Central serotonergic activity after neurogenic hypertension

European Journal of Pharmacology, 86 (1983) 337-345

337

Elsevier Biomedical Press

CENTRAL SEROTONERGIC

ACTIVITY AFTER NEUROGENIC

HYPERTENSION

SILVIA GIARCOVICH-MARTiNEZ *, MABEL FERNANDEZ, ELBA CHEMERINSKI and MARIA AMELIA ENERO lnstituto de lnvestigaciones FarmacolDgicas, CONICET, C(Jtedra de Farmacologla, Facultad de Farmacia y Bioqulmica, U,B.A. Junin 956, 5 ° Piso, Buenos Aires 1113, Argentina

Received 2 December 1981, revised MS received 13 July 1982, accepted 30 September 1982

S. GIARCOVICH-MART~NEZ, M. FERNANDEZ, E. CHEMERINSKI and M.A. ENERO, Central serotonergic activity after neurogenic hypertension, European J. Pharmacol. 86 (1983) 337-345.

Sinoaortic denervated rats, 24 h after operation, showed a 25% increase in the central (hypothalamus, frontal cortex, midbrain, pons and medulla oblongata) tryptophan content and about a 70% rise in plasma free fatty acid (NEFA) concentration. The synaptosomal serotonin (5-HT) uptake and tryptophan hydroxylase activity of slices from these central areas were not significantly different when compared to those from sham-operated rats. There was also an increase in tryptophan and N E F A concentration at 24 h in fasted sham-operated rats. Seven days after neurogenic hypertension, the central tryptophan content had returned to control values. However, a 50% increase in the tryptophan hydroxylase activity of slices from the midbrain area together with a higher turnover rate of serotonin was found in the denervated group of rats. The synaptosomal 5-HT uptake remained unchanged. These results suggest that sinoaortic denervation could induce changes in the serotonergic neurons localized in central nervous system areas during the first week after operation. Serotonergic central nervous system

Sinoaortic denervation

1. Introduction The d e v e l o p m e n t of neurogenic h y p e r t e n s i o n after a t t e n u a t i o n of the high pressure baroreflex arc b y sinoaortic d e n e r v a t i o n ( S A D ) is a c c o m p a nied b y increased activity of s y m p a t h e t i c nora d r e n e r g i c n e u r o n s ( D e Q u a t t r o et al., 1969; Chalmers, 1975). Moreover, e x p e r i m e n t s with rabbits have shown that S A D p r o d u c e s a selective increase in n o r a d r e n a l i n e turnover in the h y p o t h a l a m u s a n d spinal cord ( C h a l m e r s a n d W u r t m a n , 1971). On the other hand, Smith et al. (1979) have suggested the possible p a r t i c i p a t i o n of serotonergic n e u r o n s in the d e v e l o p m e n t of hypertension in the s p o n t a n e o u s l y hypertensive rat. C h e m e r i n s k i et al. (1980) described an increase in

Hypertensive rat

Serotonin turnover

serotonin (5-HT) and 5 - h y d r o x y i n d o l a c e t i c acid c o n t e n t in some areas of the rat brain after S A D . These changes could be related to the neurogenic h y p e r t e n s i o n i n d u c e d b y the o p e r a t i o n and suggest that there are alterations in the control of the factors that regulate 5 - H T biosynthesis. The object of this s t u d y was to analyse some m e c h a n i s m s which could be involved in the increase of the 5 - H T c o n c e n t r a t i o n observed after S A D . F o r this purpose, the serotonin uptake, t r y p t o p h a n h y d r o x y l a s e activity and turnover rate o f 5 - H T were studied in different areas of the central nervous system 24 h a n d 7 days after SAD.

