l 2 hr. E l e c t r o c h e m i c a l responses at each t i m e p o i n t were standardized to the mean basal response and expressed as the p e r c e n t of the basal value. Blood pressure data were expressed as the change f r o m the basal level. S t a t i s t i c a l analysis was done using the technique of analysis of variance for repeated measurements. Results Figure I shows a t y p i c a l in vivo v o l t a m m o g r a m taken f r o m dorsal raphe nucleus w i t h t w o d i s t i n c t peaks at +0.15-V'--~ak 1) and +0,25 V (peak 2). We have previously r e p o r t e d t h a t in dorsal raphe nucleus peak 1 is at least in p a r t derived f r o m e x t r a n e u r o n a l norepinephrine and peak 2 is produced by extraneuronaI 5 - H I A A . H i s t o l o g i c a l v e r i f i c a t i o n of e l e c t r o d e position in dorsal raphe nucleus has also been clone (8).
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Semiderivative voltammogram obtained f r o m dorsal raphe nucleus. Peak 1 appears to be produced by norepinephrine and peak 2 is produced by 5 - H I A A as described in the t e x t . E l e c t r o d e scanning was t e r m i n a t e d at + 0.5V.
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Raphe Norepinephrine in Hypertension
Vol. 34, No. 16, 1984
The effects of phenylephrine-induced hypertension on blood pressure and on electrochemical responses in dorsal raphe nucleus are shown in Figure 2. Blood pressure was raised 50 mmHg and maintained for 50 min. A f t e r discontinuation of the drug infusion, blood pressure fell below the basal level for a time but then increased to the baseline level. During the hypertensive period, peak 1 (norepinephrine) decreased during the initial 30 rain but then returned to the basal level. A f t e r discontinuation of the drug infusion, the norepinephrine peak showed a significant increase above the baseline response. By contrast, peak 2 (5-HIAA) increased soon after blood pressure was raised and this increase grew and persisted even after the drug infusion was stopped.
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FIG 2 Effects of phenylephrine-induced hypertension on peak 1 (norepinephrine) and peak 2 ((5-HIAA) responses, Changes in peak 1 and 2 are expressed as the percent change relative to baseline response, Changes in mean blood pressure are shown as the deviation from basal level. A l l points are represented by means + s.e.m. (n=6). Statistical significances are shown by *: p < 0.05 and * * : p < 0.01.
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Raphe Noreplnephrlne in Hypertension
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The e f f e c t of n i t r o p r u s s i d e - i n d u c e d hypotension on blood pressure and e l e c t r o c h e m i c a l responses is shown in Figure 3. Blood pressure was m a i n t a i n e d at a hypotensive level f o r 50 rain. A f t e r d i s c o n t i n u a t i o n of the infusion9 blood pressure rose above the basal level and r e m a i n e d e l e v a t e d . During hypotension9 n e i t h e r the norepinephrine nor the 5 - H [ A A peak showed a s i g n i f i c a n t d e v i a t i o n f r o m baseline. H o w e v e r , a f t e r nitroprusside was discontinued and transient hypertension was observed, the e x t r a e e l l u l a r norepinephrine c o n c e n t r a t i o n s i g n i f i c a n t l y decreased and e x t r a c e l l u l a r 5 - H I A A s i g n i f i c a n t l y increased.
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FIG :3 E f f e c t s of nitroprusside-induced hypotension on peak 1 (norepinephrine) and peak 2 (5-HIAA). Changes in peak 1 and 2 are expressed as the p e r c e n t change r e l a t i v e to the baseline responses. Changes in mean blood pressure are expressed as the d e v i a t i o n f r o m baseline level. A l l points are represented by mean -+ s.e.m. (n=6). S t a t i s t i c a l signifieances are shown by * : p < 0.05 and * * : p < O.01.
