European Journal of Pharmacology, 55 (1979) 43--56 43 © Elsevier/North-Holland Biomedical Press

EFFECT OF CATECHOLAMINE-RECEPTOR STIMULATING AGENTS ON BLOOD PRESSURE AFTER LOCAL APPLICATION IN THE NUCLEUS TRACTUS SOLITARII OF THE MEDULLA OBLONGATA

PIETER ZANDBERG *, WYBREN DE JONG and DAVID DE WIED Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands

Received 20 November 1978, accepted 14 December 1978

P. ZANDBERG, W. DE JONG and D. DE WIED, Effect of catecholamine-receptor stimulating agents on blood pressure after local application in the nucleus tractus solitarii of the medulla oblongata, European J. Pharmacol. 55 (1979) 43--56. The effect of various catecholamines and a-mimetics, given by microinjection in the A2-region of the nucleus tractus solitarii (NTS), on blood pressure was investigated in anesthetized male rats. A dose-dependent decrease of blood pressure and heart rate was induced by adrenaline as the most effective drug, followed by noradrenaline, dopamine, a-methylnoradrenaline and octopamine. Ablation of the rostral or caudal part of the NTS, or removal of the area postrema did not diminish the effect of a-methylnoradrenaline. Higher doses of noradrenaline and a-methylnoradrenaline caused an initial rise of blood pressure, while the blood pressure lowering effect of noradrenaline was diminished, and that of a-methylnoradrenaline and dopamine delayed. Isoprenaline and the (+)-stereoisomers of noradrenaline and a-methylnoradrenaline were ineffective. The hypotensive effect of dopamine was not prevented by systemic injection of the dopamine ~-hydroxylase inhibitor FLA 63. Prior application of haloperidol, yohimbine and phentolamine antagonized the hypotensive response to dopamine and a-methylnoradrenaline. Application of peripherally effective a-mimetics into the A2-region had no or little effect, while high doses increased blood pressure. Tyramine and clonidine caused some decrease of blood pressure. Clonidine also decreased blood pressure when it was applied in the area of the locus coeruleus. Application of isoprenaline in the locus coeruleus also decreased blood pressure while in contrast adrenaline, noradrenaline, dopamine and 0l-methylnoradrenaline increased blood pressure. The present data suggest that the catecholaminergic receptors in the A2-region of the NTS differ from the classic vascular a-receptor and that the NTS also may contain structures which can antagonize the decrease in blood pressure.

Catecholamines a-Mimetics Decrease of blood pressure Nucleus tractus solitarii Locus coeruleus

1. Introduction action of the blood pressure lowering effect of L-dopa, a-methyldopa and clonidine (for There is ample evidence that brain cate- review see Henning, 1973, 1975; Van Zwieten, cholamines play a role in cardiovascular 1975; Laubie et al., 1977). It has been sug- control (see Chalmers, 1975; De Jong et al., gested that these drugs act by direct nor- 1975; Van Zwieten, 1975). The lower brain adrenergic receptor stimulation and may stem has been proposed as the primary site of mimic baroreceptor activation (Sinha et al., 1973; Schmitt et al., 1973; Haeusler, 1975; Kobinger and Pichler, 1975). Although no * Present address: National Institute of Public Health, catecholaminergic synapses have yet been Pharmacology, Laboratory P.O.Box 1, 3720 BA shown to exist in the baroreceptor pathway Bilthoven, The Netherlands. itself (Kobinger and Pichler, 1975; Haeusler, 44 P. ZANDBERG ET AL.