2. M a t e r i a l s and methods F e m a l e W i s t a r rats ( 1 5 0 - 1 9 0 g) were used. S A D

* To whom all correspondence should be addressed: Instituto de Investigaciones Farmacol~gicas, Junln 956, 5° Piso, 1113 Buenos Aires, Argentina. 0014-2999/83/0000-0000/$03.00

L' 1983 Elsevier Biomedical Press

leading to a sustained increase in b l o o d pressure was p e r f o r m e d following the m e t h o d d e s c r i b e d b y Krieger (1964). The s h a m o p e r a t i o n consisted in

338 isolating nerves without sectioning them. SAD and sham-operated rats were also fasted in some experimental groups. In a number of animals rectal temperature was measured with a thermistor probe, Before each experiment, the systolic pressure was measured in unanesthetized animals by tailcuff plethysmographic methods using a pneumatic pulse transducer. Only those SAD rats which showed a systolic pressure of over 150 m m H g were used in the experiments. The rats were sacrificed by decapitation and central nervous system (CNS) areas were immediately dissected out at 5°C.

2.1. Subcellularfractionation Each CNS region was homogenized in 25 volumes of ice-cold 0.32 M sucrose in a Thomas glass Teflon homogenizer with a clearance of 0.025 cm. After the pellet (nuclei and cell debris) from 1 000 × g centrifugation had been discarded, the samples were again centrifuged at 12000 × g for 20 min (Gray and Whittaker, 1962). The supernatants were discarded and the pellets (not purified mitochondrial fraction) were resuspended in half of the original homogenization volume in modified Krebs solution of the following composition (mM): NaCI 118.0, KC1 4.7; CaC12 2.6, MgC12 1.2, N a H C O 3 25.0, glucose 11.1, sodium ethylenediamine tetra-acetic acid (EDTA) 0.01 and ascorbic acid 0.11. The serotonin uptake of the resuspended membranes was measured,

2.2. Synaptosomal uptake of[3H]5-HT Aliquots of the synaptosomal suspension conraining 0.2-0.3 mg protein were used to determine the uptake of [3H]5-HT (18 C i / m m o l ) . The synaptosomes were incubated in fresh Krebs oxygenated solution making a final volume of 700 ~al. The substrate concentration was 1.8 × 10 s M fitting that of the high affinity uptake system for 5-HT (Knapp and Mandell, 1973). The 4 min incubation at 37°C was terminated by dilution with 0.32 M sucrose. The synaptosomes were collected on cellulose filters, iramediately washed with 2 ml of 0.32 M sucrose and the radioactivity then counted in a Packard TriCarb 3255 liquid scintillation counting system.

Nonspecific uptake of [-~H]5-HT was determined in a similar incubation medium for 4 min at 0°C. The 5-HT uptake was referred to the synaptosomal protein content which was determined following Lowry et al. (1951).

2.3. Estimation of tryptophan After sacrifice by decapitation, some areas of the CNS were dissected out, kept on ice and quickly homogenized (5% w / v ) in trichloroacetic acid (12%) and centrifuged at 13000 × g for 10 min. Before centrifugation, a 25 /~1 sample was taken out in order to determine the protein concentration. Tryptophan content was assayed on a 200 /~1 sample of the supernatants following the method described by Denckla and Dewey (1967) with the modification suggested by Bloxam and Warren (1974). Fluorescence was read in an Aminco-Bowman spectrofluorometer using excitation and emission wavelengths of 373 and 452 nm respectively. The tryptophan content was referred to the protein content.

2.4. Estimation ofserotonin After sacrificing the animals by decapitation the brains were quickly removed. The brain areas: midbrain, pons, medulla oblongata and hypothalamus were dissected out at 4°C. All samples were immediately frozen at - 6 0 ° C until the assay was carried out (no more than 18 h later). The tissues were homogenized in a butanol-acid mixture and 5-HT was assayed fluorometrically according to the procedure described by Curzon and Green (1970).

2.5. Determination of non-esterified free fatty acids' (NEFA) After light ether anesthesia, arterial whole blood was obtained from the abdominal aorta in heparinized syringes. After centrifugation, the plasma N E F A concentration was estimated following the colorimetric method described by Duncombe (1964) and modified by Itaya and Ui (1965). Absorbance was read at 436 nm in a

339

Beckman spectrometer. Free fatty acid content was expressed as t~mol/1 of plasma.

