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Role of midbraib raphe in stress-induced renin and prolactin secretion
Brain Research, 311 (1984) 333-341
333
Elsevier BRE 10355
Role of Midbrain Raphe in Stress-Induced Renin and Prolactin Secretion LOUIS D. VAN DE KAR 1, STANLEY A. LORENS l, CRAIG R. McWILLIAMSl , KAYOKO KUNIMOTO~, JANICE H. URBAN 1and CYNTHIA L. BETHEA2
1Loyola University, Stritch School of Medicine, Departmentof Pharmacology, Maywood, IL 60153 and 20regon Regional PrimateResearch Center, Beaverton, OR 97005 (U.S.A.) (Accepted March 6th, 1984)
Key words: midbrain raphe - - renin - - prolactin - - stress
Stress-induced changes in renin and prolactin secretion were studied using a conditioned emotional response paradigm. Three minutes after being placed in a chamber, the stressed animals received a brief electric shock (1.0 mA for 10 s through the grid floor), then were returned to their home cage. This procedure was repeated for 3 consecutive days. On the fourth day, the rats were placed in the chamber for 3 min, but instead of receiving shock, they were removed and sacrificed. Control animals were treated in the same manner, except that they never received foot shock. The sham-operated stressed rats evidenced significant elevations in plasma renin activity (270%) and prolactin level (550%). Electrolytic lesions in the dorsal raphe nucleus blocked the stress-induced increase in plasma renin activity but did not affect the stress-induced increase in prolactin secretion. Electrolytic lesions in the median raphe nucleus did not affect prolactin levels in either control or stressed animals. However, median raphe lesions led to a significant increase in plasma renin activity in non-stressed rats and potentiated the stress-induced elevation in plasma renin activity. These results suggest that neurons within the dorsal and median raphe nuclei are involved in the regulation of renin but not prolactin secretion during stress. The results also suggest that median raphe neurons play a role in basal renin secretion.
INTRODUCTION Stressful conditions produce marked increases in blood pressure, plasma renin activity and catecholamine levels 9,26, as well as elevations in the plasma concentrations of prolactin and corticosteronetS, 21,22. Numerous studies, employing m a n y different methods 3,15,20, have implicated stress as an important factor in the development of hypertension. Results from several studies suggest that hypothalamic sites and pathways mediate these stress effects 8,18,21,22. The midbrain raphe is known to send fibers to hypothalamic sites2,28,29 and has been reported to mediate stress-induced prolactin secretion 6. The present study was designed to test the hypothesis that stress-induced renin and prolactin secretion is regulated by n e u r o n a l elements in the midbrain raphe. Thus sham operations were performed or electrolytic lesions were placed in either the dorsal or the median raphe nucleus. The animals were tested in
a conditioned emotional response paradigm, and their plasma assayed for renin activity and prolactin concentration. MATERIALS AND METHODS
Animals Male S p r a g u e - D a w l e y rats (250-300 g) were obtained from King A n i m a l Laboratories (Oregon, WI). The rats were housed in conventional cages (2 or 3 per cage), located in a temperature (20-22 °C), humidity (50-55 %) and illumination (12:12 h light/ dark cycle; lights on at 07.00 h) controlled room. Water and rat chow (Wayne Lab, Blox; Allied Mills, Chicago, IL) were available ad libitum. All experimental procedures were conducted between 12.00 and 15.30 h.
Surgery Electrolytic lesions were produced under pento-
Correspondence: L. D. Van de Kar, Loyola University, Stritch School of Medicine, Department of Pharmacology, 2161)S. First Avenue, Maywood IL 60153, U.S.A. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.
334 barbital anesthesia (50 mg/kg, i.p.) by passing 2.0 m A for 5 s (dorsal nucleus) or 8 s (median nucleus) through an intracranial anode and a cathode clipped to the wound margin. The anode was a stareless steel pin (0.25 mm diameter) insulated with Epoxylite except for 0.5 mm of its tip. A Kopf stereotaxic apparatus (incisor bar 3.2 mm above the rateraural line) and a Grass DC constant current lesion maker were employed. The anode was inserted midsagittally through the rostral cerebellum at an angle of 47 ° posterior to the vertical plane. Lesion coordinates were based on measurements taken from the midline, 1.0 mm rostral to the lambda skull suture: for the dorsal nucleus, 6.3 mm caudal and Y.6 mm ventral to the skull surface: and for the median nucleus, 8.0 mm caudal and 12.2 mm ventral to the skull surface. Operated control animals were treated in the same manner as the lesion rats except that the electrode was not lowered into the brain. At the start of surgery all rats received both atropine sulfate 10.4 mg/kg) and ampicillin (200 mg/kg) intramuscularly
Biochemical analyses Serotonin (5-HT) and 5-hydroxyindole acetic acid ( 5 - H I A A ) concentrations in the hippocampus and caudate-putamen were determined by high-performance liquid chromatography with electro-chemical detection. A Bioanalytic Systems LC-4 amperometer, TL-5 glassy carbon electrode (applied potential + 0.67 V) and RYT-ptotter were employed along with a Chromanetics Partisil ODS-3 column and a Waters M-45 pump (flow rate 2.0 ml/minl. The mobile phase consisted of 0.15 M monochloroacetic acid, 0.1 mM E D T A , and 25 mg/l sodium octanylsulfonic acid in 12.5% v/v methanol. The brain parts were weighed then homogenized in 0,1 N perchloric acid (4 #l/mg) containing 1.0 mM E D T A . 0.3 mM thioglycolic acid, and the internal standard. N-methyl serotonin (10 ng/100 ~1). The homogenates were centrifuged at 15,000 g for 15 min. The supernatants were millipore (0.2 ~m) filtered and injected into the chromatograph via a Rheodyne valve outfitted with a 50 td injection loop. Retention times were 5 min 30 s for 5-HT, 6 rain 50 s for N-methyl-5-HT, and 8 min for 5-H1AA. The intra-assay variability for both 5-HT and 5 - H I A A is less than 5%. All the samples were determined in one continuous assay to avoid rater-assay variability.