1977), catecholamines appear to modulate ducer (Model P23 Db) connected to a Grass the tonic inhibitory control and reflex regula- polygraph (model 79). The blood pressure tion of cardiovascular functions. The nucleus was calculated as diastolic pressure + 1/3 tractus solitarii (NTS) of the medulla oblon- (systolic pressure minus diastolic pressure). gata is one of the brain-stem sites which appear The heart rate was computed from the blood to play a role in the effects of catecholamines pressure pulse wave by means of a biotachom- on blood pressure. This nucleus, which receives eter (Narco-Bio systems, BT 1200). the major part of the afferent sino-aortic baro- The rats were placed in a stereotactic receptor fibres (see Palkovits and Zfiborszky, apparatus and the head flexed to an angle of 1977), is densely innervated by catechol- 45 ° downward for the injections into the aminergic terminals and contains catechol- NTS. After a midline incision through the aminergic cell bodies (DahlstrSm and Fuxe, doral neck muscles and opening of the atlanto- 1964; Fuxe, 1965; Palkovits and Jacobowitz, occipital membrane the dorsal surface of the 1974). Previous studies showed that local lower brain-stem was exposed via a limited bilateral application of adrenaline, noradrena- occipital craniotomy using forceps. Micro- line and a-methylnoradrenaline in the NTS injections in the area of the NTS were given caused a decrease of blood pressure and heart through a glass cannula (outer diameter 60 rate (De Jong, 1974; Nijkamp and De Jong, pm) in a volume of 0.4 pl, which was delivered 1975; De Jong and Nijkamp, 1976). The A2- in 10 sec with an Agla micrometer syringe and region (described by DahlstrSm and Fuxe a Sharlow micrometer. Except where other- 1964) was the part of the NTS in which injec- wise indicated, the injections in the A2-region tions of a-methylnoradrenaline were found to were made unilaterally at the right side of the be most effective (Zandberg and De Jong, medulla at the level of the caudal tip of the 1977a). This region contains catecholaminergic area postrema, just medial of the tractus cell bodies (DahlstrSm and Fuxe, 1964; Pal- solitarius (0.5 mm lateral of the midline and kovits and Jacobowitz, 1974) and has high 0.9 mm below the dorsal surface of the concentrations of dopamine, noradrenaline medulla oblongata) as described previously (Versteeg et al., 1976) and adrenaline (Van (Zandberg and De Jong, 1977a). A localiza- der Gugten et al., 1976). tion study with a-methylnoradrenaline In the present study, microinjections into showed that this was the most effective region the As-region were used to study the effect of of the NTS for producing a decrease in blood dopamine, noradrenaline, adrenaline and other pressure. This plane, which correspopds to catecholaminergic substances on blood P 7.4 mm of the atlas of Palkovits an, ~o- pressure and heart rate, in order to obtain bowitz (1974) and which is about ~m more precise information about the receptor caudal of the level of the obex, was the. ~'ore involved in this effect. Preliminary experi- taken as the rostro-caudal zero. Each rat ments with local microinjections of catechol- received only one injection, unless otherwise aminergic agents in the locus coeruleus are indicated. reported as well. In one experiment a microinjection of 0.3 nmoles 14C-noradrenaline (39,000 dpm) in 0.4 pl saline was given. The distribution of the 2. Materials and methods radioactivity was determined 5 min after the Male rats of an inbred Wistar strain (Wi/ injection. The rats were decapitated, the brain cpb TNO, Zeist The Netherlands), weighing removed from the skull and directly frozen on 200--250 g were anesthetized with urethane dry ice. Frozen sections (100 pm) were cut in (1.2 g/kg i.p.). Blood pressure was recorded a cryostat. The radioactivity of the sections from the femoral artery with a polyethylene was determined in a liquid scintillation (PEso) cannula and a Statham pressure trans- counter. More than 80% of the radioactivity NUCLEUS TRACTUS SOLITARII AND BLOOD PRESSURE 45 recovered in the medulla oblongata was (--)-noradrenaline as 100, the relative poten- within the rostro-caudal distribution of 1200 cies were (-)-adrenaline 400; (-)-a-methyl- pm. A very similar distribution along the NTS noradrenaline 33; oxymetazoline 8; clonidine has been observed with 2 nmoles 14C-clonidine 3; ( +-)-phenylephrine 3; dopamine 1; (-+)-octo- (35.800 dpm) in 0.4 pl and with methylene pamine 0.25; tyramine 0.5 and (+)-methox- blue (Zandberg and De Jong, 1977a,b). amine 0.08. Bilateral lesions with a diameter of about After termination of the experiments the 800 pm were made in the NTS with a stainless rats were killed by decapitation. The brain steel electrode (outer diameter 200 pm) with was removed from the skull and fixed in 4% a bare tip of 200 pm. Current was applied formalin. Frozen sections (60 pm) were cut through a radio frequency lesion generator and stained with 0.1% thionine, after which (Radionics, model RFG-4) (13 V for min). the lesions and needle tracks were observed Surgical removal of the area postrema was microscopically. The lesions and injections in performed under magnification in rats anesthe- the NTS and in the locus coeruleus were tized with ether, 48 h before the experiment. projected in an atlas, modified according to A curved fine needle tip was used to excise Palkovits and Jacobowitz (1974). the area postrema in a rostral direction. Care Data are reported as means + S.E.M. was taken to avoid damage to the cerebellum Student's t-test was used for statistical analy- and the surrounding medullary structures. sis of the results. For the injections in the locus coeruleus The following drugs, dissolved or diluted in the heads of the rats were placed in the ho- saline, were used for local injections into the rizontal plane, as described by KSnig and brain: (--)-adrenaline bitartrate (Sigma); (--)- Klippel (1963). A hole was drilled on the amidephrine mesylate; clonidine HC1 (Boeh- right and left side in the skull and bilateral ringer Ingelheim), dopamine HC1 (Koch-- injections in a volume of 1.0 pl were made via Light); haloperidol (Serenase ®, Janssen Phar- a stainless steel injection needle (outer diam- maceutica); (--)-isoprenaline sulphate (Sigma); eter 200pm). The coordinates used were (+)-methoxamine HC1 (Burroughs Wellcome); 2.5 mm posterior of lambda and 1.2 mm ( -)-a-methylnoradrenaline HC1 (Sterling- lateral of the midline and the depth was 7.0 Winthrop); (+)-a-methylnoradrenaline (Ster- mm below the surface of the skull. ling-Winthrop); (--)-noradrenaline bitartrate For comparison the peripheral a-mimetic (Sigma); (+)-noradrenaline HC1 (Sterling Win- activity of the a-adrenergic drugs was deter- throp); L-methyl-14C-noradrenaline D-bitar- mined in the pithed rat. The rats were anesthe- trate (Radiochemical Centre, Amersham); tized with ether and pithed through the orbit. (+)-octopamine HC1 (Sigma); oxymetazoline Artificial respiration with a Palmer pump (68 HC1 (Merck); phentolamine (Regitine@, Ciba); strokes/min, tidal volume 3 ml) was applied (+)-phenylephrine HC1 (Koch-Light); tyramine and blood pressure was recorded (see above) HC1 (Koch--Light); yohimbine HC1 (Sigma). from a carotid artery. Drugs were injected FLA 63, bis-( 4-methyl-l-homopiperoizinyl into the jugular vein in a volume of 0.1 ml thiocarbonyl)disulphate (Labkemi AB) and and immediately washed in with 0.1 ml of urethane were injected intraperitoneally. 0.9% NaC1. Pressor responses were calculated as the increase in mean blood pressure at the peak of the pressor response. The body tem- 3. Results perature of all rats was kept at 35--37°C with 3.1. Comparison of unilateral and bilateral an infrared heating lamp. For the reference microinjections of a-methylnoradrenaline drug, (--)-noradrenaline, the dose causing an increase of 50 mm Hg in mean blood pressure Unilateral administration of (-)-a-methyl- was 0.23 nmoles. Taking the potency of noradrenaline in the A2-region of the NTS TABLE 1 Effect of unilateral injection of various doses of (--)-adrenaline, (--)-noradrenaline, dopamine and (--)~-methylnoradrenaline in the A2-region of the nucleus tractus solitarii (NTS) on mean blood pressure and heart rate. n = number of rats.

