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Pressor responses to electrical and chemical stimulation of the rat brain A10 dopaminergic system
ELSEVIER
Neuroscience Letters 176 (1994) 142..146
Pressor responses to electrical and chemical stimulation of the rat brain A 10 dopaminergic system Jennifer L. Cornish*, Maarten van den Buuse Neuropharmacology Laboratory, Baker Medical Research Institute, PO Box 348, Prahran, Vic. 3181, Australia Received 2 February 1994; Revised version received 3 June 1994; Accepted 6 June 1994
Abstract Central dopaminergic systems have been implicated in the regulation of blood pressure. We examined the effect on blood pressure of electrical or chemical stimulation of the rat brain ventral tegmental area (VTA) which is the region of origin of the A10 dopaminergic system. Electrical stimulation in urethane-anaesthetised rats (10-120 Hz, 80/IA) produced frequency-dependent increases in blood pressure (max 30-35 mmHg). These pressor responses could be significantlyattenuated by pretreatment with the dopamine D2 receptor antagonist haloperidol, but not the D~ receptor antagonist SCH 23390. Chemical stimulation of the VTA, by microinjection of 10 nmol of the substance P analogue DiMe-C7, produced a sustained increase in blood pressure (max 10-15 mmHg), which could be completely prevented by pretreatment with haloperidol. These results suggest that stimulation of dopaminergic neurons in the VTA induces pressor responses and that projections from midbrain dopaminergic neurons, acting on dopamine D2 receptors, play a role in the regulation of blood pressure. Key words: Dopamine; Blood pressure; Ventral tegmental area; Dopamine D2 receptor; Haloperidok SCH 23390; DiMe-C7; Sprague-Dawley rat; Central nervous system
There is increasing evidence for a role of central dopaminergic systems in blood pressure regulation and the development of hypertension. Systemic administration of dopamine D2 receptor agonists caused centrallymediated increases in blood pressure [13]. Several authors have observed differential changes of indices of central dopaminergic activity in the spontaneously hypertensive rat (SHR) (for review, see [14]). Moreover, the development of hypertension in these rats can be inhibited by depletion of brain dopamine, either with the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) or with electrolytic lesions in the substantia nigra [15]. One major central dopaminergic system that has been implicated in cardiovascular regulation is the mesolimbic (A10) system which originates in the midbrain ventral tegmental area (VTA). Electrical stimulation of the VTA in both rabbits and cats has been shown to produce an increase in blood pressure [9,12]. A number of authors
* Corresponding author. Fax: (61) (3) 521 1362. 0304-3940194/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00450-0
have found that dopamine D2 receptor density in brain regions innervated by this system were different between hypertensive and normotensive rats [5,6]. The VTA provides main ascending dopaminergic efferents to the nucleus accumbens, olfactory tubercle, amygdala and habenular nucleus and descending efferents to the locus coeruleus [7,11]. The aim of the present study was to investigate cardiovascular responses to either electrical or chemical stimulation of the VTA in the rat brain. We used the substance P analogue DiMe-C7 for chemical stimulation of the VTA as this brain region is densely innervated by substance P containing neurons, such as those descending from the habenular nucleus and striatum [1]. DiMeC7 is a metabolically stable analogue of substance P which produces prolonged stimulation of A10 dopaminergic cell bodies [3]. Effects of micro-injection of DiMeC7 into the VTA include an increase in locomotor and exploratory behaviour in rats [2] and a stimulation of dopamine turnover in the nucleus accumbens [4]. All experiments followed the guidelines of the Austra-
143
J.L. Cornish, M. van den Buuse/Neuroscience Letters 176 (1994) 142 146