Plasma renin activity was measured by radioimmunoassay of generated angtotensin 125,30. The antiserum (Reid No. 11 was used at a dilution of 1:100.000 and 35% total binding. The intra-assay variability is below 2% and the interassav variability is 9.4%. All the samples were assayed m one assay. Plasma prolactin concentrations were determined bv radioimmunoassay with reagents provided by the N I A D D K . Anti-rat prolactin serum-S-8 was used at a dilution of 1:12.500 and rat prolactin t-5 was radioiodinated. Rat prolactin RP-2 served as the reference preparation. The intra-assav variability was 6.8% and the interassav variability was I3.5g~ All the samples were analyzed in one assay
Apparatus The conditioned emotional or stress response was elaborated in a rectangular chamber C19 in. long x 9 in. wide x 11 in. high) with a grid floor composed of stainless steel rods (0.3 in. diameter) spaced 0.5 in. apart. The walls and ceiling of the apparatus were made of Plexiglas. and it was illuminated by a fluorescent lamp (20 W) m o u n t e d outside the rear walt. Scrambled constant current (1.0 mA) shock was delivered through the grid floor bv a Grayson-Stadler shock generator (Model E 1064 GS). The stress chamber was located m a sound attenuated room. separate from the animal quarters and the sacrifice area.
Procedure Six groups of animals were studied: dorsal raphe (DR) lesion stressed (n = 10); median raphe (MR) [esion stressed (n = 8); sham-operated stressed (n = 7); D R lesion control (n = 10); MR lesion control (n = 8); sham-operated control (n = 7). The rats were assigned at random before surgery to a particular treatment group, such that cage mates were members of the same group. Cage mates were tested sequentially and at the same time each day. The order of testing of the different cage members (group members) was randomized Three weeks post-operatively, the rats were subjected to the stress paradigm All testing was conducted between 12.00 and 15,30 h on 4 consecutive days (Tuesday-Friday), On each day the rats were transported individually in a plastic cage identical to their home cage to the stress room located 20 ft from
335
A
C
B
D
MP
STRESS
CONTROL 3 ~
~ ,
10
18
27 " ~ !
37
35 A
B
C
D 39
40 A
B
C
D
Fig. 1. Reconstruction of the dorsal raphe lesions (blackened area) in the accepted control (n = 6) and stressed (n = 8) rats on 4 coronal planes ( A - D ) , each separated from the adjacent by about 0.3 mm. Numbers identify individual rats (for abbreviations, see Fig. 2).
336 their living quarters. Three min following placement in the chamber the experimental animals received an inescapable foot shock (1.0 mA for 10 s). Immediately thereafter, the rats were returned to the plastic cage and transported to their home cage. This procedure was repeated for 3 consecutive days. It was quite apparent that the stressed rats learned that their placement in the chamber would be followed by a painful shock. By the third day, in contrast to control animals, the stressed rats defecated, urinated, and alternated between freezing and jumping behavior. On the fourth day the rats were placed in the box for 3 rain. but instead of receiving shock, they were removed, transported to a third area 10 ft. away and immediately sacrificed by decapitation (within 15 s of the termination of the stress-test). The control rats were treated identically except that shock was not administered at any time. The blood of the decapitated rats was collected into centrifuge tubes containing 0.5 ml 0,3 M E D T A (pH 7.4). The plasma was stored at - 4 0 °C until assayed for plasma renin activity and prolactin level The brains were removed and dissected on a brass plate over ice. The neostriata and hippocampi were wrapped in aluminium foil. frozen in liquid nitrogen, then stored at -70 °C for no more than 3 weeks prior to assay for 5-HT and 5 - H I A A content. Brainstems were kept in 10% formalin for at least 2 weeks prior to histological analysis, Coronal frozen sections were cut at 50 ~m and every third section saved and stained by the cresyl violet technique. Lesion cavitation and glial scarring were determined microscopically and plotted diagramatically (Figs. 1 and 2). Prior to analysis of the hormonal data, lesion accuracy was determined histologically. In order for a lesion animal to be accepted, the tissue damage had to be localized midsagittally in either the MR or DR. Lesions centered 0.75 mm or more laterally or lesions which were placed too caudally, were rejected.
as were those which damaged both the MR and D R m the same animal Statistical analysis of the data was performed by analysis of variance (ANOVA) and Duncan's multiple range test24. RESULTS
All the rats survived the surgery and appeared healthy. At the time of testing, no significant differences in group body weights were observed.