Doses n Mean A Blood pressure after blood pressure 2 rain 5 rain 10 min 15 min (mm Hg)

Saline 13 116±3 1 --2± 1 --2±1 --2±1 --2±1

(--)-Adrenaline 0.02 nmoles 7 117+4 --7+ 3 --5+2 --4_+3 --3+4 0.08 nmotes 5 120+4 --24+ 64 --17 +54 --8+6 --2+4 0.32 nmoles 6 127+_5 "--23+ 54 --21+_34 --23+44 --18_+44 1.25 nmoles 5 114+9 --19+ 44 --18+14 --17+54 --11+4 3

(--)-Noradrenaline 0.08 nmoles 4 106+3 --10 + 4 3 --1_+2 +2+2 +2+2 0.32 nmoles 6 132_+4 --27+ 44 --13_+34 --9_+33 --2+2 1.25 nmoles 8 120_+ 2 --20 + 14 --15+24 --11_+34 --9+23 5 nmoles 5 113+ 5 +2+ 11 --7_+8 --12+6 2 --15_+44

Dopamine 0.32 nmoles 6 125+4 --15-+ 53 --11+33 --6+_4 --4_+5 1.25 nmoles 7 121+2 --15-+ 2 --20+34 --14+44 --11+2 5 nmoles 5 109+5 --19+ 34 --22+_44 --21_+54 --13+62 20 nmoles 7 121+6 O+ 2 --17+44 --32+34 --30+34

(--)-a-Methylnoradrenaline 0.32 nmoles 8 110 + 3 --14+ 34 --8+23 0+2 +3+2 1.25 nmoles 7 110 _+ 2 --18+ 24 --20+_44 --16+44 --10+33 5 nmoles 9 111 + 3 --19+ 34 --27+34 --25_+34 --21+34 20 nmoles 13 129 _+ 2 --3+ 2 --25+24 --31+14 --32+24 80 nmoles 5 114 _+ 5 +7+ 3 --8+6 --34+4 '~ --34+44

Doses n Heart rate A Heart rate after (bpm) 2 min 5 rain 10 min 15 rain

Saline 13 444 + 12 --14+10 --9 + 8 --1 + 4 +2-+ 4

(--)-Adrenaline 0.02 nmoles 7 431 + 18 --26+13 --17 + 7 --14 + 7 --16-+ 7 0.08 nmoles 5 421 + 21 --85+224 --40+102 --8+ 9 --11+11 0.32 nmoles 6 404 _+ 9 --67-+173 --45+ 83 --38+-74 --36+ 74 1.25 nmoles 5 445 + 12 --77 + 26 3 --53 _+ 12 3 --54 + 19 ~ --27 _+ 13 3

(--)-Noradrenaline 0.08 nmoles 4 409 -+ 18 --36-+13 --18+10 --11-+ 8 --16+ 11 0.32 nmoles 6 453 -+ 18 --78+18 3 --53+14 3 --24_+ 6 3 --8+ 6 1.25 nmoles 8 416 + 12 --35+12 --15+ 6 --11_+ 7 --11+ 6 5 nmoles 5 410 -+ 6 --16-+ 2 --18+ 2 --22+- 33 --25-+ 44

Dopamine 0.32 nmoles 6 424 -+ 25 --14+ 7 --18+-10 --12+ 6 --18+ 72 1.25 nmoles 7 421 +- 15 --21_+ 5 --22+ 4 --18+ 72 --12+ 73 5 nmoles 5 447 -+ 10 --23+_ 4 --18+ 6 --19+ 72 --19+_82 20 nmoles 7 439 + 14 --31 + 12 --46 _+ 14 --57 + 12 4 --56 + 16 4 NUCLEUS TRACTUS SOLITARII AND BLOOD PRESSURE 47

TABLE 1 (continued)

Doses n Heart rate A Heart rate after (bpm) 2 min 5 min 10 rain 15 min

(--)-ct-Methylnoradrenaline 0.32 nmoles 8 444 +_15 --49 + 13 2 --21+- 7 --1+_ 4 +4_+ 6 1.25 nmoles 7 411 +- 9 --33 + 15 --20+11 --1+ 6 +9+_ 7 5 nmoles 9 416 + 12 --39 + 12 --50 +_ 11 3 --43 _+ II 4 --35 +- 13 3 20 nmoles 13 457 + 9 --6 + 3 --25+ 9 --40_+13 2 --45+-153 80 nmoles 5 422 + 11 --31 +- 16 --35 -+ 14 --72 + 18 4 --91 +- 17 4

I Mean + S.E.M. 2 p < 0.05. 3 p < 0.01. 4 p < 0.001.

caused a decrease of blood pressure (table 1). which decreased blood pressure was 0.32 Maximal effects were reached within 10 min nmoles. With the 20 nmoles dose a slight and were dose-dependent. The lowest dose increase in blood pressure (6 +_ 1 mm Hg) was

TABLE 2 Effect of bilateral injections of various doses of (--)~-methylnoradrenaline in the A2-region of the nucleus tractus solitarii (NTS) on mean blood pressure and heart rate. n = number of rats.

Doses n Mean A Blood pressure after blood pressure 2 min 5 min 10 rain 15 min (mm Hg)

Saline 8 108+-4 I --3+-2 0+_2 --1+_3 --1+_3

(--)-ol-Methylnoradrenaline 0.02 nmoles 5 115 +_4 --5±2 +I±I 0±2 --11±1 0.08 nmoles 5 116 +_ 5 --29±5 4 --15±3 3 --7±2 --2±3 0.32 nmoles 7 113 + 3 --31±6 4 --19±4 3 --7±4 --4±2 1.25 nmoles 6 115 +_ 4 --36±3 4 --26±2 4 --16±4 3 --8±5

Doses n Heart rate A Heart rate after (bpm) 2 min 5 min 10 min 15 min

Saline 8 446+ 13 --46+17 --28+10 --18+_ 8 --16_+ 9

(--)-ct-Methylnoradrenaline 0.02 nmoles 5 436 + 26 --23 -+ 24 --2 + 13 +2 + 10 --6 + 16 0.08 nmoles 5 421 _+ 20 --80 + 12 --42 + 17 --8 + 11 --6 + 12 0.32 nmoles 7 458 + 13 --57 + 18 --22-+ 15 --1 + 10 +5 + 8 1.25 nmoles 6 408 + 19 --96 + 18 --81 + 8 4 --59 __. 10 3 --53 _ 92

