European Journal of Pharmacology, 182 (1990) 63-72
63
Elsevier EJP 51338
Cardiovascular effects of 5-HT1A receptor agonists injected into the dorsal raphe nucleus of conscious rats H e l e n E. C o n n o r a n d G u y A. H i g g i n s 1 Department of Neuropharmacology, Glaxo Group Research Ltd., Ware, Herts SG12 ODJ, U.K.
Received 18 January 1990, revised MS received 13 March 1990, accepted 20 March 1990
The aim of this study was to investigate the cardiovascular effects of the 5-HTIA receptor agonists, 8-OH-DPAT (8=hydroxy-2-(di-n-propylamino)tetralin), flesinoxan and 5-carboxamidotryptamine (5-CT) following injection into the dorsal raphe nucleus of conscious rats. 8-OH-DPAT (0.5-2.5 /~g), flesinoxan (2.5-5.0 ~tg) and 5-CT (0.05 /.tg) caused hypotension, bradycardia and flat body posture. In contrast, injection of 8-OH-DPAT (0.5/~g) into the median raphe nucleus caused no cardiovascular changes or flat body posture. (-)Pindolol (0.5/~g dorsal raphe nucleus) had little effect on cardiovascular parameters, but significantly attenuated the cardiovascular effects of 8-OH-DPAT (0.5 /.tg dorsal raphe nucleus). N-Methylatropine (1 mg/kg i.v.) antagonised the cardiovascular effects of 8-OH-DPAT (0.5/.tg dorsal raphe nucleus), suggesting these were vagally mediated. Both pretreatments also appeared to reduce 8-OHDPAT-induced flat body posture. The results suggest that 8-OH-DPAT activates 5-HT1Areceptors in the dorsal raphe nucleus to cause hypotension and bradycardia. 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino) tetralin); Flesinoxan; Dorsal raphe nucleus; Hypotension; Bradycardia; 5-HTIA receptors; (Conscious rat)
1. I n t r o d u c t i o n
8 - H y d r o x y - 2 - ( d i - n - p r o p y l a m i n o ) t e t r a l i n (8O H - D P A T ) is a potent, effective, centrally acting hypotensive agent in a variety of anaesthetised and conscious animal models (Gradin and Lis, 1985; Fozard et al., 1987; Ramage and Fozard, 1987; Di Francesco et al., 1988; H o f and Fozard, 1989; Laubie et al., 1989). These cardiovascular effects are attributed to stimulation of 5-HT1A receptors since they can be mimicked by other highly selective 5-HT1A receptor agonists such as flesinoxan and ipsapirone (Ramage and Fozard,
Present address: Addiction Research Foundation, 33 Russell St., Toronto, Ontario, Canada M5S 2S1. Correspondence to: H.E. Connor, Department of Neuropharmacology, Glaxo Group Research Ltd., Ware, Herts SG12 0DJ, U.K.
1987; Wouters et al., 1988), and specifically blocked by the 5 - H T receptor antagonists metergoline and methiothepin (Fozard et al., 1987). In addition, pindolol, a c o m p o u n d with some selectivity for 5-HTIA and 5-HTIB receptors, compared to other 5-HT receptor subtypes (Hoyer et al., 1985; Schoeffter and Hoyer, 1988), has been shown to antagonise the hypotensive effects of 8 - O H - D P A T and flexinoxan in anaesthetised cats (Doods et al., 1988; Wouters et al., 1988). Autoradiographic studies have demonstrated a high density of 5-HTIA binding sites within the midbrain dorsal and median raphe nuclei (Pazos and Palacios, 1985; Verge et al., 1986; WeissmanNanopoulos et al., 1985). Together, these nuclei provide a major source of central 5-HT neuronal pathways (Azmitia and Segal, 1978). A physiological role for 5-HT1A binding sites within the dorsal raphe nucleus has been suggested by electrophysi-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
64 ological (Sprouse and Aghajanian, 1987; Sinton and Fallon, 1988), biochemical (Hjorth and Magnusson, 1988; Hutson et al., 1989) and behavioural (Hutson et al., 1986; Higgins et al., 1988) experiments. Together these studies suggest that 5-HT1A receptor activation suppresses raphe cell firing, producing a consequent reduction in 5-HT turnover within raphe innervated regions. There are various lines of evidence to suggest a role for the dorsal raphe nucleus in blood pressure control. For instance, in anaesthetised rats, electrical stimulation of the dorsal raphe nucleus causes pressor responses which are mediated via serotonergic neurones (Kuhn et al., 1980; Robinson et al., 1985). Conversely, inhibition of dorsal raphe nucleus cell firing by the G A B A agonist muscimol (Gallager, 1978) has been shown to produce vasodepressor responses (Robinson et al., 1986). Therefore, the aim of the present study was to examine the cardiovascular effects of 8-OHD P A T and flexinoxan when injected directly into the dorsal raphe nucleus, and to determine whether these effects were due to 5-HTIA receptor stimulation. In order to exclude any influence of anaesthesia and to allow the simultaneous assessment of any behavioural changes, experiments were performed in conscious rats. A preliminary account of these findings has been presented in abstract form (Connor and Higgins, 1989).