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in Hypertension
Vol. 34, No. 16, 1984
Discussion Our results indicate that drug-induced hypertension causes an increase in extracellular fluid 5-HIAA concentration and a biphasic reduction, then increase in extracellular fluid norepinephrine concentration. By contrast, drug-induced hypotension is not associated with a simultaneous change in the concentration of either compound in the anesthetized rat but does lead to a subsequent reflex hypertensive period during which the 5-HIAA concentration is increased and norepinephrine release is reduced. The changes in neurotransmitter metabolism which we have observed could be caused by direct pharmacological effects of phenylephrine and nitroprusside. However, since phenylephrine and nitroprusside are polar compounds, they are unlikely to penetrate the blood brain barrier. White phenylephrine is an alpha-1 receptor agonist, nitroprusside is not known to stimulate monoaminergic receptors (10). Moreover, the trend of changes in peak 1 and peak 2 responses were similar during phenylephrine induced hypertension and during the reflex hypertension which followed the cessation of the nitroprusside infusion. Therefore, the observed changes in serotonin and norepinephrine metabolism in dorsal raphe nucleus are likely to be related to the increase in blood pressure and not to direct drug effects. It is possible that changes in local cerebral blood flow during hypertension have caused the observed changes in neurotransmitter levels. Norepinephrine-induced hypertension has been associated with regional changes in cerebral blood flow and glucose metabolism (11). We cannot say for certain whether such changes in cerebral blood flow and glucose utilization could have caused the changes in neurotransmitter turnover we have observed. However, since brain stem nuclei respond to baroreceptor input from many sites, it is more likely that the changes in dorsal raphe are related to changes in systemic pressure. Ascorbic acid is known to exist in high concentrations in brain and has been shown to be released in vitro and in vivo by neuronal depolarization (12). On the other hand, the extracellular fluid ascorbie acid concentration remains constant even after severe depletion of tissue ascorbic acid (13). Others have described amplification of the catecholamine electrochemical signal by ascorbic acid (1/4). Therefore, it is possible that a complex interaction between ascorbic acid and norepinephrine may have influenced the magnitude of the electrochemical changes we have observed. A detailed understanding of such potential interaction requires further study. Evidence from several sources suggests that serotonergic neurons in dorsal raphe nucleus are involved in maintaining blood pressure. Electrical stimulation of dorsal raphe nucleus ]eads to an increase in blood pressure (15) presumably due to increased release of serotonin from nerve terminal areas such as hypotha]amus. This possibility is supported by the fact that direct application of serotonin to anterior hypothalamus will raise blood pressure (16). On the other hand, serotonin will inhibit firing of dorsal raphe nucleus when directly applied to the nucleus (17,18). Pharmacologic studies indicate that the inhibitory action of serotonin on brainstem raphe nuclei may be the more important action in overall regulation of blood pressure. When the serotonin precursor 5-hydroxytryptophan is administered systemically, blood pressure is reduced (19,20). The site of action of the effect appears to be in the brainstem since Tadepalli et al. (21) observed hypotension in animals given 5-hydroxytryptophan in the lateral ventricle, and the hypotension could be blocked by draining the drug-containing CSF at the level of the aqueduct. In light of the link between serotonergic neuronal activity and blood pressure described above, the increased serotonin turnover which we have seen during druginduced and reflex hypertension allows us to speculate that firing of serotonin neurons in dorsal raphe nucleus is inhibited during hypertension.
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in Hypertension