1 Mean + S.E.M. 2 p < 0.05. 3 p < 0.01. 4 p < 0.001. 48 P. ZANDBERG ETAL.

observed directly after the injection (maximal 3.2. Effective part of the NTS after about lmin) and became more pronounced after 80 nmoles (11 + 2 mm Hg). Lesions of the NTS just rostral of the obex Bilateral injections of (--)-a-methylnoradrena- level, which caused an increase in blood pres- line were more effective than unilateral ones sure did not diminish the blood pressure in decreasing blood pressure (table 2). There response to a unilateral application of a-me- were no pressor responses to a-methylnor- thylnoradrenaline in the A2- region compared adrenaline after bilateral application of to the control group (table 3). After lesions in 0.02-.-1.25 nmoles doses. The minial effective the nucleus commissuralis, which did not dose which significantly decreased blood change blood pressure, a-methylnoradrenaline pressure after bilateral application was 0.08 caused a decrease in blood pressure as well. nmoles. A decrease of heart rate was observed Removal of the area postrema also did not after 5.0 nmoles given unilaterally and after affect basal blood pressure or the blood pres- 1.25 nmoles given bilaterally. Although the sure lowering effect. In rats bearing lesions or hypotensive response was more pronounced with the area postrema excised there was no after bilateral injections (especially in the first change in basal and drug-induced alteration of 5--10 min) and bradycardia was observed heart rate. with lower doses than after unilateral administration, there was no essential difference in 3.3. Effects of adrenaline, noradrenaline and the pattern of the response. Unilateral injec- dopamine tions were used in subsequent experiments because the unilateral injection sites could be Injection of (--)-adrenaline, (-)-noradrena- localized histologically more precisely. No line and dopamine into the A2-region also difference was found in responses to injec- decreased blood pressure (table 1). A dose of tions (20 nmoles) given into the NTS in the (-)-adrenaline as low as 0.08 nmoles lowered left (--25+ 5mmHg; n =5) or right side blood pressure and heart rate. These decreases (--28 + 4 mm Hg; n = 5) of the medulla. became more prolonged after 0.32 and 1.25 nmoles. An 0.08 nmole dose of (--)-nor-

TABLE 3

Effect of unilateral injection of 20 nmoles (--)-(~-methylnoradrenaline in the A2 -region of the nucleus tractus solitarii (NTS) on blood pressure and heart rate after bilateral lesions 1.0 mm rostral or caudal of zero level in the NTS or after surgical removal of the area postrema, n = number of rats.

n Mean blood A blood A blood pressure pressure pressure (mm Hg) (mm Hg) (%)

Control 5 120 -+ 3 i --24 + 2 20.0 Lesion 1.0 mm rostral of zero level 2 7 139 _+ 7 --35 + 7 25.2 Lesion 1.0 mm caudal of zero level 2 5 112 + 4 --18 _+ 5 16.1 Sham removal of area postrema 3 4 105 _+ 8 --20 _+ 4 19.0 Removal of area postrema 3 6 103 + 3 --20 _+ 2 19.4

I Mean + S.E.M. Changes in blood pressure were measured 10 rain after the injection. 2 The lesions were made 30 min before the injections. a The injections after removal of the area postrema were given 700/lm caudal of zero level; in these rats the removal of the area postrema or the sham operation were performed 48 h prior to the experiment. TABLE 4 Effect of unilateral injection of various doses of (--)-isoprenaline, (+)-noradrenaline, (+)-o~-methylnoradrenaline, tyramine, (+-)-octopamine, clonidine, (--)-amidephrine, (+)-methoxamine, (+)-phenylephrine and oxymetazoline in the A2-region of the nucleus tractus solitarii (NTS) on mean blood pressure and heart rate. n = number of rats.

Doses n A Blood pressure A Heart rate (mm Hg) (bpm)

Saline 7 --2 + 1 1 --7 + 6

(--)-Isoprenaline 1.25 nmoles 5 ----6 + 2 --28 +- 13 5 nmoles 6 --9 + 7 --31 + 11 2 20 nmoles 6 --11 + 4 --33 +- 9 2 80 nmoles 5 +7 + 4 --54 +- 13 3

( + )-Noradrenaline 20 nmoles 4 +12 + 2 3 --21 + 4 80 nmoles 9 --8 + 3 --54 -+ 10 3

( + )-a-Methylnoradrenaline 20 nmoles 4 +1 _+ 2 --1 _+ 2 200 nmoles 8 +7 _+ 3 2 --3 + 5

Tyramine 5 nmoles 5 --9 + 6 --25 +_ 5 20 nmoles 5 --16 + 3 3 --43 _+ 21 80 nmoles 6 --11 _+ 4 2 --36 + 4 3

( + )-Octopamine 1.25 nmoles 3 --4 _+ 5 --25 + 9 5 nmoles 6 --11 _+ 4 2 --48 + 21 20 nmoles 5 --21 _+ 3 4 --60 +_ 21 2

Clonidine 1.25 nmoles 7 --4 _+ 4 --31 + 20 5 nmoles 11 --20 + 6 2 --76 + 19 2 20 nmoles 11 --11 _+ 6 --47 + 10 2 80 nmoles 3 +30 + 5 4 --45 + 35

(--)-A midephrine 5 nmoles 7 --7 + 2 --16 +- 10 20 nmoles 6 --13 -+ 4 3 --53 + 12 3 80 nmoles 5 +5 -+ 6 --72 + 27 2

(+)-Methoxamine 5 nmoles 3 +5 + 5 --18 + 11 20 nmoles 6 --14 +_ 6 --35 _+ 9 2 80 nmoles 6 +29 -+ 7 4 --45 + 16 2

( +-)-Phenylephrine 5 nmoles 8 --5 + 4 --1 + 7 20 nmoles 5 --2 + 3 --26 + 5 2 80 nmoles 8 +9 + 8 --52 _+ 12 3

Oxymetazoline 5 nmoles 9 +2 + 4 --26 + 5 2 20 nmoles 9 +4 _+ 2 --49 + 9 3

I Mean + S.E.M. Changes in blood pressure were measured 10 min after the injection. Basal values for blood pressure and heart rate of the groups were not significantly different and were in the same range as those shown in tables 1--3. 2 p < 0.05. 3 p < 0.01. 4 p < 0.001. 50 P. ZANDBERG ET AL.