techniques described by Higgins et al. (1988). The co-ordinates (A + 1.2 mm, L + 0 mm, V - 3.0 mm) resulted in the cannula tip being sited 3.2 m m and 5.5 mrn above the centre of the dorsal raphe nucleus and median raphe nucleus respectively (Paxinos and Watson, 1982). Following surgery, the rats were handled daily and sham injections (needle tip terminating 2 m m above the dorsal raphe nucleus) were performed at least once before the initial experiment; these procedures minimised any injection trauma.
2.3. Implantation of carotid arterial cannulas At least 7 days later, rats were anaesthetised with a volatile anaesthetic (enflurane in 84% nitrous oxide and 16% oxygen). A small incision was made in the neck and the right carotid artery was located. A polythene cannula (PP10 tubing, internal diameter 0.28 m m connected to a length of PP50 tubing, internal diameter 0.58 mm), filled with sterile heparinised saline (250 U / m l ) , was inserted into the carotid artery according to the method of Popovic and Popovic (1960). The PP50 end of the cannula was exteriorised at the back of the neck. The carotid cannula was flushed with heparinised saline and then sealed with a stainless steel pin. Experiments were performed 24-48 h later.
2.4. Experimental procedure 2. Materials and methods
2.1. Animals Male hooded normotensive Lister rats (270 + 30 g starting weight) were used for the study. Animals were housed in groups of four until the carotid arterial cannula was implanted when animals were housed individually. All animals were allowed free access to food and water.
2.2. Implantation of CNS guide cannulas Under chloral hydrate anaesthesia (400 m g / k g i.p.; 10 m l / k g dose volume), the rats were chronically implanted with a stainless steel guide cannula (23 gauge, 7 m m length) according to tne
2.4.1. General Rats were placed individually in an observation cage (34 cm x 14 cm x 14 cm). The arterial cannula was connected to a Bell and Howell pressure transducer (type 4-422) via a length of polythene tubing (PP50) filled with heparinised saline for the measurement of blood pressure. Heart rate was derived from the blood pressure signal using a Devices instantaneous ratemeter; blood pressure and heart rate were displayed on a Devices MX8 chart recorder. Animals were allowed 10-15 min for blood pressure and heart rate to stabilise and then the injection needle (30 gauge, 10.5 m m in length) was lowered into the dorsal raphe nucleus via the guide cannula. Approximately 5 min later, when blood pressure and heart rate were stable,
65 drug or vehicle (0.9% saline) was slowly injected (dose volume of 0.5 ~1 over 1 rain) directly into the dorsal raphe nucleus. During this procedure, animals were allowed to move freely around the holding box. The injection needle was left in place for 2-3 min after injection. Blood pressure and heart rate were measured for up to 40 rain after injection; changes were expressed as % change from pre-injection levels. Each animal received only one dose of agonist. Body posture was assessed continually before and after injection and described as normal or flattened (flattened body posture).