lian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Alfred Hospital/Baker Medical Research Institute Animal Experimentation Committee. We used male SpragueDawley rats (250 350 g). In the case of electrical stimulation of the VTA, the rats were anaesthetised with urethane (1.4 g/kg, i.p.) and implanted with femoral artery catheters for the measurement of blood pressure and heart rate. Jugular vein catheters were implanted for the intravenous administration of drugs. The rats were then placed in a stereotaxic frame (David Kopf) with the nosebar set at zero. A unipolar electrode was inserted unilaterally at an angle of 12° , through a burr hole in the left hand side of the skull, at c°-°rdinates ° f 5 3 mm p°steri°r' 2"5 mm lateral and 8"5 mm ventral of the bregma. The electrical circuit was completed by attaching an earth lead to the trapezius muscle of the animal. Frequency-response curves were then obtained by applying 5 s stimulus trains (5 ms block pulse duration) at a current of 80 #A, using a Grass $88 stimulator via a Grass voltage~zurrent converter (model PSIU6). Ascending frequency-levels (10-120 Hz) were tested at 1 min intervals. Five min after the completion of the first curve, haloperidol (0.5 mg/kg; Serenace, Searle), the dopamine Dl receptor antagonist SCH 23390 (0.1 mg/kg; Research Biochemicals) or vehicle (0.9% saline, 1 ml/kg) was administered i.v., and 15 rain later a second frequency-response curve was obtained. Each animal underwent only one experimental procedure. The animals were killed with an i.v. overdose of pentobarbitone sodium (60 mg/ml, Nembutal, Bomac Laboratories, Australia) and their brains were dissected out and fixed in 4% formalin. For experiments involving chemical stimulation of the VTA, the rats underwent two surgical procedures under anaesthesia induced with a mixture of methohexitone sodium (40 mg/kg, Brietal Sodium, Eli Lilly), pentobarbitone sodium (30 mg/kg) and atropine sulphate (0.5 mg/kg, Sigma). The first procedure involved the implantation of a metal guide cannula (C313-G, Plastic Products Company, Roanoke, VA, USA) into the region of the VTA using the co-ordinates previously mentioned, These cannulas were secured with dental cement and three small screws placed in the skull. The cannulas were kept patent with a chronically placed dummy cannula (C313-DC) cut to the appropriate length. After 5 7 days recovery, the animals underwent surgery for implantation of chronic catheters into the abdominal aorta and jugular vein. These animals were then allowed another 5 7 days recovery before being studied while conscious, We pretreated the rats 15 min before central injections with either haloperidol (0.5 mg/kg, i.v.) or saline (1 ml/ kg, i.v.), and then microinjected 10 nmol of [pGlu s, MePhe8, Sarg]-substance P5 L~ (DiMe-C7, Auspep, Vic., Australia) unilaterally into the VTA. The central injection was in 1.0 #1 of saline which was infused over 1 min
f-------"---'-~. ~
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Fig. 1. Typical examples of midbrainsectionsof rats withan electrode (top panel) or guide cannula (bottom panel) placed into the region of the VTA. Right panels show histological sections and left panels represent corresponding diagrams modified from lhe atlas of Paxinos and Watson [8]. Thick arrows and shaded areas show the site of the electrode tip or guide cannula. Animals which showed placement outside of this region were discardedfrom lhe analyses.
with a Hamilton 10 #1 micro-syringe and a Harvard Apparatus syringe infusion pump (Model 22, South Natick, MA, USA). Each of the seven rats used in this part of the study was tested with both treatments in random order on two separate occasions, wilh at least 48 h between the experiments. At the completion of both experiments, the animals were killed and their brains taken as for the acute experiments. In all experiments, pulsatile blood pressure and mean arterial pressure (MAP) were recorded with a Statham P23XL pressure transducer and displayed on a Neomedix Systems Neotrace recorder. Heart rate was derived off the pressure pulse by tachographs. For histological verification of the stimulation and injection sites, 30 #m sections of the midbrain were cut using a Leitz freezing microtome and were stained with Cresyl violet. Electrode and guide cannula placement in the VTA were verified by the comparison of these sections with the brain atlas of Paxinos and Watson [8]. Only results from animals with electrode or cannula placement inside the VTA (Fig. 1) were analysed. Results are expressed as mean values _+ standard error