adrenaline decreased blood pressure while a sure, reaching a maximum within 5 min, while 0.32 nmole dose also decreased heart rate. the effect of a-methylnoradrenaline and With a 5.0 nmole dose there was an initial rise dopamine developed more slowly. of blood pressure (12 + 5 mm Hg) directly 3.4. Effects of various adrenergic substances after the injection (maximal after about 1 rain) and the subsequent decrease of blood pressure As shown in table 4, various doses of (--)- was less marked. Dopamine was about as isoprenaline were ineffective. The (+)-stereo- effective as a-methylnoradrenaline in decreas- isomers of noradrenaline and a-methylnor- ing blood pressure. There was a decrease in adrenaline either did not alter the blood pres- blood pressure with the 0.32 nmole dose. sure or increased it. With the higher doses of dopamine the decrease Tyramine, 20 nmoles, induced a significant became more prolonged and more pro- decrease in blood pressure and heart rate and nounced and the heart rate also decreased. 80 nmoles did not induce a significantly The 20 nmole dose decreased blood pressure greater change in blood pressure. (+)-Octo- after a delay of 2--5 min. pamine, 5 and 20 nmoles, lowered blood pres- The pattern of the decrease in blood pressure. The response pattern was similar to that sure after dopamine and a-methylnoradrena- with dopamine and a-methylnoradrenaline. line was different from that following adrena- Oxymetazoline and (+)-phenylephrine were line and noradrenaline. Adrenaline and nor- ineffective in changing blood pressure. Only adrenaline caused a sharp drop in blood pres- the 20 nmole dose of (_+)-methoxamine and

TABLE 5 Effect on mean blood pressure and heart rate of unilateral injection of 5 nmoles dopamine and 5 nmoles (--)-(~- methylnoradrenaline in the A2-region of the nucleus tractus solitarii (NTS) after pretreatment with FLA 63, or following administration of phentolamine, yohimbine and haloperidol at the same injection site. n -- number of rats.

n A Blood pressure A Heart rate (ram Hg) (bpm)

Dopamine Saline 3 --21-+1 i --16-+ 7 FLA 63 (25 mg/kg i.p.) 5 --31 + 3 --35 + 17 Phentolamine (5 nmoles) 6 --1+2 4 --30-+ 6 Yohimbine (1.25 nmoles) 4 --4+4 3 --23 + 6 Haloperidol (5 nmoles) 6 --3 + 5 2 --9 -+ 5 Haloperidol (0.32 nmoles) 5 --11+4 --8 + 6

(-- )-ovMe th y ln oradre naline Saline 4 --28 -+ 2 --11 + 12 FLA 63 (25 mg/kg i.p.) 5 --27-+2 --8 + 8 Phentolamine (5 nmoles) 4 --3+5 4 --5+ 8 Yohimbine (1.25 nmoles) 5 +6 + 6 4 +4 + 5 Haloperidol (5 nmoles) 4 +3 + 2 4 +2 + 6 Haloperidol (0.32 nmoles) 5 --14 + 6 --4 + 2

t Mean -+ S.E.M. Changes in blood pressure were measured 10 rain after the injection. Basal values for blood pressure and heart rate of the groups were not significantly different and were in the same range as those shown in Tables 1--3. 2 p < 0.05. a p < 0.01. 4 p < 0.001. NUCLEUS TRACTUS SOLITARII AND BLOOD PRESSURE 51

( -)-amidephrine somewhat lowered the blood 3.5. Effects of a-adrenoceptor blocking agents pressure while 5 nmoles clonidine could lower and of systemic pretreatment with FLA 63 it. However, a higher dose, 80 nmoles, of clonidine and methoxamine only increased The a-adrenergic blocking agents phentol- blood pressure. The maximal rise of blood amine (5 nmoles) and yohimbine (1.25 pressure occurred within 5 min. The heart nmoles), locally applied to the A2-region rate was somewhat decreased by all these sub- 10 min previously blocked the effect of both stances, especially with the higher doses (table 5 nmoles a-methylnoraclrenaline and 5 nmoles 4). dopamine. Haloperidol (5 nmoles) also

TABLE6 Effect on blood pressure and heart rate of bilateral injections of various doses of (--)-isoprenaline, (--)-adrenaline, (--)-a-methylnoradrenaline, (--)-noradrenaline, dopamine and clonidine in the locus coeruleus, n = number of rats.

Doses n A Blood pressure A Heart rate (mm Hg) (bpm)

Saline 4 --1+- 3 --7 + 3 I

(--)-Isoprenaline 1.25 nmoles 5 --3 + 1 --20 + 5 5 nmoles 5 --13+ 10 --19 + 6 20 nmoles 6 --17 +- 62 --40 + 5 4

(--)-Adrenaline 1.25 nmoles 6 +9 + 3 --4 + 3 5nmoles 6 +13+ 5 --16+ 7 20nmoles 5 +5-+ 8 --37+ 83

(--)-(~-Methylnoradrenaline 5nmoles 3 +14_+ 22 --7+ 7 20 nmoles 5 +14 + 7 --14 + 6

(--)-Noradrenaline 5 nmoles 6 +22 + 4 3 --17 + 6 20nmoles 6 +24_+ 53 --29_+ 43

Dopamine 5nmoles 3 +8-+ 5 --7-+ 7 20nmoles 6 +14_+ 52 --28+11

Clonidine 1.25nmoles 2 --18+ 32 --25+ 52 5 nmoles 5 --13+ 23 --30 _+ 11 20 nmoles 5 --30+ 102 --66 -+ 21 2