2.6. Assay oftryptophan hydroxylase (TPH)activity T P H [EC 1.14.16.4, L-tryptophan, tetrahydropterin, oxygen oxidoreductase (5-hydroxylating)] is the rate-limiting enzyme of the first step in the biosynthesis of 5-HT. It catalyzes the hydroxylation of tryptophan into 5-hydroxytryptophan. This is in turn decarboxylated by a non-specific decarboxylase with the production of one molecule of CO2 for each molecule of 5-HT obtained. T P H was assayed following a modification of the method described by Ichiyama et al. (1970) which measures the radioactive CO 2 liberated when the tissue is incubated with 1-[14C]L-tryptophan. After decapitation of the animals, some CNS areas were dissected out at 4°C, cut into 1 m m slices with a double-bladed knife and put into 5 ml of cold. oxygenated Krebs solution. The slices were first incubated for 30 rain with bubbling 95% 02-5% CO~; they were then transferred to small flasks with fresh Krebs solution and incubation (30 rain, 37°C) was started by the addition of 1-[~4C]Ltryptophan (specific activity 31.8 m C i / m m o l ) , Each flask was sealed with a rubber cap supporting a small plastic ladle containing 200 /~1 of Protosol NEN. The final incubation volume was about 700/~1 for each 50 mg of CNS tissue with an initial tryptophan concentration of 13 /~M. T P H activity was stopped by injecting perchloric acid 2 N (0.5 ml each 700 #1) through the rubber cap. The flasks were left at 37°C with shaking for 3 h

longer to ensure the fixation of the radioactive CO 2 in the Protosol. The ladles were then placed in vials containing liquid scintillator and the radioactivity measured 24 h afterwards in a Packard Tri-Carb 3255 liquid scintillation counting system. T P H activity was referred to the protein content. 2.7. Turnover rate of5-HT Rats were killed at 0, 2, 5, 6.5 and 8 h after 300 m g / k g i.p. of DL-p-chlorophenylalanine methyl ester hydrochloride in saline solution which inhibited the enzyme tryptophan hydroxylase. Some CNS areas were removed as soon as possible and stored in a freezer at - 6 0 ° C (no longer than 18 h) until the assay was carried out. Brain 5-HT was determined by the fluorometric procedure of Curzon and Green (1970). The 5-HT values were transformed logarithmically for calculating the linearity of regression coefficients.

3. Results Sinoaortic denervation induced a marked increase in blood pressure (24 h after operation: sham-operated 110.0 + 4.3 mmHg, SAD 160.2 + 5.4 mmHg, n = 12 P < 0.001; 7 days after operation: sham-operated 112.5 + 3.7 mmHg, SAD 163.0 + 4.8 mmHg, n = 15, P < 0.001). The blood pressure of the fasted sham-operated and fasted SAD groups (sham 110.0 + 2.0 m m H g and SAD: 162.5 + 5.2 mmHg, n = 4) was similar to that of the non-fasted animals.

TABLE 1 H a e m a t o c r i t , non-esterified fatty acids ( N E F A ) and body weight after sinoaortic denervation. n H a e m a t o c r i t (%) ~ NEFA (~mol/l) ~ Rectal t e m p e r a t u r e ( ° C ) ~ Variation in body weight (g) ;' 24h 7days

Sham-operated 4 5 6

50.7_+ 0.4 242.5 _+42.7 37.6_+ 0.2

14 16

+0.3_+ 1.1 +9.6_+ 2.6

Hypertensive 49.1 _+ 2.2 416.0_+47.6 " 36.9_+ 0.2 -20.0_+ -13.6_+

2.1'1 3.9 '~

~ H a e m a t o c r i t , N E F A and rectal t e m p e r a t u r e 24 h after the operation, b Shows the changes in body weight, from the day of the operation, for each animal, t-test: ~ P < 0.05: '~ P < 0.001 as c o m p a r e d to s h a m - o p e r a t e d rats. n = n u m b e r of experiments. Values are given as m e a n s _+S.E.M.