2.4.2. Antagonist experiments In some animals, the antagonist effects of ( - ) p i n d o l o l were studied. ( - ) P i n d o l o l or vehicle (0.9% saline) was injected into the dorsal raphe nucleus and, 5 min later, 8 - O H - D P A T or vehicle (0.9% saline) was injected into the dorsal raphe nucleus. Changes (%) in blood pressure and heart rate were measured from basal levels recorded just before the first injection.
2.4. 3. Median raphe nucleus injections 8 - O H - D P A T was injected directly into the median raphe nucleus as described above, but using an injection needle 12.8 m m in length.
2.4. 4. Intravenous administration of drugs In some animals, 8 - O H - D P A T or N-methylatropine was administered intravenously. These animals were implanted with a cannula inserted into the right jugular vein, at the same time as the arterial cannula was implanted. Drugs were administered intravenously in a volume of 1 m l / k g and flushed in with 0.2 ml heparinised saline.
atlas of Paxinos and Watson (1982). Any animal in which the location of the injection site was found to be incorrectly sited ( < 10%) was excluded from the study.
2.6. Drugs The following drugs were used: N-methylatropine bromide (MSD), flesinoxan hydrochloride (Duphar), ( - ) p i n d o l o l (Sandoz). 5-Carboxamidotryptamine maleate (5-CT) and ( _ ) 8 - h y d r o x y 2-(di-n-propylamino)tetralin h y d r o b r o m i d e (8O H - D P A T ) were synthesised in the Chemical Research Department, Glaxo G r o u p Research Ltd., Ware. ( - ) P i n d o l o l was dissolved in a couple of drops of 0.2 N HC1 and made up to volume with 0.9% isotonic saline. All other drugs were dissolved and diluted in 0.9% isotonic sodium chloride with concentrations expressed as that of the free base. The p H was adjusted to 7.4 with N a H C O 3.
2. 7. Statistics Statistical differences between drug and vehicle treated animals were assessed using Student's t-test (unpaired); values of P < 0.05 were considered to be statistically significant. Statistical differences between resting levels of blood pressure and heart rate in the various groups of animals were assessed using a one way analysis of variance.
3. Results
3.1. Effects of injection of agonists into the dorsal raphe nucleus
2.5. Histology U p o n completion of the study the rats were anaesthetised with chloral hydrate (400 m g / k g i.p.) and then injected with 0.5 ~1 of India ink into either the dorsal raphe nucleus or median raphe nucleus as appropriate. The locations of injection sites were identified from coronal sections (10/~m) prepared on a microtome (Leitz, model 1321) and histological placements were identified using the
Resting mean blood pressure and heart rate were not significantly different between the various groups of animals (one way analysis of variance; table 1). Injection of vehicle (0.5/~l) into the dorsal raphe nucleus caused no significant change in blood pressure or heart rate over a 30 min observation period. The behaviour of the animals (as assessed by visual observation) was similarly unaffected.