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Others have shown that noradrenergic and serotonergic neurons in brainstem i n t e r a c t both e l e c t r i c a l l y and neurochemically (22-24). Etectrophysiological studies have revealed that: reduction of noradrenergic tone by blockade of alpha-receptors, by inhibition of catecholamine synthesis, and by inhibition of noradrenergic celt firing w i l l all lead to reduced serotonergic cell firing (16,25). Neurochemica] approaches have demonstrated that reduced concentrations of norepinephrine are associated with increased turnover of 5 - H I A A and that high concentrations of norepinephrine inhibit serotonin release. Norepinephrine w i l l inhibit serotonin release from rat brain slices and synaptosomal preparations (26,27). When the dopamine-beta-hydroxylase inhibitor fusaric acid is administered to rats, brain norepinephrine concentration falls while serotonin turnover increases (28). We have confirmed this result using in vivo e l e c t r o c h e m i c a l recording in dorsal raphe nucleus (8). This evidence suggests that norepinephrine may t o n i c a l l y inhibit serotonin release and turnover in dorsal raphe nucleus. Because of the inhibitory action of serotonin on autoreceptors in raphe nucleus, enhanced norepinephrine turnover may be a marker for enhanced raphe cell firing. Conversely, reduced norepinephrine release may be associated with enhanced serotonin turnover in raphe but reduced serotonergic cell firing. We have observed that norepinephrine release i n i t i a l l y fel~ then increased during phenytephrine-induced hypertension and was persistently reduced during the compensatory hypertension that followed nitroprusside infusion. Since norepinephrine has an inhibitory action on serotonin turnover as described above, the reduction in e x t r a cellular fluid norepinephrine concentration may be the cause of the increased 5 - H I A A concentration during hypertension. We hypothesize a neural network in dorsal raphe nucleus with noradrenergic neurons making synaptic connections on inhibitory recurrent c o l l a t e r a l serotonergic dendrites as shown in Figure 4. The physiological meaning of the changes in serotonin and norepinephrine which we have observed remain conjectural. We have seen increases in 5 - H I A A and reductions in norepinephrine in association with two very d i f f e r e n t forms of hypertension. Since drug-induced hypertension is accompanied by a reduction in sympathetic o u t f l o w f r o m the brain while r e f l e x hypertension is the result of increased sympathetic a c t i v i t y , it is unlikely that the changes we have seen in dorsal raphe nucleus are a measure of central sympathetic a c t i v i t y . Since similar chemical changes were seen with similar changes in blood pressure, it is likely that the dorsal raphe nucleus responses are related to the increase in pressure alone. As such, the n e u r o t r a n s m i t t e r responses may r e f l e c t baroreceptor a c t i v i t y . Since dorsal raphe nucleus is not known to be linked to baroreceptor inputs, it is possible that the noradrenergic input is a primary signal carrying i n f o r m a t i o n about peripheral blood pressure changes. The i n i t i a l decline in norepinephrine release which we observed at the onset of drug-induced hypertension might be the trigger for increased local serotonin release leading to decreased cell firing in dorsal raphe nucleus. This t h e o r e t i c a l chain of events fits with the facts that increased f i r i n g of dorsal raphe nucleus leads to increased blood pressure and that increased serotonin turnover in brainstem reduces blood pressure. The changes we have seen in dorsal raphe nucleus during hypertension may be interpreted as part of the central hypotensive response i n i t i a t e d by the baroreceptors. In summary, we have seen that drug-induced and r e f l e x hypertension decrease norepinephrine release and increase serotonin turnover in dorsal raphe nucleus of anesthetized normotensive rats. These n e u r o t r a n s m i t t e r changes may represent a baroreceptor-mediated compensatory response to induced hypertension.
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f
Vol. 34, No. 16, 1984
Hypothalamus
Dorsal Raphe Nucleus
FIG 4
Hypothetical interaction between norepinephrine and serotonin in the regulation of firing of seretonergic neurons in the dorsal raphe nucleus. Recurrent collateral serotonergic dendrites inhibit ceil firing. They in turn are inhibited by noradrenergic neurons. Acknowledgment This work was supported by U.S. Public Health Service Grants NS 09199, NS 18639, H I 30722; a grant from Merck Sharp & Dohme, and an RCDA HL 00782 (CRF-).
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References
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A. DAHLSTROM and K. FUXE, Aeta Physiol. Scand. 6_44suppl. 247, 1-35 (1965). D.M. KUHN, W.A. WOLF and W. LOVENBERG, Hypertension 2243-255 (1981). 3.M. B A R A B A N and Q.K. A Q H A J A N I A N , Brain Res. 2041-11-(1981). A. PHILIPPU, H. DIETL and 3.N. SINHA, Naunyn-Schmiedeberg's Arch. Pharmaeol. 310237-240 (1980). A. PHILIPPU,--H. DIETL and A. EISERT, Eur. 3. Pharmaeol. 69519-523 (1981). P.T. KISSINQER, 3.B. HART and R.N. ADAMS, Brain Res. 55--2119-213 (1973). R.F. LANE, A.T. HUBBARD and C.D. BLAHA, Bioelectrochem. Bioenergt. 5_ 504525 (1978)). H. ECHIZEN and C.R. FREED, Brain Res. 27755-62 (1983). L. 3. PELLEQRINO, A.S. PELLEQR]NO and A.O. CUSHMAN, A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York (1979). R.F. PALMER and K.C. LASSETER, N. Eng], 3. Med. 292294-297 (1975). W. KUSCHINSKY and M. WAHL, Pharm. Reviews 58656-689 (1978) K.H. MILBY, I.N. MEFFORD, W. CHEY, and R.N. ADAMS, Brain Res. Bull, 7_237242 (1981). 3.O. SCHENK, E. MILLER, R. QODDES, and R.N. ADAMS, Brain Res. 253353-356 (1982). R.D. O'NEILL, R.A. GRUNEWALD, M. FILLENZ, and W.3. ALBERY, Neurosciences 71945-1954 (1982). D.M. KUHN, W~A. WOLF and W. LOVENBERG, J. Pharmacol. Exp. Ther. 214403-