Histological and biochemical analysis of lesions According to the histological criteria elaborated above, 6 rats in the median raphe lesion (MR) group and 6 rats in the dorsal raphe (DR) lesion group were eliminated from the study The lesions in the animals accepted for analysis of the hormonal data are presented in Figs. 1 and 2. The D R lesions were well confined to the ventromedial central grey between the trochlear nucleus and the rostral tip of the dorsal tegmental nucleus of Gudden and destroyed at least 65% of the D R nucleus The medial longitudinal fasciculus, trochlear nucleus, and the intermediate linear nucleus were damaged in most of the animals. The MR lesions extended from the mid-portion of the interpeduncular nucleus to the level of the trigeminal motor nucleus, and destroyed at least 90% of the MR nucleus and ventral tegmental nucleus of Gudden. The brachium conjunctivum, medial longitudinal fasciculus, tectospinal tracts, interpeduncular nucleus, and pontine raphe nucleus were damaged in all the animals. The lesions extended unilaterally into the reticular formation in several rats. The dorsal tegmental nucleus of Gudden and the pontine reticular tegmental nucleus, however, were spared in all animals. As seen in Table I. there were no significant differences between the sham-operated stressed and con-
Fig. 2. Median raphe lesions (blackened area) in the accepted control (n = 5) and stressed (n = 5) rats are shown in 8 coronal planes ( A - H ) , each separated from the adjacent by approximately 0.3 ram. BC. brachium conjunctivum: BP, brachium pontis; CP, cerebral peduncle; DBC, decussation of the BC: ML. medial lemniscus: MLF. medial longitudinal fasciculus: MP. mammillary peduncle; TTS. tectospinal tracts; cnlII, oculomotor nucleus: cnIV, trochlear nucleus; dr. dorsal raphe nucleus; dtg, dorsal tegmental.nucleus of Gudden; gp, griseum pontis; ic. inferior colliculus; ipn, interpeduncular nucleus; mr, median raphe nucleus; turf, midbrain reticular formation; inV. motor nucleus of trigeminal nerve; pag, periaqueductal gray; prf, pontine reticular formation; rt, rostral linear nucleus', rn. red nucleus: rp, pontine raphe nucleus: rtp, pontine tegmental reticular nucleus; sc, superior colliculus: sn, substantia nigra: vta. ventral tegmental area of Tsai: vtg, ventral tegmental nucleus of Gudden.
337
CONTROL
mP
15
STRESS
MI.
338 trol rats with respect to their hippocampal or neo-
DISCUSSION
striatal 5-HT and 5 - H I A A concentrations. In agreement with previous studies 1~,2s the D R lesions
Plasma renin actwity and plasma prolactin concen-
produced significant falls in the 5-HT and 5 - H I A A
tration are elevated during stress. Electrolytic lesions
concentrations in the c a u d a t e - p u t a m e n without af-
in the dorsal raphe nucleus block the stress-induced increase in plasma renin activity while median raphe
fecting hippocampal 5-HT or 5 - H I A A content. The M R lesions, in contrast, significantly reduced the 5-HT and 5 - H I A A concentrations in the hippocampus, but failed to affect neostriatal 5-HT or 5 - H I A A level. These lesion-induced decreases m the 5-HT
lesions cause an increase m plasma renin activity in unstressed rats and potentiate the stress induced increase in plasma renin activity. Neither lesion affects
and 5 - H I A A content of the c a u d a t e - p u t a m e n and
the stress induced rise in plasma prolactin level These observations suggest that renin but not prolac-
hippocampus confirm the accuracy of the lesions.
tin secretion during stress is regulated by midbrain
Plasma renin activity ( P R A ) and prolactin level
raphe neurons. It is unlikely that the prevention of the stress-in-
control
duced increase in plasma renin activity was due to im-
group, the sham-operated stressed animals showed a significant 270% increase in P R A (Fig. 3) and 550~
paired learning since Srebro and Lorens 23 have demonstrated that dorsal raphe lesions do not affect the
in plasma prolactin concentration (Fig. 4). The P R A of the D R lesion control rats did not dif-
acquisition of a conditioned avoidance response.
In
comparison
to
the
sham-operated
fer from that of the sham-operated controls. However, the D R lesions blocked the stress induced mcrease in P R A (Fig, 3). The P R A of the M R lesion control rats was significantly higher than that of the sham-operated control group. In addition, the MR lesions e n h a n c e d the stress induced elevation in P R A (stress: F(1, 32) = 14.05, P < 0.0008, lesion F ( 2 . 3 2 ) = 22.13, P < 0.0001. stress x lesion: F(2, 32~ 14.59 P < 0.0001; (Fig. 3)). Neither the D R nor the M R lesions significantly affected the control or stress induced increase in plasma prolactin concentration (stress: F(1, 32) = 23.8 P < 0,0001, lesion: F(2, 32) -- 0.39, n.s. stress x lesion F(2, 32) = 0.13 n.s (Fig. 4)).
Furthermore. the dorsal raphe lesions did not affect the prolactin response to stress. Eljarmak et al. 6 have reported that median raphe radio frequency (thermal) lesions a t t e n u a t e d the increase in plasma prolactin 15 min after immobilization stress, and that dorsal raphe lesions enhanced the prolactin response to immobilization stress. The authors suggest that two separate populations of serotonergic neurons have oppositeeffects on prolactin secretion. The present results are inconsistent with these conclusions, but it should be pointed out that the present stress paradigm is different both in the absence of a noxious somatic stHnulus (immobilization) on the test day and duration (3 min in the present study vs 30 mini. The stress-induced increase
TABLE I Effect of electrolytic dorsal or median raphe lesions on the serotonin and 5-HIAA content of the caudate nucleus and hippocampus Data represent mean ___S.E.M.. ng/g wet weight. The number of samples is in parentheses. DR, dorsal raphe; MR, median raphe. Hippocampus
Neostriatum
Sham control (7) Sham stress (7) DRcontrol(6) DRstress(8) MR control (5) MR stress (5)
5-HT
5-HIAA
5-HT
520± 36 478 _~ 39
445 + 30 444 +__36
315_+ 8 305 ± 18
123 z 27* 127 ± 21"
267 z 12 283 z 27
99 ~ 39* 98 ± 20* 433 *-- 42 472 ___17
459 _+ 24 497 _+ 17
46 + 10" 50 ~ "~*
5-HIAA -
"
196± o 193 z 8 167 z 8 173 z 8 104 *__ 12" 92 -- ""
* Significant difference from the corresponding sham group P < 0.0t (ANOVA and Duncan's multiple range testL
339 CONTROL
°1
~U'sTRESS"
,<
3o
*-I-
>. II-
,o 20 z Z w <
s:
10
,<
SHAM
DR
LESION
MR
LESION
Fig. 3. Effect of electrolytic dorsal raphe (DR) and median raphe (MR) lesions on plasma renin activity in stressed and control rats. Data represent mean _+ S.E.M. Numbers indicate animals per group. *Significant difference from the corresponding control group P < 0.01 (two-way A N O V A and Duncan's multiple range test). +Significant difference from the sham stress groups P < 0.01 (two-way A N O V A and Duncan's multiple range test).