144
./.L. CmwLvh, '~1 van &'t~ Buu.~c/.%,uroscicm'c Lcm'rs l - 6
luO4, 142 146
blood pressure and heart rate responses to local DiMeC7 infusion were calculated w i t h a graphics computer
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program (Graphpad Inplot, San Diego, CA. USA). Electrical stimulation of the VTA in anaesthetised rats (Fig. I) consistently produced a frequency-dependent increase in blood pressure which was phase-locked with the onset and offset of the stimulation. Effects on heart rate were variable, with some animals showing moderate increases, while other animals exhibited bradycardic responses or no heart rate changes (data not shown). In all curves obtained before vehicle or antagonist treatment. maximum increases in blood pressure occurred at frequencies of 100 120 Hz (Fig. 2) with similar MAP plateau values and EFs0 values (Table 1). Treatment with haloperidol caused a small, but statistically significant reduction in basal blood pressure (P = 0.005; Table 1). After haloperidol pretreatment, the frequency-response curve was significantly depressed (Fig. 2), with a 35% inhibition of the maximal plateau (P < 0.001' Table 1). Treatment with haloperidol also caused the frequency-response curve to shift to the right, resulting in a significant increase in the EFso. values (P = 0.014: Table 1). The plateau and EFs,, values of the frequency-response curves after treatment with either saline or SCH 23390 were not significantly different from those obtained before these respective treatments (Table l ). Microinjection of DiMe-C7 into the VTA of conscious rats (Fig. 1) produced an increase in blood pressure with little effect on heart rate (Fig. 3). The pressor responses were of 10-15 mmHg in magnitude, were maximal 10-15 min after the infusion of DiMe-C7 (Fig. 3) and remained elevated above baseline for much of the remaining pcriod. Pretreatment with haloperidol did not alter basal MAP; however, it completely blocked the pressor response elicited by chemical stimulation of the VTA
Fig. 2. Changes in blood pressure produced by electrical stimulation of the VTA with increasing frequencies in urethane-anaesthetised rats•
Table 1
The top, middle and b o t t o m panels show the effect of vehicle (n = 6), haloperidol (n = 9) and SCH 23390 (n = 6) pretreatment, respectively, after the initial control curve• Treatment with haloperidol produced a marked reduction in pressor responses. F o r baseline values, see Table l. *P < 0.05 for difference between the plateau values of responses
Frequency response curves to electrical stimulation of the VTA in anaesthetised rats .......................... Treatment Basal M A P AMAP plateau EFs0 (Hz) (mmHg) (mmHg)
before and alter haloperidol treatment.
Control
85.8 _+ 2.0
34.5 _+ 2.7
3916 + 4.0
Saline ( I ml/kg)
87.0 _+ 2.1
29.6 _+ 3.2
41.6 +_ 3.2
Control Haloperidol ~0.5 mg/kg)
83.1 + 2.8 74.2 _+ 2.3*
33.0 _+ 1.7 21.3 + 1•2"
46.2 +_ 3.3 55.3 _+ 4.1'
Control SCH 23390 (0.1 mg/kg)
85.3 + 5.1 84.2 + 3.4
32•0 + 5.0 33.9 _+ 4.7
43.2 _+ 2.9 46.3 _+ 4.0
of the mean (S.E.M.) and were statistically analysed with
analysis of variance (ANOVA) for repeated
measures.
Blood pressure responses to electrical stimulation of the VTA were frequency-dependent and reached a plateau at higher frequencies. Thus, frequency-response curves obtained from the experiments with electrical stimulation were analysed by sigmoidal curve fitting (SIGMOID, Baker Medical Research Institute), which yielded plateau values and effective frequency values that produced 50% of the maximal pressor response (EF50) for each curve. The area under the curve (AUC) of the
d M A P plateau is the maximal increase in M A P seen with electrical stimulation. EFs0 value is the frequency required to induce 50% o f the
maximalpressor response. *P < 0.05 for difference between
treated and control values. The number of animals was 6 (saline group), 9 (haloperidol) and 6 (SCH 23390),
J.L. Cornish, M. van den Buuse/Neuroscience Letters 176 (1994) 142 146
--O--Control + DiMe-C7 ~Haloperidol
+ DiMe-C7
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.
.