66 TABLE 1 Resting mean blood pressure and heart rate in the various groups of animals measured just before dorsal raphe nucleus injection of agonist or vehicle. Values are means + S.E.M. from n observations. Also shown is the incidence of flattened body posture following dorsal raphe nucleus injection. Treatment
Vehicle 8-OH-DPAT 8-OH-DPAT 8-OH°DPAT Flesinoxan Flesinoxan 5-CT
0.5 ~1 0.1 #g 0.5 #g 2.5 ~g 2.5 ~g 5.0 ~g 0.05 ~g
Resting mean BP (mm Hg)
Resting HR (bpm)
n
109 + 11 113_+ 9 131 _+ 3 125 _+ 9 119 + 4 117-+ 5 114-+ 4
408 + 46 359_+33 447_+ 16 421 _+31 405 -+28 426-+ 25 373_+28
4 3 5 4 5 4 3
3.1.1. Effects of 8-OH-DPA T 8 - O H - D P A T (0.5 o r 2.5 /~g) caused m a r k e d decreases in b l o o d p r e s s u r e a n d h e a r t rate when injected d i r e c t l y i n t o the d o r s a l r a p h e nucleus of c o n s c i o u s rats. Effects were r a p i d in onset ( < 30 s p o s t injection), a l t h o u g h the decrease in h e a r t rate t e n d e d to p r e c e d e the decrease in b l o o d pressure; i n d e e d , in some animals, heart rate s t a r t e d to decline b e f o r e the injection of 8 - O H - D P A T was complete. P e a k changes were r e a c h e d a b o u t 10 m i n after injection a n d within 15 min b l o o d pressure a n d h e a r t rate were starting to r e t u r n t o w a r d s p r e - i n j e c t i o n levels. A lower d o s e (0.1 ~tg) h a d little effect o n c a r d i o v a s c u l a r p a r a m e t e r s . A representative tracing to illustrate the effects o f 8 - O H D P A T (0.5 ktg) o n b l o o d pressure a n d h e a r t rate is shown in fig. 1; m e a n results are shown in fig. 2. In these animals, 8 - O H - D P A T also c a u s e d a d o s e - d e p e n d e n t i n c i d e n c e of flattened b o d y posture (table 1) with the highest d o s e tested causing f l a t t e n e d b o d y p o s t u r e in all animals. A m a r k e d b e h a v i o u r a l h y p o a c t i v i t y was also n o t e d with locomotion and rearing being markedly reduced a l t h o u g h these b e h a v i o u r a l o b s e r v a t i o n s were n o t q u a n t i f i e d . Behavioural changes were a p p a r e n t w i t h i n 1-2 m i n after d o r s a l r a p h e nucleus injection a n d lasted for a b o u t 15-20 min. I n contrast, w h e n injected directly into the m e d i a n r a p h e nucleus 8 - O H - D P A T (0.5/~g) caused n o d e c r e a s e in b l o o d p r e s s u r e or h e a r t rate ( - 3 + 5 a n d 0 + 6% respectively, m e a s u r e d 10 m i n after
Incidence of flattened body posture 0/4 0/3 4/5 4/4 1/5 4/4 3/3
injection, n = 5). F u r t h e r m o r e , these a n i m a l s showed no f l a t t e n e d b o d y posture. I n d e e d , alt h o u g h activity was n o t q u a n t i f i e d in these animals, they a p p e a r e d h y p e r a c t i v e c o m p a r e d to pre-injection b e h a v i o u r (see fig. 1). I n t r a v e n o u s injection of 8 - O H - D P A T (1.5 /~g/kg), a d o s e a p p r o x i m a t e l y equivalent to 0.5/~g into the d o r s a l r a p h e nucleus, c a u s e d little or n o c h a n g e in b l o o d pressure or h e a r t rate (4 + 2 a n d 2 + 3% respectively, 10 rain after dosing, n = 4).
3.1.2. Effects of flesinoxan Flesinoxan caused a dose-dependent decrease in b l o o d pressure a n d h e a r t rate w h e n injected d i r e c t l y i n t o the d o r s a l r a p h e nucleus, a l t h o u g h higher doses (2.5-5.0/~g) were r e q u i r e d to p r o d u c e these effects. R e s p o n s e s were i m m e d i a t e in o n s e t a n d p e a k c h a n g e s were seen 10-15 m i n after injection (fig. 3). F l e s i n o x a n also c a u s e d a d o s e - d e p e n d e n t i n c i d e n c e of f l a t t e n e d b o d y p o s t u r e in these rats (table 1).
3.1.3. Effects of 5-CT Injection of 5 - c a r b o x a m i d o t r y p t a m i n e (5-CT, 0.05/~g) i n t o the d o r s a l r a p h e nucleus also caused m a r k e d decreases in b l o o d p r e s s u r e a n d h e a r t rate. Effects were i m m e d i a t e in onset b u t slow to r e a c h a m a x i m u m ; p e a k changes ( - 30 + 3 a n d - 33 + 5% respectively, n = 3) were n o t o b t a i n e d until 20-25 m i n after injection. T h e s e a n i m a l s all s h o w e d
67 8
63
Elsevier EJP 51338
Cardiovascular effects of 5-HT1A receptor agonists injected into the dorsal raphe nucleus of conscious rats H e l e n E. C o n n o r a n d G u y A. H i g g i n s 1 Department of Neuropharmacology, Glaxo Group Research Ltd., Ware, Herts SG12 ODJ, U.K.