I
CONTROL
~'STRESS" 4~
01 I
4O E z:
z
30
I-,,.I O
2o
Ill
~. ~o
SHAM
DR LESION
MR LESION
Fig. 4. Effect of electrolytic dorsal raphe (DR) and median raphe (MR) lesions on plasma prolactin levels in stressed and control rats. Data represent mean + S.E.M. Numbers indicate animals per group. *Significant difference from the corresponding control group P < 0.01 (two-way ANOVA and Duncan's multiple range test).
(550%) in plasma prolactin level observed in the present study,'however, is similar to the maximal increase (600%) in prolactin observed by Eljarmak et al. 6 in their sham-operated group. Stress has been reported to increase 5-HT content s and turnover in various brain areas 11. Brain 5-HT also is known to regulate the secretion of prolactin, corticosterone and reninl.4, 7,12A3,30-32. We also have reported previously that 5,7-dihydroxytryptamine injections into the dorsal raphe nucleus or mechanical ablation of the mediobasal hypothalamus abolished the increase in plasma renin activity produced by the administration of the serotonin releasing drug, p-chloroamphetaminel0,3L The falls in the serotonin and 5-H1AA contents of the caudate nucleus but not of the hippocampus (Table I) following the dorsal raphe lesions are similar to the decreases produced in previous studies29, 3~ by intra-raphe injections of 5,7-dihydroxytryptamine. Therefore, the present data suggest that the neurons in the dorsal raphe nucleus that mediate the effect of stress on renin secretion are serotonergic. The increase in plasma renin activity in unstressed rats after the median raphe lesions could be due to a decrease in food intake resulting in a reduction in sodium delivery to the macula densa 19. Median and dorsal raphe lesions were reported by Lorens and Yunger 17to cause a decrease in food intake and an increase in water intake, but this effect only lasted for the first 10 days post-surgery and returned to presurgery levels 15 days after the lesions. The rats in the present study were allowed to recuperate from the surgery for 21 days before the stress testing was conducted. Furthermore, at the time of sacrifice the body weight of the lesion groups did not differ from the control rats. Therefore, it is unlikely that decreased sodium delivery to the macula densa 19 could account for this effect. Another possibility is that the MR lesion may have caused hypotension and therefore stimulated renin secretion via the renal stretch receptor 19. This is not very likely because median raphe lesions were reported not to cause a change in blood pressure TM. The changes in basal and stress-induced renin secretion produced by median raphe lesions could be due to destruction of serotonergic neurons. However, in a previous study, 5,7-dihydroxytryptamine injection into the median raphe did not produce a
34(I c h a n g e in p l a s m a r e n i n activity in u n s t r e s s e d rats 32.
t r a n s m i t t e r s . In o r d e r to d e t e r m i n e w h e t h e r s e r o t o -
O n t h e o t h e r h a n d . t h e s e rats w e r e k e p t in t h e i r h o m e
nergic or other raphe neurons regulate renin secre-
cage a n d w e r e u n p e r t u r b e d u n t i l d e c a p i t a t i o n .
tion d u r i n g stress, o t h e r m e t h o d s m u s t b e e m p l o y e d .
Electrolytic
midbrain
raphe
lesions destroy
as-
S t u d i e s c u r r e n t l y are u n d e r w a y utilizing 5 , 7 - d i h y -
c e n d i n g a n d d e s c e n d i n g f i b e r s of p a s s a g e as well as
d r o x y t r y p t a m i n e a n d i b o t e n i c acid to e l u c i d a t e w h i c h
p e r i k a r y a w h i c h utilize s e r o t o n i n a n d o t h e r n e u r o -
neurons control stress-induced renin secretion.
REFERENCES
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1 Advis. J. P.. Simpkins, J. W.. Bennet. J. and Meites. J.. Serotonergic control of prolactin release in male rats. Life Sci., 24 (1979) 359-366. 2 Azmitia. E. C. and Segal, M.. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. comp. Neurol. 179 (1978) 641-668. 3 Brody, D. S.. Psychological distress and hypertension control. J. Human Stress. 6 (1980) 2-6. 4 Clemens. J A.. Roush. M. E.. Fuller, R. W.. Evidence that serotonin neurons stimulate secretion of prolactin releasing factor. Life Sci.. 22 (1978) 2209-2214. 5 Culman. J.. Kvetnansky, R.. Torda. T. and Murgas, K.. Serotonin concentration in individual hypothalamic nuclei of rats exposed to acute immobilization stress. Neuroscience, 5 f1980) 1503-1506. 6 Etjarmak. D., Charpenet. G.. Jequier. J. C. and Collu. R.. Role of midbrain raphe nuclei in stress-pentobarbital-. B-endorphin-. or TRH-induced changes in plasma PRL levels of adult male rats. Brain Res. Bull.. 8 (1982) 149-154. 7 Fuller. R. W. and Snoddy, H. D.. Effect of serotonin-releasing drugs on serum corticosterone concentration in rats. Neuroendocrinology. 31 (1980) 96-100. 8 Galosv. R. A., Clarke. L. D.. Vasko. M. R. and Crawford. I. L.. Neurophysiology and neuropharmacology of cardiovascular regulation and stress. Neurosci. Behav. Res.. 5 (1981) 137-175. 9 Januszewicz. W.. Sznajderman, M., Wocial. B., Feltynowski. T. and Klonowicz. T. The effect of mental stress on catecholamines their metabotites and plasma remn activity in patients with essential hypertension and in healthy subjects, Clin. Sci.. 57 (1979) 2295-2315. 