34O D u r i n g the first 24 h after o p e r a t i o n the b o d y w e i g h t of h y p e r t e n s i v e rats d e c r e a s e d by a b o u t 15% b u t n o such d e c l i n e was o b s e r v e d in s h a m o p e r a t e d rats. D e n e r v a t e d rats w e r e f o u n d to eat n o f o o d at all w h i l e t h e s h a m - o p e r a t e d g r o u p ate 10.8_+ 0.8 g p e r rat (n = 7) d u r i n g the first 24 h a f t e r o p e r a t i o n ; the n o r m a l f o o d c o n s u m p t i o n in n o n - o p e r a t e d rats was a b o u t 16 g daily. F a s t e d s h a m - o p e r a t e d a n i m a l s also s h o w e d a s i g n i f i c a n t d e c r e a s e in b o d y w e i g h t ( - 11.8 -+_ 3.7 g; n = 4) as c o m p a r e d to the s h a m - o p e r a t e d g r o u p ( t a b l e 1). D u r i n g the f o l l o w i n g days, the d e n e r v a t e d a n i m a l s slowly r e c o v e r e d their initial b o d y weight. T h e b l o o d levels of N E F A o f h y p e r t e n s i v e rats i n c r e a s e d , w i t h o u t c h a n g e s in the h a e m a t o c r i t or rectal t e m p e r a t u r e , 24 h a f t e r o p e r a t i o n ( t a b l e 1). T h e p l a s m a N E F A c o n c e n t r a t i o n o f the f a s t e d s h a m - o p e r a t e d rats (407.0_+ 52.3 / ~ m o l / 1 ; n = 4) was similar to t h a t of the S A D g r o u p a n d signific a n t l y h i g h e r (P < 0.05) t h a n t h a t of the s h a m - o p e r a t e d a n i m a l s ( t a b l e 1). T h e p l a s m a N E F A levels o f fasted d e n e r v a t e d rats w e r e s i m i l a r to t h o s e of d e n e r v a t e d rats.

rF~¥ •/~ ,~.

10o-

_

20o - B ~Tn_v

N

a f t e r o p e r a t i o n . F o r this p u r p o s e , the s y n a p t o s o m a l f r a c t i o n o b t a i n e d f r o m several areas of the CNS (midbrain, pons, medulla oblongata, hypot h a l a m u s , f r o n t a l c o r t e x a n d spinal c o r d ) was inc u b a t e d w i t h [ 3 H ] 5 - H T at 3 7 ° C .

-~Jl]]l[] ]llIjt

100 -

__~

3.1. [~H]Serotonin uptake in synaptosornal fraction areas of the CNS T h e u p t a k e o f 5 - H T was d e t e r m i n e d in s h a m o p e r a t e d a n d h y p e r t e n s i v e rats 24 h a n d 7 d a y s

~.

200 ~-

Hypot.

F. C o r t .

Midb.

Pons

Med. Ob.

Fig. 1. Tryptophan content in CNS areas of sham-operated, hypertensive and fasted (sham and SAD) rats 24 h after operation. Tryptophan content is expressed as pmol/mg protein. Means _+S.E.M. are given. [] Sham-operated in A and fasted sham-operated rats in B; IN SAD in A and fasted SAD rats in ]~. Asterisks indicate significant differences from the sham group, assessed by analysis of variance (Winer, t971) (* P < 0.05; ** P < 0.001). Number of experiments, n = 7 in A and n ~ 4 in B. Hypot.: hypothalamus; F. corr.: frontal cortex: Midb.: midbrain: Med. Ob.: Medulla oblongata.