Received 18 January 1990, revised MS received 13 March 1990, accepted 20 March 1990
The aim of this study was to investigate the cardiovascular effects of the 5-HTIA receptor agonists, 8-OH-DPAT (8=hydroxy-2-(di-n-propylamino)tetralin), flesinoxan and 5-carboxamidotryptamine (5-CT) following injection into the dorsal raphe nucleus of conscious rats. 8-OH-DPAT (0.5-2.5 /~g), flesinoxan (2.5-5.0 ~tg) and 5-CT (0.05 /.tg) caused hypotension, bradycardia and flat body posture. In contrast, injection of 8-OH-DPAT (0.5/~g) into the median raphe nucleus caused no cardiovascular changes or flat body posture. (-)Pindolol (0.5/~g dorsal raphe nucleus) had little effect on cardiovascular parameters, but significantly attenuated the cardiovascular effects of 8-OH-DPAT (0.5 /.tg dorsal raphe nucleus). N-Methylatropine (1 mg/kg i.v.) antagonised the cardiovascular effects of 8-OH-DPAT (0.5/.tg dorsal raphe nucleus), suggesting these were vagally mediated. Both pretreatments also appeared to reduce 8-OHDPAT-induced flat body posture. The results suggest that 8-OH-DPAT activates 5-HT1Areceptors in the dorsal raphe nucleus to cause hypotension and bradycardia. 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino) tetralin); Flesinoxan; Dorsal raphe nucleus; Hypotension; Bradycardia; 5-HTIA receptors; (Conscious rat)
1. I n t r o d u c t i o n
8 - H y d r o x y - 2 - ( d i - n - p r o p y l a m i n o ) t e t r a l i n (8O H - D P A T ) is a potent, effective, centrally acting hypotensive agent in a variety of anaesthetised and conscious animal models (Gradin and Lis, 1985; Fozard et al., 1987; Ramage and Fozard, 1987; Di Francesco et al., 1988; H o f and Fozard, 1989; Laubie et al., 1989). These cardiovascular effects are attributed to stimulation of 5-HT1A receptors since they can be mimicked by other highly selective 5-HT1A receptor agonists such as flesinoxan and ipsapirone (Ramage and Fozard,
Present address: Addiction Research Foundation, 33 Russell St., Toronto, Ontario, Canada M5S 2S1. Correspondence to: H.E. Connor, Department of Neuropharmacology, Glaxo Group Research Ltd., Ware, Herts SG12 0DJ, U.K.
1987; Wouters et al., 1988), and specifically blocked by the 5 - H T receptor antagonists metergoline and methiothepin (Fozard et al., 1987). In addition, pindolol, a c o m p o u n d with some selectivity for 5-HTIA and 5-HTIB receptors, compared to other 5-HT receptor subtypes (Hoyer et al., 1985; Schoeffter and Hoyer, 1988), has been shown to antagonise the hypotensive effects of 8 - O H - D P A T and flexinoxan in anaesthetised cats (Doods et al., 1988; Wouters et al., 1988). Autoradiographic studies have demonstrated a high density of 5-HTIA binding sites within the midbrain dorsal and median raphe nuclei (Pazos and Palacios, 1985; Verge et al., 1986; WeissmanNanopoulos et al., 1985). Together, these nuclei provide a major source of central 5-HT neuronal pathways (Azmitia and Segal, 1978). A physiological role for 5-HT1A binding sites within the dorsal raphe nucleus has been suggested by electrophysi-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
64 ological (Sprouse and Aghajanian, 1987; Sinton and Fallon, 1988), biochemical (Hjorth and Magnusson, 1988; Hutson et al., 1989) and behavioural (Hutson et al., 1986; Higgins et al., 1988) experiments. Together these studies suggest that 5-HT1A receptor activation suppresses raphe cell firing, producing a consequent reduction in 5-HT turnover within raphe innervated regions. There are various lines of evidence to suggest a role for the dorsal raphe nucleus in blood pressure control. For instance, in anaesthetised rats, electrical stimulation of the dorsal raphe nucleus causes pressor responses which are mediated via serotonergic neurones (Kuhn et al., 1980; Robinson et al., 1985). Conversely, inhibition of dorsal raphe nucleus cell firing by the G A B A agonist muscimol (Gallager, 1978) has been shown to produce vasodepressor responses (Robinson et al., 1986). Therefore, the aim of the present study was to examine the cardiovascular effects of 8-OHD P A T and flexinoxan when injected directly into the dorsal raphe nucleus, and to determine whether these effects were due to 5-HTIA receptor stimulation. In order to exclude any influence of anaesthesia and to allow the simultaneous assessment of any behavioural changes, experiments were performed in conscious rats. A preliminary account of these findings has been presented in abstract form (Connor and Higgins, 1989).