10 Karteszi. M.. Van de Kar. L. D.. Makara. G., Stark. E. and Ganong, W F.. Evidence that the mediobasal hypothalamus is involved in serotonergic stimulation of renin secretion. Neuroendocrinology. 34 (1982) 323-326. 11 Kennet. G. A and Joseph. M. H.. The functional importance of increased brain tryptophan in the serotonergic response to restraint stress, Neuropharmacology. 20 (1981) 39-43, 12 Kordon. C.. Blake. C A.. Terkel. J. and Sawyer, C. H.. Participation of serotonin-containing neurons in the suckling-induced rise in plasma prolactin level in lactating rats, Neuroendocrinology, 13 (1974) 213-223. 13 Krulich. L., Vijayan. E., Coppings. R. J.. Giachetti, A., McCann. S. M. and Mayfield. A., On the role of the central serotoninergic system in the regulation of the secretion of thyrotropin and prolactin: thyrotropin-inhibiting and prolactin-releasing effects of 5-HT and quipazine in the male rat. Endocrinology, 105 (1979) 276-283. 14 Kuhn. D. M.. Wolf. W. A. and Lovenberg, W., Pressor et-
341 other forebrain regions by the dorsal and median raphe nuclei, Brain Research, 162 (1979) 45-54. 29 Van de Kar, L. D., Lorens, S. A., Vodraska, A., Allers, G., Green, M., Van Orden, D. E. and Van Orden, L. S. III., Effect of selective midbrain and diencephalic 5,7-dihydroxytryptamine lesions on serotonin content in individual preopticohypothalamic nuclei and on serum luteinizing hormone level, Neuroendocrinology, 31 (1980) 309-315. 30 Van de Kar, L. D., Wilkinson, C. W. and Ganong, W. F., Pharmacological evidence for a role of brain serotonin in the maintenance of plasma renin activity in unanesthetized
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333
Elsevier BRE 10355
Role of Midbrain Raphe in Stress-Induced Renin and Prolactin Secretion LOUIS D. VAN DE KAR 1, STANLEY A. LORENS l, CRAIG R. McWILLIAMSl , KAYOKO KUNIMOTO~, JANICE H. URBAN 1and CYNTHIA L. BETHEA2
1Loyola University, Stritch School of Medicine, Departmentof Pharmacology, Maywood, IL 60153 and 20regon Regional PrimateResearch Center, Beaverton, OR 97005 (U.S.A.) (Accepted March 6th, 1984)
Key words: midbrain raphe - - renin - - prolactin - - stress
Stress-induced changes in renin and prolactin secretion were studied using a conditioned emotional response paradigm. Three minutes after being placed in a chamber, the stressed animals received a brief electric shock (1.0 mA for 10 s through the grid floor), then were returned to their home cage. This procedure was repeated for 3 consecutive days. On the fourth day, the rats were placed in the chamber for 3 min, but instead of receiving shock, they were removed and sacrificed. Control animals were treated in the same manner, except that they never received foot shock. The sham-operated stressed rats evidenced significant elevations in plasma renin activity (270%) and prolactin level (550%). Electrolytic lesions in the dorsal raphe nucleus blocked the stress-induced increase in plasma renin activity but did not affect the stress-induced increase in prolactin secretion. Electrolytic lesions in the median raphe nucleus did not affect prolactin levels in either control or stressed animals. However, median raphe lesions led to a significant increase in plasma renin activity in non-stressed rats and potentiated the stress-induced elevation in plasma renin activity. These results suggest that neurons within the dorsal and median raphe nuclei are involved in the regulation of renin but not prolactin secretion during stress. The results also suggest that median raphe neurons play a role in basal renin secretion.
INTRODUCTION Stressful conditions produce marked increases in blood pressure, plasma renin activity and catecholamine levels 9,26, as well as elevations in the plasma concentrations of prolactin and corticosteronetS, 21,22. Numerous studies, employing m a n y different methods 3,15,20, have implicated stress as an important factor in the development of hypertension. Results from several studies suggest that hypothalamic sites and pathways mediate these stress effects 8,18,21,22. The midbrain raphe is known to send fibers to hypothalamic sites2,28,29 and has been reported to mediate stress-induced prolactin secretion 6. The present study was designed to test the hypothesis that stress-induced renin and prolactin secretion is regulated by n e u r o n a l elements in the midbrain raphe. Thus sham operations were performed or electrolytic lesions were placed in either the dorsal or the median raphe nucleus. The animals were tested in
a conditioned emotional response paradigm, and their plasma assayed for renin activity and prolactin concentration. MATERIALS AND METHODS
Animals Male S p r a g u e - D a w l e y rats (250-300 g) were obtained from King A n i m a l Laboratories (Oregon, WI). The rats were housed in conventional cages (2 or 3 per cage), located in a temperature (20-22 °C), humidity (50-55 %) and illumination (12:12 h light/ dark cycle; lights on at 07.00 h) controlled room. Water and rat chow (Wayne Lab, Blox; Allied Mills, Chicago, IL) were available ad libitum. All experimental procedures were conducted between 12.00 and 15.30 h.
Surgery Electrolytic lesions were produced under pento-
Correspondence: L. D. Van de Kar, Loyola University, Stritch School of Medicine, Department of Pharmacology, 2161)S. First Avenue, Maywood IL 60153, U.S.A. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.