TABLE 2 Serotonin uptake in the synaptosomal fraction of different areas of the CNS from sham-operated or hypertensive rats (24 h or 7 days after operation), n = number of experiments. Values are given as means + S.E.M. Area

Serotonin uptake (pmol/mg protein per min) 24 h

Spinal cord Midbrain Pons Medulla oblongata Hypothalamus Frontal cortex

7 days

n

Sham

n

Hypertensive

n

Sham

n

Hypertensive

7 7 7 7 7 7

1.50 ± 0.25 2.15 4-_0.37 1.87 ± 0.12 1.89 ± 0.25 1.78 ± 0.24 1.42 -4-_0.23

7 7 7 7 7 7

1.60 ± 0.35 2.12 _+0.32 1.56 ± 0.35 2.06 ± 0.33 2.02 ± 0.18 1.65 ± 0.31

4 5 5 5 4 5

1.66 ± 0.16 2.07 ± 0.18 2.22 ± 0.21 2.33 ± 0.14 2.25 +_0.18 1.70 + 0.14

4 5 5 5 5 5

1.69 ± 0.14 1.98 ± 0.22 1.84 ± 0.16 1.91 ± 0.06 1.75 ± 0.20 1.99 + 0.12

341

TABLE3 Tryptophan content in CNS areas of sham-operated and hypero tensive rats, 7 days after operation, n = number of experiments, Values are given as means _+S.E.M.

Area

n

Midbrain Pons Medulla oblongata Hypothalamus Frontal cortex

4 4 4

Tryptophan(pmol/mgprotein)

Sham

Hypertensive

178.3_+ 17.1 194.5_+ 1 8 . 1 219.8_+ 26.5

198.0_+22.5 212.3_+21.3 201.8_+ 15.6

5

193.2_+12.3

182.6_+16.6

5

160.6 -+ 13.2

160.0 _+20.7

There were not significant differences between the uptake of 5-HT of sham-operated and hypertensive rats at 24 h and 7 days after operation (table 2). 3.2. Tryptophan and serotonin content in areas of the C N S

Tryptophan was determined in areas of the CNS in sham-operated, fasted sham-operated,

:~ _ ~-

•~

3.3. Tryptophan hydroxylase activity in areas of the CNS

Tryptophan hydroxylase activity was assayed in sham-operated and hypertensive rats, 24 h and 7 days after the operation, by incubating slices of areas of the CNS with L-[~4C]tryptophan. The activity of the enzyme was not modified 24 h after surgery in the structures under study (data not shown). However, 7 days after denervation there was a 50% increase in the T P H activity of the midbrain area, while no changes were detected in hypothalamus, frontal cortex and medulla oblongata (fig. 3). The mean value for enzyme activity in the pons of SAD animals was almost 75% higher than that of the sham-operated group but this difference was not statistically different.

5-HI

2

fasted denervated and denervated rats. Twentyfour h after SAD, it was significantly increased in the central areas under study except in the medulla oblongata where the increase w a s n o t significantly different from the values of the sham-operated group (fig. IA). However, the tryptophan concentration of the fasted sham-operated and fasted SAD animals was almost identical 24 h after denervation (fig. 1B). There w e r e n o significant differences in the tryptophan content of the CNS areas between sham-operated and hypertensive rats 7 days after the operation (table 3). Serotonin concentration was also determined in some central areas of fasted sham-operated and fasted denervated rats. Twenty-four h after SAD, it was significantly increased in midbrain, pons and medulla oblongata when compared with fasted sham-operated animals. Only the hypothalamus of the fasted sham-operated group showed a 5-HT content similar to that of fasted SAD rats (fig. 2).

~

1

3.4. Turnover rate of 5oHT in areas of the C N S

0

Mi0h.

Pons

Meal. Oh.

Hypot.