techniques described by Higgins et al. (1988). The co-ordinates (A + 1.2 mm, L + 0 mm, V - 3.0 mm) resulted in the cannula tip being sited 3.2 m m and 5.5 mrn above the centre of the dorsal raphe nucleus and median raphe nucleus respectively (Paxinos and Watson, 1982). Following surgery, the rats were handled daily and sham injections (needle tip terminating 2 m m above the dorsal raphe nucleus) were performed at least once before the initial experiment; these procedures minimised any injection trauma.
2.3. Implantation of carotid arterial cannulas At least 7 days later, rats were anaesthetised with a volatile anaesthetic (enflurane in 84% nitrous oxide and 16% oxygen). A small incision was made in the neck and the right carotid artery was located. A polythene cannula (PP10 tubing, internal diameter 0.28 m m connected to a length of PP50 tubing, internal diameter 0.58 mm), filled with sterile heparinised saline (250 U / m l ) , was inserted into the carotid artery according to the method of Popovic and Popovic (1960). The PP50 end of the cannula was exteriorised at the back of the neck. The carotid cannula was flushed with heparinised saline and then sealed with a stainless steel pin. Experiments were performed 24-48 h later.
2.4. Experimental procedure 2. Materials and methods
2.1. Animals Male hooded normotensive Lister rats (270 + 30 g starting weight) were used for the study. Animals were housed in groups of four until the carotid arterial cannula was implanted when animals were housed individually. All animals were allowed free access to food and water.
2.2. Implantation of CNS guide cannulas Under chloral hydrate anaesthesia (400 m g / k g i.p.; 10 m l / k g dose volume), the rats were chronically implanted with a stainless steel guide cannula (23 gauge, 7 m m length) according to tne
2.4.1. General Rats were placed individually in an observation cage (34 cm x 14 cm x 14 cm). The arterial cannula was connected to a Bell and Howell pressure transducer (type 4-422) via a length of polythene tubing (PP50) filled with heparinised saline for the measurement of blood pressure. Heart rate was derived from the blood pressure signal using a Devices instantaneous ratemeter; blood pressure and heart rate were displayed on a Devices MX8 chart recorder. Animals were allowed 10-15 min for blood pressure and heart rate to stabilise and then the injection needle (30 gauge, 10.5 m m in length) was lowered into the dorsal raphe nucleus via the guide cannula. Approximately 5 min later, when blood pressure and heart rate were stable,
65 drug or vehicle (0.9% saline) was slowly injected (dose volume of 0.5 ~1 over 1 rain) directly into the dorsal raphe nucleus. During this procedure, animals were allowed to move freely around the holding box. The injection needle was left in place for 2-3 min after injection. Blood pressure and heart rate were measured for up to 40 rain after injection; changes were expressed as % change from pre-injection levels. Each animal received only one dose of agonist. Body posture was assessed continually before and after injection and described as normal or flattened (flattened body posture).