I Mean -+ S.E.M. Changes in blood pressure were measured 10 rain after the injection. Basal values for blood pressure and heart rate of the groups were not significantly different and were in the same range as those shown in tables 1--3. 2 p < 0.05. 3 p < 0.01. 4 p < 0.001. 52 P. ZANDBERG ET AL. blocked the blood pressure lowering response did not change the blood pressure lowering to 5 nmoles dopamine as well as to 5 nmoles effect of a-methylnoradrenaline when the a-methylnoradrenaline and 0.32 nmoles still same injection technique was used. Taken reduced the effect of both drugs to a similar together, these results support the hypothesis extent (table 5). The blocking agents them- that the A2-region is the site in the NTS where selves did not significantly change blood catecholamines cause a decrease in blood pressure and heart rate. pressure and heart rate. The present micro- The blood pressure lowering effect of a-me- injection studies did not allow a more precise thylnoradrenaline and dopamine was not localization since there was an elongated blocked by administration of FLA 63, given rostro-caudal distribution of the injection i.p. 1 h before the local injection (table 5). volume (0.4 pl) along the NTS over a distance However there was some delay (2--5 min) of approximately 1200 pm, as shown with before the decrease of blood pressure radioactive noradrenaline and, previously, occurred. with methylene blue and ~4C-clonidine (Zand- berg and De Jong, 1977a,b). Interestingly 3.6. Effects of catecholamines and clonidine these results indicate a differentiation after injection into the area of the locus between the area from which hypertension coeruleus can be evoked by lesions and the area which, when injected with catecholamines, evokes a As shown in table 6 (--)-adrenaline, (--)- decrease of blood pressure, because only noradrenaline, dopamine and (-)-a-methyl- lesions of the NTS located rostral of the A2- noradrenaline caused an increase in blood region elicited hypertension (Zandberg et al., pressure after bilateral local application into 1978). the locus coeruleus. An injection of 5 nmoles The relative potencies of the substances noradrenaline into the fourth ventricle did which, when applied to the A2-region, not change the blood pressure or heart rate. decreased blood pressure, were adrenaline > (--)-Isoprenaline (20 nmoles) and clonidine, noradrenaline > dopamine > a-methylnor- however, decreased blood pressure. Clonidine adrenaline > octopamine. Although bilateral and the higher doses of adrenaline and nor- injections were more effective than unilateral adrenaline also caused bradycardia. ones, the unilateral microinjection technique as used in the present study with a smaller cannula size and injection volume was appar- 4. Discussion ently more sensitive than the bilateral injection technique used earlier (De Jong and Nij- Our previous localization study with local kamp, 1976). The more prolonged effect of injections of a-methylnoradrenaline into the ~-methylnoradrenaline and the fact that the NTS of rats showed that the most effective maximal decrease of blood pressure induced region for causing a decrease in blood pressure by adrenaline and noradrenaline, occurred was at the level of the caudal tip of the area within 5 min may explain the fact that De postrema (Zandberg and De Jong, 1977a). Jong and Nijkamp (1976) had previously Injections into the NTS just rostral of the found a-methylnoradrenaline somewhat more obex level were ineffective, while injections effective than adrenaline 5 min after adminis- caudally in the commissural part of the NTS tration. were only slightly effective. These findings The finding, that isoprenaline and the (+)- are in accordance with the present results, stereoisomers of noradrenaline and a-methyl- showing that bilateral lesions in the NTS just noradrenaline were ineffective is consistent rostral of the obex, or in the nucleus com- with the idea that the blood pressure lowering missuralis, or removal of the area postrema effect of the various catecholamines may be NUCLEUS TRACTUS SOLITARII AND BLOOD PRESSURE 53 caused by direct stimulation of postsynaptic injection into the anterior hypothalamus stereospecific a-adrenoceptors in the brain (Struyker Boudier and Bekers, 1975). How- (see Introduction). However, dopamine was ever this effect may also reflect the more effective in a dose-range similar to that of lipophylic character of adrenaline. a-methylnoradrenaline. Apart from an action An initial pressor response was caused by on dopamine receptors, dopamine may higher doses of noradrenaline and a-methyl- stimulate a- and ~-receptors directly or be noradrenaline and by the different a-mimetics converted into noradrenaline (Hornykiewicz, following their application into the A2-region. 1966; Goldberg, 1972). Systemic injection of After higher doses of dopamine the blood FLA 63 with subsequent local injections of pressure decrease was delayed as was also seen dopamine at the time of maximal inhibition with a-methylnoradrenaline. A higher dose of of dopamine-~-hydroxylase (Svensson and noradrenaline induced a delayed and less Waldeck, 1969; Corrodi et al., 1970) did not pronounced decrease of blood pressure. In affect the decrease of blood pressure caused previous studies we found that the pressor by dopamine or a-methylnoradrenaline. This response to the high doses of a-methylnor- suggests a direct effect of dopamine on a adrenaline was enhanced following the local catecholaminergic receptor. The prior admin- application of phentolamine (De Jong and istration of haloperidol, which blocks dopa- Nijkamp, 1976). The pressor effect, however, mine receptors preferentially to noradrena- appears not to be explainable by ~-mimetic line receptors (Van Rossum, 1966; And~n effects since isoprenaline had no pressor et al., 1970; Day and Roach, 1976), failed to activity. Although the increase of blood pres- separate the effect of dopamine from that sure may be caused in part by leakage to the a-methylnoradrenaline. Also, the a-lyric drugs circulation, no correlation -- either positive phentolamine and yohimbine both blocked or negative --with the peripheral vasoconstric- the effect of dopamine as well as that of tor activity could be found; e.g. methoxamine