The turnover rate of 5-HT was determined 7 days after operation since by this time the 5-HT

Fig. 2. Serotonin levels in CNS areas of fasted sham-operated and fasted hypertensive rats 2 4 h after operation. The results are expressed as #g of 5 - H T / g of tissue. Means _+S.E.M. are given. [] Fasted sham-operated and [] fasted SAD rats. Statistical analysis as in fig. I (* P < 0.01). N u m b e r of experiments, n = 4, Midb.: midbrain, Med. Oh.: Medulla oblongata and

steady state as was observed in previous studies (Chemerinski et al., 1980). T h e t u r n o v e r r a t e o f 5-HT could thus be measured 1 week after opera-

Hypot.: hypothalamus,

tion

l e v e l s in t h e

by

CNS

injecting

areas

had

already

reached

p-chlorophenylalanine

a

(300

342

Hy pot. SH

SAD

F. Cort. SH

SAO

Midb. SH

Pons

SAD

SH

SAD

Med. Ob. SH

SAD

•

} .,t ~

•

'° t

~

•

t ~ •

t

'.

t ° =

°

t

Fig. 3. Tryptophan hydroxylase activity ? days after SAD. Enzyme activity is expressed as pmol CO;/mg protein per 30 rain. Each circle represents the value for an individual experiment. Circles with bars indicate the group means ~ S.E.M. The asterisk indicates a significant difference (P < 0.05, t-test)between groups. Hypot.: hypothalamus; F.Cort.: frontal cortex; Midb.: midbrain; Med.Ob.: medulla oblongata; SH: sham-operated rats; SAD: sinoaortic denervated rats.

TABLE 4 Turnover rate of serotonin in some regions of the CNS from sham-operated and hypertensive rats (7 days after operation). Area

Sham-operated turnover rate (~g/g per h)

Hypertensive turnover rate (~g/g per h)

Hypothalamus Midbrain Pons Medu]]a oblongata Spinal cord Cervical Thoracic Lumbar

0.364 ~ 0.028 0.124 ~ 0.021 0.1 ] 0 + 0.009 0.120 + 0.005

0.463 + 0.074 0.286 ~ 0.051 " 0.200 ~ 0.028 " 0.104 ~ 0.014

0.154 ~ 0.021 0.081 ~ 0.007 0.405 ~ 0.037

0.143 + 0.012 0.071 ~ 0.009 0.398 ~ 0.028

Values are given as means ~ S.E.M. of 4 experiments, a p < 0.025 (t-test).

m g / k g ) . T h e 5 - H T levels were d e t e r m i n e d at 0, 2, 5, 6.5 and 8 h after a d m i n i s t r a t i o n o f the drug. T h e t u r n o v e r rate in b o t h g r o u p s was higher in areas rich in nerve endings (e.g. h y p o t h a l a m u s and l u m b a r spinal c o r d ) than in areas rich in cell bodies. As seen in table 4 s i n o a o r t i c d e n e r v a t e d rats showed a h i g h e r t u r n o v e r rate o f 5 - H T in m i d b r a i n and ports than d i d the s h a m - o p e r a t e d a n i m a l s w h i l e there were no s i g n i f i c a n t differences in the o t h e r areas,

4. Diseussion T r y p t o p h a n levels were s i g n i f i c a n t l y increased in h y p o t h a l a m u s , f r o n t a l cortex, m i d b r a i n and pons 24 h after S A D w h e n c o m p a r e d w i t h shamo p e r a t e d animals. The t r y p t o p h a n c o n t e n t in tissues of the fasted s h a m - o p e r a t e d rats was a l m o s t i d e n t i c a l to that of the fasted-denervated and denervated groups. Seven days after o p e r a t i o n there were no s i g n i f i c a n t differences in the t r y p t o p h a n