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W.A. WOLF, O.M. KUHN and W. LOVENBERG, Brain Res. 208192-197 (1981). D.W. GALLAGER and G.K. A G H A J A N I A N , Neuropharmacol. 155149-156 (1976). H.3. HAIGLER and G.K. A G H A J A N I A N , 3. Pharmacol. Exp. Ther. 188688-699 (1974). M. HENNING and A RUBENSON, Acta Pharmacol. Toxicol. 2_99145-154 (1971). H. ECHIZEN and C.R. FREED, J. Pharmacol. Exp. Ther. 220579-584 (1982). E. TADEPALLI, E. MILLS and S.M. SHANBE£G, 3. Pharmacol. Exp. Ther. 202 510-319 (1977). W. KOSTOWSKI, R. SAMANIN, S.R. BAREGGI, V. MARC, S. GARATTINI and L. VALZELLI, Brain Res. 82178-182 (1974). A. McRAE-DEGUERCE, A. BEROD, A. MERMET G. KELLER, T.H. CHOUVET, T.H. 3OH and J.F. PUJOL, Brain Res. 235285-301 (1982). 3.F. REINHARD and R.H. ROTH, 3. Pharmacol. Exp. Ther. 221541-546 (1982). T.H. SVENSSON, B.S. BUNNEY and G.K. A G H A J A N I A N , Brain Res. 9__22291-308 (1975). A.L. F R A N K H U Y Z E N and A.H. MULDER, Eur. J. Pharmacol. 3179-182 (1980). (3. M A U R A , A GEMINQNANI and M. RAITERI, Naunyn-Schmiedeberg's Arch. PharmacoL 320272-274 (1982). G.A. JOHNSON, E.G. KIM and S.3. BOUKMA, J. Pharmacol. Exp. Ther. 180539546 (1972).
ALTERED
SEROTONIN AND NOREPINEPHRINE METABOLISM RAT DORSAL RAPHE NUCLEUS AFTER DRUG-INDUCED HYPERTENSION
Pergamon Press
IN
Hirotoshi Echizen and Curt R. Freed I University of Colorado School of Medicine Departments of Medicine and Pharmacology, Box C237 b.200 East Ninth Avenue Denver, Colorado 80262 (Received in final form February 17, 1984) Summary The e f f e c t of drug-induced hypertension on neurotransmitter release f r o m dorsal raphe nucleus was studied by in vivo electrochemical electrodes in urethane anesthetized male Sprague-Dawley rats. Carbon paste electrodes were stereotaxically placed into dorsal raphe nucleus and neurot r a n s m i t t e r release was estimated electrochemically. Blood pressure was recorded f r o m a femoral a r t e r i a l catheter. Voltammograms taken f r o m dorsal raphe nucleus showed two distinct peaks corresponding to norepinephrine and 5-hydroxyindole acetic acid (5-HIAA). A f t e r basal blood pressure and neurotransmitter release were monitored for 30 min, blood pressure was raised 50 mmHg by continuous intravenous infusion of I_phenylephrine hydrochloride. Drug infusion was discontinued a f t e r 50 min, but blood pressure and neurotransmitter release were measured for an additional 2 hr. Results showed that the 5-HIAA response increased i m m e d i a t e l y a f t e r the i n i t i a t i o n of hypertension and remained elevated. By contrast, norepinephrine release i n i t i a l l y decreased, then returned to the basal level and then rose in parallel with 5-HIAA to a level above baseline as drug-induced hypertension was discontinued. The same experimental protocol was used to study the electrochemical response to drug-induced hypotension. Blood pressure was lowered 20 mmHg by intravenous infusion of sodium nitroprusside dihydrate. During hypotension, no changes were seen in either t r a n s m i t t e r response. However, as reflex hypertension appeared following discontinuation of the sodium nitroprusside infusion, the 5-HIAA response increased and the norepinephrine response decreased. These results show that drug-induced and reflex hypertension reduce norepinephrine release and increase serotonin turnover in dorsal raphe nucleus in anesthetized normotensive rats. These reciprocal changes appear to be a part of the neural response to hypertension. Central norepinephrine and serotonin are believed to have an i m p o r t a n t role in regulating blood pressure. Since Dahlstrom and Fuxe (i) revealed that serotonergic neurons were located in brain stem nuclei known to be cardioregulatory centers, pharmacological and electrophysiological studies have shown a relationship between neuronal activity and blood pressure changes. These data have been recently reviewed (2). The dorsal raphe nucleus is an important locus of serotonergic neurons and also receives dense noradrenergic innervation (3). W e therefore felt that this nucleus would be a useful site for studying changes in noradrenergic and serotonergic neurotransmitter activity that might occur in association with changes in blood pressure.