a-methylnoradrenaline. These findings may be caused a pronounced pressor response, while interpreted as indicating that the catechol- peripherally it is not a very active vasocon- amines act on an a-receptor site. This could strictor drug. It should be noted that a dose- also fit with the finding that dopamine was dependent increase in blood pressure can be less effective than were noradrenaline and evoked from the same area of the NTS by adrenaline. The a-mimetic substances inves- local injections of physostigmine (Zandberg tigated, however, did not decrease blood and De Jong, 1977b). There are thus struc- pressure dose-dependently after their micro- tures in the NTS which can cause an increase injection into the A2-region. Neither the in blood pressure. a-mimetics with a relatively preferential post- The results with clonidine show that a synaptic effect (methoxamine and phenyl- variable decrease in blood pressure could be ephrine) or that with a relatively presynaptic observed only with relatively high doses effect (oxymetazoline) (Starke et al., 1975; applied to the A2-region, while still higher Berthelsen and Pettinger, 1977) had a marked doses only increased blood pressure. Philippu effect. From these data it is not possible to et al. (1973) showed that, in the cat, super- define the catecholaminergic structures in the fusion of the NTS with clonidine had no As-region responsible for the decrease of effect on blood pressure while it diminished blood pressure. Adrenaline was the most po- the pressor response evoked by stimulation of tent of the catecholamines and this may the posterior hypothalamus. Lipski et al. indicate an adrenoceptor type. Similarly (1976) reported that, in rats, the decrease in adrenaline was reported to be more effective blood pressure caused by the intravenous than noradrenaline in decreasing the blood injection of clonidine was significantly pressure of anesthetized rats following an diminished after ablation of the NTS. Laubie 54 P. ZANDBERG ET AL. and Schmitt (1977) and Antonaccio and sis (Cedarbaum and Aghajanian, 1975). These Halley (1977) found that bilateral destruction results may be explained by a selective activa- of the NTS did not significantly change the tion of presynaptic a-receptors by clonidine blood pressure response of clonidine in cats (Starke et al., 1975). and dogs. These authors have shown that in contrast to the hypotensive effect the NTS Acknowledgements was the main site of the vagally mediated bradycardia of clonidine in these species. In The authors are indebted to H. de Lang, C. Creutz- rats, however, we only found limited changes burg and A.J.M. Seegers for their excellent assistance. in heart rate after the local injection of clonidine. Thus, with respect to the effect on heart rate, there may be a difference between References cats and dogs on one hand and rats on the other hand in the response to clonidine. It And6n, N.-E., H. Corrodi, K. Fuxe, B. ttSkfelt, C. Rydin and T. Svensson, 1970, Evidence for a is possible that in rats the bradycardia caused central noradrenaline receptor stimulation by by agents like a-methylnoradrenaline and clonidine, Life Sci. 9,513. clonidine is mediated via a decrease in sym- Antonaccio, M.J. and J. Halley, 1977, Clonidine hy- pathetic tone more than via an increase in potension: lack of effect of bilateral lesions of the vagal tone. De Jong and Nijkamp (1976) nucleus tractus solitary tract in anaesthetized cats, Neuropharmacol. 16,431. found that the bradycardia induced by Berthetsen, S. and W.A. Pettinger, 1977, A functional a-methylnoradrenaline applied into the A2- basis for classification of a-adrenergic receptors, region of the NTS was only reduced by Life Sci. 21,595. atropine and bilateral vagotomy and the Cedarbaum, J.M. and G.K. Aghajanian, 1976, Nor- blood pressure lowering effect was enhanced. adrenergic neurons of the locus coeruleus: inhibi- It can be concluded that in the rat the NTS is tion by epinephrine and activation by the a-antag- onist piperoxane, Brain Res. 112,413. not sensitive to clonidine and appears to be of Chalmers, J.P., 1975, Brain amines and models of limited importance for the bradycardia caused experimental hypertension, Circulation Res. 36, by clonidine. It is however possible that cloni- 469. dine does not act primarily on the catechol- Corrodi, H., K. Fuxe, B. Hamberger and fl~. Ljung- aminergic structures in the NTS but on other dahl, 1970, Studies on central and peripheral noradrenaline neurons using a new dopamine-~- catecholaminergic cell groups in the pons- hydroxylase inhibitor, European J. Pharmacol. medulla which project to the NTS. Szreniaw- 12,145. ski et al. (1977) found that, in rats, bilateral DahlstrSm, A. and K. Fuxe, 1964, Evidence for the lesions of the locus coeruleus inhibited the existence of monoamine containing neurones in blood pressure lowering effect of clonidine. the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neu- Our results also suggest that the locus rons, Acta Physiol. Scand. 62, Suppl. 232, 1. coeruleus may be one of the sites where Day, M.D. and A.G. Roach, 1976, Cardiovascular clonidine is capable of decreasing blood pres- effects of dopamine after central administration sure. The catecholamines, however, increased into conscious cats, Brit. J. PHarmacol. 58, 505. De Jong, W., 1974, Noradrenaline, central inhibitory blood pressure at this site while isoprenaline control of blood pressure and heart rate, European decreased it. And~n et al. (1970) reported J. Pharmacol. 29,179. that clonidine reduced noradrenaline turnover De Jong, W., P. Zandberg and B. Bohus, 1975, Cen- in the brain. A reduction of spontaneous tral inhibitory noradrenergic cardiovascular con- firing of the noradrenaline containing neurons trol, in: Hormones, Homeostasis and the Brain, Progr. Brain Res. 42, eds. W.H. Gispen, Tj.B. van of the locus coeruleus has been observed fol- Wimersma Greidanus, B. Bohus and D. de Wied lowing i.v. injection of clonidine in cats (Elsevier, Amsterdam) p. 285. (Svensson et al., 1975) or local administration De Jong, W. and F.P. Nijkamp, 1976, Centrally in the locus coeruleus using micro-iontophore- induced hypotension by a-methylnoradrenaline in NUCLEUS TRACTUS SOLITARII AND BLOOD PRESSURE 55