334 barbital anesthesia (50 mg/kg, i.p.) by passing 2.0 m A for 5 s (dorsal nucleus) or 8 s (median nucleus) through an intracranial anode and a cathode clipped to the wound margin. The anode was a stareless steel pin (0.25 mm diameter) insulated with Epoxylite except for 0.5 mm of its tip. A Kopf stereotaxic apparatus (incisor bar 3.2 mm above the rateraural line) and a Grass DC constant current lesion maker were employed. The anode was inserted midsagittally through the rostral cerebellum at an angle of 47 ° posterior to the vertical plane. Lesion coordinates were based on measurements taken from the midline, 1.0 mm rostral to the lambda skull suture: for the dorsal nucleus, 6.3 mm caudal and Y.6 mm ventral to the skull surface: and for the median nucleus, 8.0 mm caudal and 12.2 mm ventral to the skull surface. Operated control animals were treated in the same manner as the lesion rats except that the electrode was not lowered into the brain. At the start of surgery all rats received both atropine sulfate 10.4 mg/kg) and ampicillin (200 mg/kg) intramuscularly
Biochemical analyses Serotonin (5-HT) and 5-hydroxyindole acetic acid ( 5 - H I A A ) concentrations in the hippocampus and caudate-putamen were determined by high-performance liquid chromatography with electro-chemical detection. A Bioanalytic Systems LC-4 amperometer, TL-5 glassy carbon electrode (applied potential + 0.67 V) and RYT-ptotter were employed along with a Chromanetics Partisil ODS-3 column and a Waters M-45 pump (flow rate 2.0 ml/minl. The mobile phase consisted of 0.15 M monochloroacetic acid, 0.1 mM E D T A , and 25 mg/l sodium octanylsulfonic acid in 12.5% v/v methanol. The brain parts were weighed then homogenized in 0,1 N perchloric acid (4 #l/mg) containing 1.0 mM E D T A . 0.3 mM thioglycolic acid, and the internal standard. N-methyl serotonin (10 ng/100 ~1). The homogenates were centrifuged at 15,000 g for 15 min. The supernatants were millipore (0.2 ~m) filtered and injected into the chromatograph via a Rheodyne valve outfitted with a 50 td injection loop. Retention times were 5 min 30 s for 5-HT, 6 rain 50 s for N-methyl-5-HT, and 8 min for 5-H1AA. The intra-assay variability for both 5-HT and 5 - H I A A is less than 5%. All the samples were determined in one continuous assay to avoid rater-assay variability.
Plasma renin activity was measured by radioimmunoassay of generated angtotensin 125,30. The antiserum (Reid No. 11 was used at a dilution of 1:100.000 and 35% total binding. The intra-assay variability is below 2% and the interassav variability is 9.4%. All the samples were assayed m one assay. Plasma prolactin concentrations were determined bv radioimmunoassay with reagents provided by the N I A D D K . Anti-rat prolactin serum-S-8 was used at a dilution of 1:12.500 and rat prolactin t-5 was radioiodinated. Rat prolactin RP-2 served as the reference preparation. The intra-assav variability was 6.8% and the interassav variability was I3.5g~ All the samples were analyzed in one assay
Apparatus The conditioned emotional or stress response was elaborated in a rectangular chamber C19 in. long x 9 in. wide x 11 in. high) with a grid floor composed of stainless steel rods (0.3 in. diameter) spaced 0.5 in. apart. The walls and ceiling of the apparatus were made of Plexiglas. and it was illuminated by a fluorescent lamp (20 W) m o u n t e d outside the rear walt. Scrambled constant current (1.0 mA) shock was delivered through the grid floor bv a Grayson-Stadler shock generator (Model E 1064 GS). The stress chamber was located m a sound attenuated room. separate from the animal quarters and the sacrifice area.
Procedure Six groups of animals were studied: dorsal raphe (DR) lesion stressed (n = 10); median raphe (MR) [esion stressed (n = 8); sham-operated stressed (n = 7); D R lesion control (n = 10); MR lesion control (n = 8); sham-operated control (n = 7). The rats were assigned at random before surgery to a particular treatment group, such that cage mates were members of the same group. Cage mates were tested sequentially and at the same time each day. The order of testing of the different cage members (group members) was randomized Three weeks post-operatively, the rats were subjected to the stress paradigm All testing was conducted between 12.00 and 15,30 h on 4 consecutive days (Tuesday-Friday), On each day the rats were transported individually in a plastic cage identical to their home cage to the stress room located 20 ft from
335
A
C
B
D
MP
STRESS
CONTROL 3 ~
~ ,
10
18
27 " ~ !
37
35 A
B
C
D 39
40 A
B
C
D
Fig. 1. Reconstruction of the dorsal raphe lesions (blackened area) in the accepted control (n = 6) and stressed (n = 8) rats on 4 coronal planes ( A - D ) , each separated from the adjacent by about 0.3 mm. Numbers identify individual rats (for abbreviations, see Fig. 2).
336 their living quarters. Three min following placement in the chamber the experimental animals received an inescapable foot shock (1.0 mA for 10 s). Immediately thereafter, the rats were returned to the plastic cage and transported to their home cage. This procedure was repeated for 3 consecutive days. It was quite apparent that the stressed rats learned that their placement in the chamber would be followed by a painful shock. By the third day, in contrast to control animals, the stressed rats defecated, urinated, and alternated between freezing and jumping behavior. On the fourth day the rats were placed in the box for 3 rain. but instead of receiving shock, they were removed, transported to a third area 10 ft. away and immediately sacrificed by decapitation (within 15 s of the termination of the stress-test). The control rats were treated identically except that shock was not administered at any time. The blood of the decapitated rats was collected into centrifuge tubes containing 0.5 ml 0,3 M E D T A (pH 7.4). The plasma was stored at - 4 0 °C until assayed for plasma renin activity and prolactin level The brains were removed and dissected on a brass plate over ice. The neostriata and hippocampi were wrapped in aluminium foil. frozen in liquid nitrogen, then stored at -70 °C for no more than 3 weeks prior to assay for 5-HT and 5 - H I A A content. Brainstems were kept in 10% formalin for at least 2 weeks prior to histological analysis, Coronal frozen sections were cut at 50 ~m and every third section saved and stained by the cresyl violet technique. Lesion cavitation and glial scarring were determined microscopically and plotted diagramatically (Figs. 1 and 2). Prior to analysis of the hormonal data, lesion accuracy was determined histologically. In order for a lesion animal to be accepted, the tissue damage had to be localized midsagittally in either the MR or DR. Lesions centered 0.75 mm or more laterally or lesions which were placed too caudally, were rejected.
as were those which damaged both the MR and D R m the same animal Statistical analysis of the data was performed by analysis of variance (ANOVA) and Duncan's multiple range test24. RESULTS
All the rats survived the surgery and appeared healthy. At the time of testing, no significant differences in group body weights were observed.