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60
Fig. 3. Changes in mean arterial pressure ( M A R top panel) and heart rate (bottom panel) after local infusion of DiMe-C7 into the VTA of conscious rats. Baseline blood pressures were 100.3 + 3.0 and 98.8 _+ 2.1 m m Hg, and baseline heart rates were 381.0 + 12.8 and 381.9 +_ 11.5 beats/min for vehicle (n = 7) and haloperidol (n = 7) pretreated animals, respectively. Pretreatment with haloperidol caused a complete inhibition of the pressor response produced with DiMe-C7 infusion and also revealed a marked bradycardia,
(P = 0.001). After this pretreatment, a marked bradycardia was observed with a maximum of approximately 65 beats/rain at 20 min after injection (Fig. 3). Heart rate had returned to near resting levels by the end of the 60 min monitoring period. There was a statistically significant overall difference between heart rate values obtained with or without haloperidol pretreatment (P < 0.001). The AUC values for the blood pressure responses to DiMe-C7 infusion were 602+125 units for the saline-treated rats, which was significantly different from - 8 6 + 134 units calculated for the haloperidol treated animals (P < 0.001). Similarly, the A U C value for the heart rate responses to DiMe-C7 infusion of the salinetreated animals (1522+ 517 units) was significantly greater than that calculated for the haloperidol treated rats (-2743 + 595 units, P = 0.001). In the present study, we observed that both electrical and chemical stimulation of the VTA caused an increase
145
in blood pressure. In both cases the involvement of dopamine D 2 receptors was suggested by the finding that pretreatment with haloperidol produced a 35% inhibition of electrical stimulation and a complete blockade of the pressor response produced after DiMe-C7 infusion into the VTA. The pressor response to electrical stimulation of the VTA began immediately upon stimulation and lasted only as long as the pulse duration. This suggests that the effects elicited by this stimulation were most likely sympathetic in origin. Indeed, in preliminary experiments we observed that pretreating the animals with the ganglion blocking agent, pentolinium, cornpletely inhibited any pressor effect of electrical stimulation (data not shown). Approximately 35% of the response elicited with electrical stimulation appears to be produced by stimulation of dopamine D2 receptors, which could be mediated by dopaminergic projections from the VTA to brain regions such as the nucleus accumbens or the amygdala, or by descending projections to regions such as the locus coeruleus. The latter possibility may be considered as lesions of the posterior hypothalamus had little effect on the pressor response elicited by electrical stimulation of the cat VTA [9]. Since this lesion was anterior to the VTA this suggests the involvement of lower brainstem regions, such as the locus coeruleus, in the pressor response elicited. The antagonist results from this study imply that approximately 65% of the pressor response produced by electrical stimulation of the VTA is not mediated by either dopamine D Lor D2 receptors. This response m a y be caused by stimulation of non-dopaminergic efferents of the VTA as approximately one third of the n e u r o n s in this brain area are non-dopaminergic cells which a r e intermixed with dopaminergic cell bodies [11]. There is also the possibility that the electrical stimulation technique has affected fibres of passage in the ventral midbrain, such as ascending noradrenergic neurones, to increase blood pressure. The metabolically stable substance P analogue DiMe-C7 is thought to directly stimulate dopaminergic neuronal cell bodies by actiw~ting substance P receptors in this area [2,3,4]. In addition, we used conscious rats for this part of the study, avoiding any influence of anaesthesia on the results. Chemical stimulation of the rabbit VTA with glutamate produced pressor and bradycardic responses [12]. In the present study, chemical stimulation of the rat VTA with DiMe-C7 also induced a long-lasting pressor response but failed to produce a decrease in heart rate. The complete inhibition of the pressor response by haloperidol suggests that dopamine De receptors are the major mediators of this effect. In recent preliminary experiments, we observed that the pressor effect of intra-VTA injections of DiMe-C7 could also be blocked by pretreatment with the selective dopamine De receptor antagonist raclopride (unpublished data). The bradycardia revealed
146
J.L. ('ornish, M. van den Buuse / Neur oscicnce Letters 176 ~ 1994 j 142 146
after haloperidol treatment could be either dopamine D I receptor-mediated or non-dopaminergic in origin. The latter effect may be mediated through non-dopaminergic neurones that originate in the VTA and project to structures involved in the regulation of heart rate, such as the locus coeruleus [ 10,11 ]. H e a r t r a t e in rats injected with DiMe-C7 is obviously under the influence of a number of mechanisms: on the one hand, the increase in blood pressure observed in
these animals would normally produce a baroreflexmediated bradycardia, and central non-dopaminergic mechanisms (see above) also induce a decrease in heart
rate, which becomes apparent when dopamine receptors are blocked with haloperidol. On the other hand, there could be a cardio-acceleration concomittant with the increase in blood pressure, possibly mediated by an increased sympathetic outflow, which is largely compensated for by the other two mechanisms, In conclusion, the present study shows that electrical or chemical stimulation of the A 10 dopaminergic cells in the VTA elicit a pressor response. The effects are most
likely mediated by dopamine D2 receptors. These results suggest that the A10 dopaminergic system is involved in cardiovascular control and that the projections from midbrain dopaminergic neurons may be associated with cardiovascular homeostasis, [1] Brownstein, M.J., Mroz, E., Kizer, J.S., Palkovits, M. and Leeman, S.E., Regional distribution of substance P in the brain of the rat, Brain Res., 116 (1976) 299-305. [2] Eison, A.S., Eison, M.S. and Iversen, S.D., The behavioural effects of a novel substance P analogue following infusion into the ventral tegmental area or substantia nigra of rat brain, Brain Res., 238 (1982) 137-152. [3] Eison, A.S. and Iversen, S.D., Substance P analogue, DiMe-CV: evidence for stability in rat brain and prolonged central actions, Science, 215 (1982) 188-190.