normotensive and renal hypertensive rats, Brit. Palkovits, M. and D.M. Jacobowitz, 1974, Topographic J. Pharmacol. 58,593. atlas of catecholamine and acetylcholinesterase Fuxe, K., 1965, Evidence for the existence of mono- containing neurons in the rat brain. II. Hindbrain amine neurons in the central nervous system. IV. (mesencephalon, rhombencephalon), J. Comp. Distribution of monoamine nerveflerminals in the Neurol. 157, 29. central nervous system, Acta Physiol. Scand. 64, Palkovits, M. and L. Zfiborszky, 1977, Neuroanatomy Suppl. 247, 37. of central cardiovascular control. Nucleus tractus Goldberg, L.I., 1972, Cardiovascular and renal actions solitarii: afferent and efferent neuronal connec- of dopamine, Pharmacol. Rev. 24, 1. tions in relation to the baroreceptor reflex arc, in: Haeusler, G., 1975, Cardiovascular regulation of cen- Hypertension and Brain Mechanisms, Progr. Brain tral adrenergic mechanisms and its alteration by Res. 47, eds. W. De Jong, A.P. Provoost and A.P. hypotensive drugs, Circulation Res., Suppl. I to Shapiro (Elsevier, Amsterdam) p.9. Vols. 36 and 37,223. Philippu, A., W. Roensberg and H. Przuntek, 1973, Haeusler, G., 1977, Neuronal mechanisms influencing Effects of adrenergic drugs on pressor responses transmission in the baroreceptor reflex arc., in: to hypothalamic stimulation, N.S. Arch. Pharma- Hypertension and Brain Mechanisms, Progr. Brain col. 278,373. Res. 47, eds. W. De Jong, A.P. Provoost and A.P. Schmitt, H., H. Schmitt, S. F6nard and M. Laubie, Shapiro (Elsevier, Amsterdam) p. 95. 1973, Evidence for a sympathomimetic compo- Henning, M., 1973, Receptor regulation studies: nent inhibiting the sympathetic centres: nature significance of central catecholamine receptors of the receptor, in: Symposium on Pharmacological in cardiovascular control, in: Frontiers in Cate- Agents and Biogenic Amines in the Central Nervous cholamine Research, ed. E. Usdin and S.H. Snyder System, eds. J. Knoll and K. Magyar (Akademiai (Pergamon Press, Oxford--London) p. 595. Kiad6, Budapest) p. 177. Henning, M., 1975, Central sympathetic transmitters Sinha, J..N., J.M. Atkinson and H. Schmitt, 1973, Ef- and hypertension, Clin. Sci. Mol. Med. 48, 195s. fects of clonidine and 1-dopa on spontaneous and Hornykiewicz, O., 1966, Dopamine and brain func- evoked splanchnic nerve discharges, European J. tion, Pharmacol. Rev. 18,925. Pharmacol. 24,113. Kobinger, W. and L. Pichler, 1975, The central Starke, K., T. Endo and H.D. Taube, 1975, Relative modulatory effects of clonidine on the cardio- pre- and postsynaptic potencies of a-adrenoceptor depressor reflex after suppression of synthesis and agonists in the rabbit pulmonary artery, N.S. storage of noradrenaline, European J. Pharmacol. Arch. Pharmacol. 291, 55. 30, 56. Struyker Boudier, H.A.J. and A. Bekers, 1975, KSnig, J.F.R. and R.A. Klippel, 1963, The rat brain: Adrenaline-induced cardiovascular changes after a stereotaxic atlas of the forebrain and lower parts intrahypothalamic administration to rats, Euro- of the brainstem (The Williams and Wilkins Com- pean J. Pharmacol. 31,153. pany, Baltimore). Svensson, T., B.S. Bunney and G.K. Aghajanian, 1975, Laubie, M., B. Delbarre, D. Bogaievsky, Y. Bogaiev- Inhibition of both noradrenergic and serotonergic sky, D. Tsoucaris-Kupfer, D. Senon, H. Schmitt neurons in brain by the c~-adrenergic agonist and H. Schmitt, 1977, Pharmacological evidence clonidine, Brain Res. 92,291. for a central a-sympathomimetic mechanism con- Svensson, T. and B. Waldeck, 1969, On the signifi- trolling blood pressure and heart rate, Circulation cance of central noradrenaline for motor activity: Res. 38, Suppl. IIp. 35. experiments with a new dopamine ~-hydroxylase Laubie, M. and H. Schmitt, 1977, Sites of action of inhibitor, European J. Pharmacol. 7,278. clonidine: Centrally mediated increase in vagal Szreniawski, Z., W. Kostowski and E. Widy-Tyszkie- tone, centrally mediated hypotensive and sympa- wicz, 1976, Effects of lesions of monoaminergic tho-inhibitory effects, in: Hypertension and Brain systems in the brain on the hypotensive effects of Mechanisms, Progr. Brain Res. 47, eds. W. De Jong, clonidine in rats, Pol. J. Pharmacol. Pharm. 28, A.P. Provoost and A.P. Shapiro (Elsevier, Amster- 605. dam) p. 337. Van der Gugten, J., M. Palkovits, H.L.J.M. Wijnen Lipski, J., J. Przybylski and E. Solnicka, 1976, Re- and D.H.G. Versteeg, 1976, Regional distribution duced hypotensive effect of clonidine after lesions of adrenaline in rat brain, Brain Res. 107,171. of area of nucleus of tractus solitarii in rats, Euro- Van Rossum, J.M., 1966, The significance of dopa- pean J. Pharmacol. 38, 19. mine-receptor blockade for the mechanism of Nijkamp, F.P. and W. De Jong, 1975, ~-Methyl- action of neuroleptic drugs, Arch. Intern. Pharma- noradrenaline induced hypotension and brady- codyn. 160,492. cardia after administration into the area of the Van Zwieten, P.A., 1975, Antihypertensive drugs nucleus tractus solitarii, European J. Pharmacol. with a central action, Progr. Pharmacol. 1, 1. 32,361. 56 P. ZANDBERG ET AL.

Versteeg, D.H.G., J. van der Gugten, W. De Jong and tractus solitarii involved in the regulation of M. Palkovits, 1976, Regional concentrations of arterial blood, pressure, in: Hypertension and noradrenaline and dopamine in rat brain, Brain Brain Mechanisms, Progr. Brain Res. 47, ed. W. De Res. 113,563. Jong, A.P. Provoost and A.P. Shapiro (Elsevier, Zandberg, P. and W. De Jong, 1977a, ~-Methylnor- Amsterdam) p. 117. adrenaline-induced hypotension in the nucleus Zandberg, P., M. Palkovits and W. De Jong, 1978, tractus solitarii of the rat: a localization study, Effect of various lesions in the nucleus tractus Neuropharmacol. 16,219. solitarii of the rat on blood pressure, heart rate Zandberg, P. and W. De Jong, 1977b, Localization of and cardiavascular reflex responses Clin. Exptl. catecholaminergic receptor sites in the nucleus Hypertension (in press).