1 Address reprint requests to Curt R. Freed, M.D. 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Until recently, the push-pull cannula was the only technique available for detecting neurotransmitter release in vivo. Using this method, Philippu et al. (4,5) revealed that anterior hypothalamus released norepinephrine in response to druginduced hypotension, while posterior hypothalamus released norepinephrine during druginduced hypertension. The recent development of Ln viv___£oelectrochemistry has made it feasible to measure extraneuronal catecholamines and indoleamines (6,7). We have previously reported that the in vivo electrochemical electrode measures 5-hydroxyindole acetic acid (5-HIAA) and norepinephrine in rat dorsal raphe nucleus (8). Since 5HIAA concentration is a direct index of serotonin turnover in dorsal raphe nucleus and since noradrenergic neurons are known to synapse on dorsal raphe nucleus, we decided to study both serotonin and norepinephrine release in this nucleus during drug-induced hypertension and hypotension. Methods Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA), weighing 350-450 g, were anesthetized with urethane (1.25 g/kg) and were implanted with polyethylene catheters (PE-50) in the femoral artery for blood pressure monitoring and in the femoral vein for drug infusion. Stereotaxie coordinates for electrode implantation in dorsal raphe nucleus were taken from the atlas of Pellegrino et al. (9). The upper incisor bar of the stereotaxic frame was raised 5 mm above the interaural line. Bregma was used as the rostral-cauda] zero point. Coordinates used were A, -6.5 ram; L, +1.22 ram; V, -5.87 mm from the surface of dura. Electrodes were lowered at an angle of 12 degrees from vertical to avoid the sagittal sinus. Histological verification was done to confirm electrode placement. Carbon paste electrodes were fabricated accoding to Kissinger et al. (6), using a teflon coated 32-gauge stainless steel wire ( l e i c o Industries, Inc., New York, NY) filled with carbon paste (CP-O, Bioanalytieal Systems, Inc., West Lafayette, IN). Ag/AgCI electrodes and 27-gauge stainless steel needles were used as reference and auxiliary electrodes, respectively. A DCV-5 cyclic voltammetry amplifier with semiderivative signal processing (Bioanalytical Systems, Inc.) was used to measure the electrochemical responses. The working electrodes were scanned at the rate of either 10 mV/sec or 5 mV/sec. The slower sweep enhanced resolution of electrochemical peaks. Electrodes were scanned from -0.2 V to +0.5 V every 5 rain. Peak height was measured from trough-to-trough baseline. We have shown in previous experiments that carbon paste electrodes implanted in dorsal raphe nucleus measure two peaks in the voltage range -0.2 to +0.5 V. Pharmacological studies have indicated that peak 1 is related to extracellular norepinephrine while peak 2 represents primarily 5-HIAA (8). Briefly, in in vitro experiments, buffer solutions containing indoles (5-HTP, serotonin, 5-HIAA) or catecholamines (dopamine and norepinephrine) were scanned over the range of -0.2 to +0.5 V and showed well defined peaks at +0.25 and +0.15 V, respectively. No other precursors or major metabolites showed distinct peaks in this potential range. Tested compounds included tryptophan, tyrosine, 3,4-dihydroxyphenyla]anine(E)OPA), 3,/4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxymandelie acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3-methoxytyramine, normetanephrine and ascorbic acid. Some of those compounds, most particularly DOPAC and ascorbic acid, did produce continuously increasing current over the voltage range scanned but no peaks were observed for these compounds. Since peaks were measured from trough-to-trough baseline, the increasing baseline current did not interfere with peak height measurement. In vivo experiments were also performed using various pharmacologic treatments. The peak 1 response decreased after tyrosine hydroxylase inhibition with alpha-methyl-p-tyrosine and after dopamine-beta-hydroxylase inhibition with fusaric acid. Monoamine oxidase inhibition with pargyline did not change the peak 1 response. The peak 2 response decreased after tryptophan hydroxylase inhibition with p-chlorphenylalanine and monoamine oxidase