2.4.2. Antagonist experiments In some animals, the antagonist effects of ( - ) p i n d o l o l were studied. ( - ) P i n d o l o l or vehicle (0.9% saline) was injected into the dorsal raphe nucleus and, 5 min later, 8 - O H - D P A T or vehicle (0.9% saline) was injected into the dorsal raphe nucleus. Changes (%) in blood pressure and heart rate were measured from basal levels recorded just before the first injection.
2.4. 3. Median raphe nucleus injections 8 - O H - D P A T was injected directly into the median raphe nucleus as described above, but using an injection needle 12.8 m m in length.
2.4. 4. Intravenous administration of drugs In some animals, 8 - O H - D P A T or N-methylatropine was administered intravenously. These animals were implanted with a cannula inserted into the right jugular vein, at the same time as the arterial cannula was implanted. Drugs were administered intravenously in a volume of 1 m l / k g and flushed in with 0.2 ml heparinised saline.
atlas of Paxinos and Watson (1982). Any animal in which the location of the injection site was found to be incorrectly sited ( < 10%) was excluded from the study.
2.6. Drugs The following drugs were used: N-methylatropine bromide (MSD), flesinoxan hydrochloride (Duphar), ( - ) p i n d o l o l (Sandoz). 5-Carboxamidotryptamine maleate (5-CT) and ( _ ) 8 - h y d r o x y 2-(di-n-propylamino)tetralin h y d r o b r o m i d e (8O H - D P A T ) were synthesised in the Chemical Research Department, Glaxo G r o u p Research Ltd., Ware. ( - ) P i n d o l o l was dissolved in a couple of drops of 0.2 N HC1 and made up to volume with 0.9% isotonic saline. All other drugs were dissolved and diluted in 0.9% isotonic sodium chloride with concentrations expressed as that of the free base. The p H was adjusted to 7.4 with N a H C O 3.
2. 7. Statistics Statistical differences between drug and vehicle treated animals were assessed using Student's t-test (unpaired); values of P < 0.05 were considered to be statistically significant. Statistical differences between resting levels of blood pressure and heart rate in the various groups of animals were assessed using a one way analysis of variance.
3. Results
3.1. Effects of injection of agonists into the dorsal raphe nucleus
2.5. Histology U p o n completion of the study the rats were anaesthetised with chloral hydrate (400 m g / k g i.p.) and then injected with 0.5 ~1 of India ink into either the dorsal raphe nucleus or median raphe nucleus as appropriate. The locations of injection sites were identified from coronal sections (10/~m) prepared on a microtome (Leitz, model 1321) and histological placements were identified using the
Resting mean blood pressure and heart rate were not significantly different between the various groups of animals (one way analysis of variance; table 1). Injection of vehicle (0.5/~l) into the dorsal raphe nucleus caused no significant change in blood pressure or heart rate over a 30 min observation period. The behaviour of the animals (as assessed by visual observation) was similarly unaffected.