Histological and biochemical analysis of lesions According to the histological criteria elaborated above, 6 rats in the median raphe lesion (MR) group and 6 rats in the dorsal raphe (DR) lesion group were eliminated from the study The lesions in the animals accepted for analysis of the hormonal data are presented in Figs. 1 and 2. The D R lesions were well confined to the ventromedial central grey between the trochlear nucleus and the rostral tip of the dorsal tegmental nucleus of Gudden and destroyed at least 65% of the D R nucleus The medial longitudinal fasciculus, trochlear nucleus, and the intermediate linear nucleus were damaged in most of the animals. The MR lesions extended from the mid-portion of the interpeduncular nucleus to the level of the trigeminal motor nucleus, and destroyed at least 90% of the MR nucleus and ventral tegmental nucleus of Gudden. The brachium conjunctivum, medial longitudinal fasciculus, tectospinal tracts, interpeduncular nucleus, and pontine raphe nucleus were damaged in all the animals. The lesions extended unilaterally into the reticular formation in several rats. The dorsal tegmental nucleus of Gudden and the pontine reticular tegmental nucleus, however, were spared in all animals. As seen in Table I. there were no significant differences between the sham-operated stressed and con-
Fig. 2. Median raphe lesions (blackened area) in the accepted control (n = 5) and stressed (n = 5) rats are shown in 8 coronal planes ( A - H ) , each separated from the adjacent by approximately 0.3 ram. BC. brachium conjunctivum: BP, brachium pontis; CP, cerebral peduncle; DBC, decussation of the BC: ML. medial lemniscus: MLF. medial longitudinal fasciculus: MP. mammillary peduncle; TTS. tectospinal tracts; cnlII, oculomotor nucleus: cnIV, trochlear nucleus; dr. dorsal raphe nucleus; dtg, dorsal tegmental.nucleus of Gudden; gp, griseum pontis; ic. inferior colliculus; ipn, interpeduncular nucleus; mr, median raphe nucleus; turf, midbrain reticular formation; inV. motor nucleus of trigeminal nerve; pag, periaqueductal gray; prf, pontine reticular formation; rt, rostral linear nucleus', rn. red nucleus: rp, pontine raphe nucleus: rtp, pontine tegmental reticular nucleus; sc, superior colliculus: sn, substantia nigra: vta. ventral tegmental area of Tsai: vtg, ventral tegmental nucleus of Gudden.
337
CONTROL
mP
15
STRESS
MI.
338 trol rats with respect to their hippocampal or neo-
DISCUSSION
striatal 5-HT and 5 - H I A A concentrations. In agreement with previous studies 1~,2s the D R lesions
Plasma renin actwity and plasma prolactin concen-
produced significant falls in the 5-HT and 5 - H I A A
tration are elevated during stress. Electrolytic lesions
concentrations in the c a u d a t e - p u t a m e n without af-
in the dorsal raphe nucleus block the stress-induced increase in plasma renin activity while median raphe
fecting hippocampal 5-HT or 5 - H I A A content. The M R lesions, in contrast, significantly reduced the 5-HT and 5 - H I A A concentrations in the hippocampus, but failed to affect neostriatal 5-HT or 5 - H I A A level. These lesion-induced decreases m the 5-HT
lesions cause an increase m plasma renin activity in unstressed rats and potentiate the stress induced increase in plasma renin activity. Neither lesion affects
and 5 - H I A A content of the c a u d a t e - p u t a m e n and
the stress induced rise in plasma prolactin level These observations suggest that renin but not prolac-
hippocampus confirm the accuracy of the lesions.
tin secretion during stress is regulated by midbrain
Plasma renin activity ( P R A ) and prolactin level
raphe neurons. It is unlikely that the prevention of the stress-in-
control
duced increase in plasma renin activity was due to im-
group, the sham-operated stressed animals showed a significant 270% increase in P R A (Fig. 3) and 550~
paired learning since Srebro and Lorens 23 have demonstrated that dorsal raphe lesions do not affect the
in plasma prolactin concentration (Fig. 4). The P R A of the D R lesion control rats did not dif-
acquisition of a conditioned avoidance response.
In
comparison
to
the
sham-operated
fer from that of the sham-operated controls. However, the D R lesions blocked the stress induced mcrease in P R A (Fig, 3). The P R A of the M R lesion control rats was significantly higher than that of the sham-operated control group. In addition, the MR lesions e n h a n c e d the stress induced elevation in P R A (stress: F(1, 32) = 14.05, P < 0.0008, lesion F ( 2 . 3 2 ) = 22.13, P < 0.0001. stress x lesion: F(2, 32~ 14.59 P < 0.0001; (Fig. 3)). Neither the D R nor the M R lesions significantly affected the control or stress induced increase in plasma prolactin concentration (stress: F(1, 32) = 23.8 P < 0,0001, lesion: F(2, 32) -- 0.39, n.s. stress x lesion F(2, 32) = 0.13 n.s (Fig. 4)).