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i n h i b i t i o n w i t h pargy]ine. A l l of these results suggest t h a t peak 1 p r i m a r i l y represents norepinephrine and peak 2 is p r i m a r i l y produced by 5 - H I A A . A f t e r i n i t i a l s t a b i l i z a t i o n of the e l e c t r o c h e m i c a l response and the blood pressure, 3 0 - m i n u t e recordings of basal e l e c t r o c h e m i c a l and blood pressure data were obtained. Blood pressure was then e i t h e r raised or lowered w i t h drugs, in one group of animals, blood pressure was increased 50 m m H g above baseline f o r 50 rain by infusion of L phenylephrine hydrochloride at a dose of 1-10 p g / m i n ( A l d r i c h C h e m i c a l Company, Inc., M i l w a u k e e , WI). In another group of animals blood pressure was lowered 20 mmHg below the basal level f o r 50 min by infusion of sodium nitroprusside d i h y d r a t e at a dose of 0.25-1.24 p g / m i n (Roche L a b o r a t o r i e s , Nutiey, N0). Because the urethane anesthet i z e d animals had low baseline blood pressures (75+5 m m H g ) , only a 20 m m H g reduction in pressure could be sustained. Both drugs were continuously infused using a p e r i s t a l t i c infusion pump (Gilson Medical E l e c t r o n i c s , Inc., M i d d i e t o n , W]) at a rate to m a i n t a i n the desired blood pressure. A f t e r 50 rain, drug infusions were discontinued, but blood pressure and n e u r o t r a n s m i t t e r release were m o n i t o r e d for an a d d i t i o n a l 2 hr. E l e c t r o c h e m i c a l responses at each t i m e p o i n t were standardized to the mean basal response and expressed as the p e r c e n t of the basal value. Blood pressure data were expressed as the change f r o m the basal level. S t a t i s t i c a l analysis was done using the technique of analysis of variance for repeated measurements. Results Figure I shows a t y p i c a l in vivo v o l t a m m o g r a m taken f r o m dorsal raphe nucleus w i t h t w o d i s t i n c t peaks at +0.15-V'--~ak 1) and +0,25 V (peak 2). We have previously r e p o r t e d t h a t in dorsal raphe nucleus peak 1 is at least in p a r t derived f r o m e x t r a n e u r o n a l norepinephrine and peak 2 is produced by extraneuronaI 5 - H I A A . H i s t o l o g i c a l v e r i f i c a t i o n of e l e c t r o d e position in dorsal raphe nucleus has also been clone (8).
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FIG 1
¢t) I~1
Semiderivative voltammogram obtained f r o m dorsal raphe nucleus. Peak 1 appears to be produced by norepinephrine and peak 2 is produced by 5 - H I A A as described in the t e x t . E l e c t r o d e scanning was t e r m i n a t e d at + 0.5V.
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Vol. 34, No. 16, 1984
The effects of phenylephrine-induced hypertension on blood pressure and on electrochemical responses in dorsal raphe nucleus are shown in Figure 2. Blood pressure was raised 50 mmHg and maintained for 50 min. A f t e r discontinuation of the drug infusion, blood pressure fell below the basal level for a time but then increased to the baseline level. During the hypertensive period, peak 1 (norepinephrine) decreased during the initial 30 rain but then returned to the basal level. A f t e r discontinuation of the drug infusion, the norepinephrine peak showed a significant increase above the baseline response. By contrast, peak 2 (5-HIAA) increased soon after blood pressure was raised and this increase grew and persisted even after the drug infusion was stopped.
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FIG 2 Effects of phenylephrine-induced hypertension on peak 1 (norepinephrine) and peak 2 ((5-HIAA) responses, Changes in peak 1 and 2 are expressed as the percent change relative to baseline response, Changes in mean blood pressure are shown as the deviation from basal level. A l l points are represented by means + s.e.m. (n=6). Statistical significances are shown by *: p < 0.05 and * * : p < 0.01.