343 levels between SAD and sham-operated rats. The activity of the rate-limiting enzyme in the biosynthesis of 5-HT, tryptophan hydroxylase, was not affected 24 h after SAD while it was significantly increased in midbrain 7 days after operation. The turnover rate of 5-HT was increased in midbrain and pons areas 7 days after SAD. Therefore, the half-life of the 5-HT store in these areas was lower in hypertensive animals than in shamoperated rats. These results suggest that SAD should induce changes in the serotonergic neurons localized in CNS areas 7 days after operation. It has been reported that changes in the nutritional state markedly affect the availability of tryptophan and also to a lesser degree the activity of serotonergic neurons (Fernstrom and Wurtman, 1971; Curzon, 1978). Ordinarily, as much as 75-80% of non-peptide tryptophan in the blood is albumin-bound (Pardridge, 1979; Madras et al., 1974). N E F A molecules may compete with tryptophan for binding sites on plasma albumin and may increase free tryptophan by displacing it from albumin. Consequently, more free precursor may be available to the CNS (Curzon, 1978). This hypothesis agrees with our results. The denervated rats did not eat (voluntarily) for the first 24 h after operation and had consequently augmented levels of plasma NEFA, as did both of the fasted groups (sham and SAD). Therefore, the availability of free tryptophan to the CNS would be enhanced (as previously described)accounting for the increased levels of this amino acid found in the central areas. On the other hand, the increased sympathetic discharge resulting from SAD could have increased the plasma catecholamines which in turn could increase the plasma free fatty acids. However, the fasted sham-operated group showed a concentration of plasma N E F A similar to that of the denervated rats, suggesting that the fast was the most important reason for the rise in free fatty acid concentration, Another possibility is that the animals were fasting for 24 h and that this reduced the plasma concentration of other neutral amino acids which compete with tryptophan for entry into the CNS, thus facilitating the transport of tryptophan across the blood-brain barrier (Fernstrom and Wurtman,

1972; Fernstrom and Failer, 1978). In any case, the increased tryptophan concentration due to the fast 24 h after SAD could have been the main reason for the marked increase in the 5-HT content. The rate-limiting step of 5-HT biosynthesis, tryptophan hydroxylase activity, was unchanged 24 h after SAD. But since central tryptophan levels were augmented in the denervated rats, dilution of the labelled tryptophan could occur thus masking a real increase in the activity of the enzyme. Seven days after SAD, when the tryptophan content in hypertensive rats had returned to control values, there was a marked increase in tryptophan hydroxylase activity in the midbrain which was coincident with the increase in turnover rate in the same area. As Costa et al. (1966) has demonstrated, the increase in turnover rate of the neurotransmitter is an indication of enhanced neurotransmission. Additionally, this increase in tryptophan hydroxylase activity also suggests that adaptive changes should have occurred in serotonergic neurons in response to SAD. The results just discussed would explain the previously observed persistent elevation of the 5-HT content in the midbrain area only (Chemerinski et al., 1980). Whether these changes are related to the maintenance of hypertension or are compensatory mechanisms that down-regulate blood pressure remains to be elucidated. It seems that the serotonergic system may be responsible for a hypertensive or hypotensive effect depending on the pathway stimulated. For example, results published previously (Ito and Schanberg, 1972; Tadepalli et al., 1977) suggested that the hypotension obtained after serotonergic stimulation is mediated by a bulbo-spinal descending serotonergic pathway whose origin is localized in the medulla oblongata. This serotonergic action would be mediated by inhibition of preganglionic sympathetic activity. On the other hand, the administration of 5-HT i.c.v. (Nahmod et al., 1978) or the stimulation of the ascending serotonergic mesencephalic nucleus (Smits et al., 1978; Kuhn et al., 1980a) induced a hypertensive effect. In the present work, an increase in TPH activity and serotonin turnover in the mesencephalic area

344

occurred 7 days after SAD. According to Kuhn et al. (1980b) 5 - H T c a n play a 'permissive', excitatory role along the midbrain-raphe-hypothalamic axis under conditions involving increased efferent sympathetic outflow (like sinoaortic denervation is) perhaps by overriding more tonic inhibitory centers that predominate under normal conditions, Thus, our results could support the idea that ascending serotonergic pathways may play a role in the maintenance of neurogenic hypertension, Acknowledgements This work was supported by grants from C O N I C E T (Consejo Nacional de Investigaciones Cientlficas y T~cnicas). The authors wish to thank Dr. Francisco J.E. Stefano for reading the manuscript and helpful criticism of the work and Mr. Mario Viggiano for his expert assistance,

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