66 TABLE 1 Resting mean blood pressure and heart rate in the various groups of animals measured just before dorsal raphe nucleus injection of agonist or vehicle. Values are means + S.E.M. from n observations. Also shown is the incidence of flattened body posture following dorsal raphe nucleus injection. Treatment
Vehicle 8-OH-DPAT 8-OH-DPAT 8-OH°DPAT Flesinoxan Flesinoxan 5-CT
0.5 ~1 0.1 #g 0.5 #g 2.5 ~g 2.5 ~g 5.0 ~g 0.05 ~g
Resting mean BP (mm Hg)
Resting HR (bpm)
n
109 + 11 113_+ 9 131 _+ 3 125 _+ 9 119 + 4 117-+ 5 114-+ 4
408 + 46 359_+33 447_+ 16 421 _+31 405 -+28 426-+ 25 373_+28
4 3 5 4 5 4 3
3.1.1. Effects of 8-OH-DPA T 8 - O H - D P A T (0.5 o r 2.5 /~g) caused m a r k e d decreases in b l o o d p r e s s u r e a n d h e a r t rate when injected d i r e c t l y i n t o the d o r s a l r a p h e nucleus of c o n s c i o u s rats. Effects were r a p i d in onset ( < 30 s p o s t injection), a l t h o u g h the decrease in h e a r t rate t e n d e d to p r e c e d e the decrease in b l o o d pressure; i n d e e d , in some animals, heart rate s t a r t e d to decline b e f o r e the injection of 8 - O H - D P A T was complete. P e a k changes were r e a c h e d a b o u t 10 m i n after injection a n d within 15 min b l o o d pressure a n d h e a r t rate were starting to r e t u r n t o w a r d s p r e - i n j e c t i o n levels. A lower d o s e (0.1 ~tg) h a d little effect o n c a r d i o v a s c u l a r p a r a m e t e r s . A representative tracing to illustrate the effects o f 8 - O H D P A T (0.5 ktg) o n b l o o d pressure a n d h e a r t rate is shown in fig. 1; m e a n results are shown in fig. 2. In these animals, 8 - O H - D P A T also c a u s e d a d o s e - d e p e n d e n t i n c i d e n c e of flattened b o d y posture (table 1) with the highest d o s e tested causing f l a t t e n e d b o d y p o s t u r e in all animals. A m a r k e d b e h a v i o u r a l h y p o a c t i v i t y was also n o t e d with locomotion and rearing being markedly reduced a l t h o u g h these b e h a v i o u r a l o b s e r v a t i o n s were n o t q u a n t i f i e d . Behavioural changes were a p p a r e n t w i t h i n 1-2 m i n after d o r s a l r a p h e nucleus injection a n d lasted for a b o u t 15-20 min. I n contrast, w h e n injected directly into the m e d i a n r a p h e nucleus 8 - O H - D P A T (0.5/~g) caused n o d e c r e a s e in b l o o d p r e s s u r e or h e a r t rate ( - 3 + 5 a n d 0 + 6% respectively, m e a s u r e d 10 m i n after
Incidence of flattened body posture 0/4 0/3 4/5 4/4 1/5 4/4 3/3
injection, n = 5). F u r t h e r m o r e , these a n i m a l s showed no f l a t t e n e d b o d y posture. I n d e e d , alt h o u g h activity was n o t q u a n t i f i e d in these animals, they a p p e a r e d h y p e r a c t i v e c o m p a r e d to pre-injection b e h a v i o u r (see fig. 1). I n t r a v e n o u s injection of 8 - O H - D P A T (1.5 /~g/kg), a d o s e a p p r o x i m a t e l y equivalent to 0.5/~g into the d o r s a l r a p h e nucleus, c a u s e d little or n o c h a n g e in b l o o d pressure or h e a r t rate (4 + 2 a n d 2 + 3% respectively, 10 rain after dosing, n = 4).
3.1.2. Effects of flesinoxan Flesinoxan caused a dose-dependent decrease in b l o o d pressure a n d h e a r t rate w h e n injected d i r e c t l y i n t o the d o r s a l r a p h e nucleus, a l t h o u g h higher doses (2.5-5.0/~g) were r e q u i r e d to p r o d u c e these effects. R e s p o n s e s were i m m e d i a t e in o n s e t a n d p e a k c h a n g e s were seen 10-15 m i n after injection (fig. 3). F l e s i n o x a n also c a u s e d a d o s e - d e p e n d e n t i n c i d e n c e of f l a t t e n e d b o d y p o s t u r e in these rats (table 1).
3.1.3. Effects of 5-CT Injection of 5 - c a r b o x a m i d o t r y p t a m i n e (5-CT, 0.05/~g) i n t o the d o r s a l r a p h e nucleus also caused m a r k e d decreases in b l o o d p r e s s u r e a n d h e a r t rate. Effects were i m m e d i a t e in onset b u t slow to r e a c h a m a x i m u m ; p e a k changes ( - 30 + 3 a n d - 33 + 5% respectively, n = 3) were n o t o b t a i n e d until 20-25 m i n after injection. T h e s e a n i m a l s all s h o w e d
67 8