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Raphe Noreplnephrlne in Hypertension
1585
The e f f e c t of n i t r o p r u s s i d e - i n d u c e d hypotension on blood pressure and e l e c t r o c h e m i c a l responses is shown in Figure 3. Blood pressure was m a i n t a i n e d at a hypotensive level f o r 50 rain. A f t e r d i s c o n t i n u a t i o n of the infusion9 blood pressure rose above the basal level and r e m a i n e d e l e v a t e d . During hypotension9 n e i t h e r the norepinephrine nor the 5 - H [ A A peak showed a s i g n i f i c a n t d e v i a t i o n f r o m baseline. H o w e v e r , a f t e r nitroprusside was discontinued and transient hypertension was observed, the e x t r a e e l l u l a r norepinephrine c o n c e n t r a t i o n s i g n i f i c a n t l y decreased and e x t r a c e l l u l a r 5 - H I A A s i g n i f i c a n t l y increased.
t~,o F
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FIG :3 E f f e c t s of nitroprusside-induced hypotension on peak 1 (norepinephrine) and peak 2 (5-HIAA). Changes in peak 1 and 2 are expressed as the p e r c e n t change r e l a t i v e to the baseline responses. Changes in mean blood pressure are expressed as the d e v i a t i o n f r o m baseline level. A l l points are represented by mean -+ s.e.m. (n=6). S t a t i s t i c a l signifieances are shown by * : p < 0.05 and * * : p < O.01.
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Discussion Our results indicate that drug-induced hypertension causes an increase in extracellular fluid 5-HIAA concentration and a biphasic reduction, then increase in extracellular fluid norepinephrine concentration. By contrast, drug-induced hypotension is not associated with a simultaneous change in the concentration of either compound in the anesthetized rat but does lead to a subsequent reflex hypertensive period during which the 5-HIAA concentration is increased and norepinephrine release is reduced. The changes in neurotransmitter metabolism which we have observed could be caused by direct pharmacological effects of phenylephrine and nitroprusside. However, since phenylephrine and nitroprusside are polar compounds, they are unlikely to penetrate the blood brain barrier. White phenylephrine is an alpha-1 receptor agonist, nitroprusside is not known to stimulate monoaminergic receptors (10). Moreover, the trend of changes in peak 1 and peak 2 responses were similar during phenylephrine induced hypertension and during the reflex hypertension which followed the cessation of the nitroprusside infusion. Therefore, the observed changes in serotonin and norepinephrine metabolism in dorsal raphe nucleus are likely to be related to the increase in blood pressure and not to direct drug effects. It is possible that changes in local cerebral blood flow during hypertension have caused the observed changes in neurotransmitter levels. Norepinephrine-induced hypertension has been associated with regional changes in cerebral blood flow and glucose metabolism (11). We cannot say for certain whether such changes in cerebral blood flow and glucose utilization could have caused the changes in neurotransmitter turnover we have observed. However, since brain stem nuclei respond to baroreceptor input from many sites, it is more likely that the changes in dorsal raphe are related to changes in systemic pressure. Ascorbic acid is known to exist in high concentrations in brain and has been shown to be released in vitro and in vivo by neuronal depolarization (12). On the other hand, the extracellular fluid ascorbie acid concentration remains constant even after severe depletion of tissue ascorbic acid (13). Others have described amplification of the catecholamine electrochemical signal by ascorbic acid (1/4). Therefore, it is possible that a complex interaction between ascorbic acid and norepinephrine may have influenced the magnitude of the electrochemical changes we have observed. A detailed understanding of such potential interaction requires further study. Evidence from several sources suggests that serotonergic neurons in dorsal raphe nucleus are involved in maintaining blood pressure. Electrical stimulation of dorsal raphe nucleus ]eads to an increase in blood pressure (15) presumably due to increased release of serotonin from nerve terminal areas such as hypotha]amus. This possibility is supported by the fact that direct application of serotonin to anterior hypothalamus will raise blood pressure (16). On the other hand, serotonin will inhibit firing of dorsal raphe nucleus when directly applied to the nucleus (17,18). Pharmacologic studies indicate that the inhibitory action of serotonin on brainstem raphe nuclei may be the more important action in overall regulation of blood pressure. When the serotonin precursor 5-hydroxytryptophan is administered systemically, blood pressure is reduced (19,20). The site of action of the effect appears to be in the brainstem since Tadepalli et al. (21) observed hypotension in animals given 5-hydroxytryptophan in the lateral ventricle, and the hypotension could be blocked by draining the drug-containing CSF at the level of the aqueduct. In light of the link between serotonergic neuronal activity and blood pressure described above, the increased serotonin turnover which we have seen during druginduced and reflex hypertension allows us to speculate that firing of serotonin neurons in dorsal raphe nucleus is inhibited during hypertension.