Furthermore. the dorsal raphe lesions did not affect the prolactin response to stress. Eljarmak et al. 6 have reported that median raphe radio frequency (thermal) lesions a t t e n u a t e d the increase in plasma prolactin 15 min after immobilization stress, and that dorsal raphe lesions enhanced the prolactin response to immobilization stress. The authors suggest that two separate populations of serotonergic neurons have oppositeeffects on prolactin secretion. The present results are inconsistent with these conclusions, but it should be pointed out that the present stress paradigm is different both in the absence of a noxious somatic stHnulus (immobilization) on the test day and duration (3 min in the present study vs 30 mini. The stress-induced increase
TABLE I Effect of electrolytic dorsal or median raphe lesions on the serotonin and 5-HIAA content of the caudate nucleus and hippocampus Data represent mean ___S.E.M.. ng/g wet weight. The number of samples is in parentheses. DR, dorsal raphe; MR, median raphe. Hippocampus
Neostriatum
Sham control (7) Sham stress (7) DRcontrol(6) DRstress(8) MR control (5) MR stress (5)
5-HT
5-HIAA
5-HT
520± 36 478 _~ 39
445 + 30 444 +__36
315_+ 8 305 ± 18
123 z 27* 127 ± 21"
267 z 12 283 z 27
99 ~ 39* 98 ± 20* 433 *-- 42 472 ___17
459 _+ 24 497 _+ 17
46 + 10" 50 ~ "~*
5-HIAA -
"
196± o 193 z 8 167 z 8 173 z 8 104 *__ 12" 92 -- ""
* Significant difference from the corresponding sham group P < 0.0t (ANOVA and Duncan's multiple range testL
339 CONTROL
°1
~U'sTRESS"
,<
3o
*-I-
>. II-
,o 20 z Z w <
s:
10
,<
SHAM
DR
LESION
MR
LESION
Fig. 3. Effect of electrolytic dorsal raphe (DR) and median raphe (MR) lesions on plasma renin activity in stressed and control rats. Data represent mean _+ S.E.M. Numbers indicate animals per group. *Significant difference from the corresponding control group P < 0.01 (two-way A N O V A and Duncan's multiple range test). +Significant difference from the sham stress groups P < 0.01 (two-way A N O V A and Duncan's multiple range test).
I
CONTROL
~'STRESS" 4~
01 I
4O E z:
z
30
I-,,.I O
2o
Ill
~. ~o
SHAM
DR LESION
MR LESION
Fig. 4. Effect of electrolytic dorsal raphe (DR) and median raphe (MR) lesions on plasma prolactin levels in stressed and control rats. Data represent mean + S.E.M. Numbers indicate animals per group. *Significant difference from the corresponding control group P < 0.01 (two-way ANOVA and Duncan's multiple range test).
(550%) in plasma prolactin level observed in the present study,'however, is similar to the maximal increase (600%) in prolactin observed by Eljarmak et al. 6 in their sham-operated group. Stress has been reported to increase 5-HT content s and turnover in various brain areas 11. Brain 5-HT also is known to regulate the secretion of prolactin, corticosterone and reninl.4, 7,12A3,30-32. We also have reported previously that 5,7-dihydroxytryptamine injections into the dorsal raphe nucleus or mechanical ablation of the mediobasal hypothalamus abolished the increase in plasma renin activity produced by the administration of the serotonin releasing drug, p-chloroamphetaminel0,3L The falls in the serotonin and 5-H1AA contents of the caudate nucleus but not of the hippocampus (Table I) following the dorsal raphe lesions are similar to the decreases produced in previous studies29, 3~ by intra-raphe injections of 5,7-dihydroxytryptamine. Therefore, the present data suggest that the neurons in the dorsal raphe nucleus that mediate the effect of stress on renin secretion are serotonergic. The increase in plasma renin activity in unstressed rats after the median raphe lesions could be due to a decrease in food intake resulting in a reduction in sodium delivery to the macula densa 19. Median and dorsal raphe lesions were reported by Lorens and Yunger 17to cause a decrease in food intake and an increase in water intake, but this effect only lasted for the first 10 days post-surgery and returned to presurgery levels 15 days after the lesions. The rats in the present study were allowed to recuperate from the surgery for 21 days before the stress testing was conducted. Furthermore, at the time of sacrifice the body weight of the lesion groups did not differ from the control rats. Therefore, it is unlikely that decreased sodium delivery to the macula densa 19 could account for this effect. Another possibility is that the MR lesion may have caused hypotension and therefore stimulated renin secretion via the renal stretch receptor 19. This is not very likely because median raphe lesions were reported not to cause a change in blood pressure TM. The changes in basal and stress-induced renin secretion produced by median raphe lesions could be due to destruction of serotonergic neurons. However, in a previous study, 5,7-dihydroxytryptamine injection into the median raphe did not produce a
34(I c h a n g e in p l a s m a r e n i n activity in u n s t r e s s e d rats 32.
t r a n s m i t t e r s . In o r d e r to d e t e r m i n e w h e t h e r s e r o t o -
O n t h e o t h e r h a n d . t h e s e rats w e r e k e p t in t h e i r h o m e
nergic or other raphe neurons regulate renin secre-
cage a n d w e r e u n p e r t u r b e d u n t i l d e c a p i t a t i o n .
tion d u r i n g stress, o t h e r m e t h o d s m u s t b e e m p l o y e d .
Electrolytic
midbrain
raphe
lesions destroy
as-
S t u d i e s c u r r e n t l y are u n d e r w a y utilizing 5 , 7 - d i h y -
c e n d i n g a n d d e s c e n d i n g f i b e r s of p a s s a g e as well as
d r o x y t r y p t a m i n e a n d i b o t e n i c acid to e l u c i d a t e w h i c h
p e r i k a r y a w h i c h utilize s e r o t o n i n a n d o t h e r n e u r o -
neurons control stress-induced renin secretion.
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