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Others have shown that noradrenergic and serotonergic neurons in brainstem i n t e r a c t both e l e c t r i c a l l y and neurochemically (22-24). Etectrophysiological studies have revealed that: reduction of noradrenergic tone by blockade of alpha-receptors, by inhibition of catecholamine synthesis, and by inhibition of noradrenergic celt firing w i l l all lead to reduced serotonergic cell firing (16,25). Neurochemica] approaches have demonstrated that reduced concentrations of norepinephrine are associated with increased turnover of 5 - H I A A and that high concentrations of norepinephrine inhibit serotonin release. Norepinephrine w i l l inhibit serotonin release from rat brain slices and synaptosomal preparations (26,27). When the dopamine-beta-hydroxylase inhibitor fusaric acid is administered to rats, brain norepinephrine concentration falls while serotonin turnover increases (28). We have confirmed this result using in vivo e l e c t r o c h e m i c a l recording in dorsal raphe nucleus (8). This evidence suggests that norepinephrine may t o n i c a l l y inhibit serotonin release and turnover in dorsal raphe nucleus. Because of the inhibitory action of serotonin on autoreceptors in raphe nucleus, enhanced norepinephrine turnover may be a marker for enhanced raphe cell firing. Conversely, reduced norepinephrine release may be associated with enhanced serotonin turnover in raphe but reduced serotonergic cell firing. We have observed that norepinephrine release i n i t i a l l y fel~ then increased during phenytephrine-induced hypertension and was persistently reduced during the compensatory hypertension that followed nitroprusside infusion. Since norepinephrine has an inhibitory action on serotonin turnover as described above, the reduction in e x t r a cellular fluid norepinephrine concentration may be the cause of the increased 5 - H I A A concentration during hypertension. We hypothesize a neural network in dorsal raphe nucleus with noradrenergic neurons making synaptic connections on inhibitory recurrent c o l l a t e r a l serotonergic dendrites as shown in Figure 4. The physiological meaning of the changes in serotonin and norepinephrine which we have observed remain conjectural. We have seen increases in 5 - H I A A and reductions in norepinephrine in association with two very d i f f e r e n t forms of hypertension. Since drug-induced hypertension is accompanied by a reduction in sympathetic o u t f l o w f r o m the brain while r e f l e x hypertension is the result of increased sympathetic a c t i v i t y , it is unlikely that the changes we have seen in dorsal raphe nucleus are a measure of central sympathetic a c t i v i t y . Since similar chemical changes were seen with similar changes in blood pressure, it is likely that the dorsal raphe nucleus responses are related to the increase in pressure alone. As such, the n e u r o t r a n s m i t t e r responses may r e f l e c t baroreceptor a c t i v i t y . Since dorsal raphe nucleus is not known to be linked to baroreceptor inputs, it is possible that the noradrenergic input is a primary signal carrying i n f o r m a t i o n about peripheral blood pressure changes. The i n i t i a l decline in norepinephrine release which we observed at the onset of drug-induced hypertension might be the trigger for increased local serotonin release leading to decreased cell firing in dorsal raphe nucleus. This t h e o r e t i c a l chain of events fits with the facts that increased f i r i n g of dorsal raphe nucleus leads to increased blood pressure and that increased serotonin turnover in brainstem reduces blood pressure. The changes we have seen in dorsal raphe nucleus during hypertension may be interpreted as part of the central hypotensive response i n i t i a t e d by the baroreceptors. In summary, we have seen that drug-induced and r e f l e x hypertension decrease norepinephrine release and increase serotonin turnover in dorsal raphe nucleus of anesthetized normotensive rats. These n e u r o t r a n s m i t t e r changes may represent a baroreceptor-mediated compensatory response to induced hypertension.
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f
Vol. 34, No. 16, 1984
Hypothalamus
Dorsal Raphe Nucleus
FIG 4
Hypothetical interaction between norepinephrine and serotonin in the regulation of firing of seretonergic neurons in the dorsal raphe nucleus. Recurrent collateral serotonergic dendrites inhibit ceil firing. They in turn are inhibited by noradrenergic neurons. Acknowledgment This work was supported by U.S. Public Health Service Grants NS 09199, NS 18639, H I 30722; a grant from Merck Sharp & Dohme, and an RCDA HL 00782 (CRF-).
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