[4] Elliott, P.J., Alpert, J.E., Bannon, M.J. and lverscn. S.D., Selectiw activation of mesolimbic and mesocortical dopamine metabolism in rat brain by infusion of a stable substance P analogue into the ventral tegmental area, Brain Res., 363 (1986) t45 147. [5] Kirouac, G.J. and Ganguly, P.K., Up-regulation of dopamme receptors in the brain of the spontaneously hypertensive rat: an autoradiographic analysis, Neuroscience, 52 (1993) 135-141. [6] Le Fur, G., Guilloux, F., Kabouche, M., Mihani. N., Ferris, O. and Uzan, U., Central dopaminergic neurons during the development of genetic and DOCA-salt hypertension in the rat, Brain Res., 227 (1981)153-163. [7] Oades, R.D. and Halliday, G.M., Ventral tegrnental (AI0) system: neurobiology. 1. Anatomy and connectivity, Brain Res. Rev., 12 (1987) 117 165. [81 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1982.
[9] Spring. A. and Winkelmiiller, W., Ventral midbrain stimulation.
blood pressure responses and their relation to the dopaminergic nigro-striatal pathways, Pflfigers Arch., 358 (1975) 339--348. [10] Sved, A.F. and Felsten, G., Stimulation of the locus coeruleus decreases arterial pressure, Brain Res., 414 (1987) 119 132. [111 Swanson, L.W., The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat, Brain Res. Bull., 9 (1982)
321 353. [12] Tan, E., Goodchild, A.K. and Dampney, R.A.L., Intense vasocon-
striction and bradycardia evokedby stimulation of neurons within the midbrain and the ventral tegmentum of the rabbit, Clin. Exp. Pharmacol. Physiol., 10 (1983) 305--309. [13l Van den Buuse, M., Central effects ofquinpirole on blood pressure of spontaneously hypertensive rats, J. Pharmacol. Exp. Ther., 262 (1992) 303-311. [14] Van den Buuse, M. and De Jong, W., Pharmacology of central dopaminergic modulation in hypertension, In H. Saito, M. Minami and S.H. Parvez (Eds.), Progress in Hypertension, Vol. 2; Antihypertensive Drugs Today, VSP, Utrecht, 1991, pp. 157 188. [15] Van den Buuse, M., Versteeg, D.G.H. and De Jong, W., Brain dopamine depletion by lesions of the substantia nigra attenuates the development of hypertension in the spontaneously hypertensive rat, Brain Res., 368 (1986) 69-78.
Neuroscience Letters 176 (1994) 142..146
Pressor responses to electrical and chemical stimulation of the rat brain A 10 dopaminergic system Jennifer L. Cornish*, Maarten van den Buuse Neuropharmacology Laboratory, Baker Medical Research Institute, PO Box 348, Prahran, Vic. 3181, Australia Received 2 February 1994; Revised version received 3 June 1994; Accepted 6 June 1994
Abstract Central dopaminergic systems have been implicated in the regulation of blood pressure. We examined the effect on blood pressure of electrical or chemical stimulation of the rat brain ventral tegmental area (VTA) which is the region of origin of the A10 dopaminergic system. Electrical stimulation in urethane-anaesthetised rats (10-120 Hz, 80/IA) produced frequency-dependent increases in blood pressure (max 30-35 mmHg). These pressor responses could be significantlyattenuated by pretreatment with the dopamine D2 receptor antagonist haloperidol, but not the D~ receptor antagonist SCH 23390. Chemical stimulation of the VTA, by microinjection of 10 nmol of the substance P analogue DiMe-C7, produced a sustained increase in blood pressure (max 10-15 mmHg), which could be completely prevented by pretreatment with haloperidol. These results suggest that stimulation of dopaminergic neurons in the VTA induces pressor responses and that projections from midbrain dopaminergic neurons, acting on dopamine D2 receptors, play a role in the regulation of blood pressure. Key words: Dopamine; Blood pressure; Ventral tegmental area; Dopamine D2 receptor; Haloperidok SCH 23390; DiMe-C7; Sprague-Dawley rat; Central nervous system
There is increasing evidence for a role of central dopaminergic systems in blood pressure regulation and the development of hypertension. Systemic administration of dopamine D2 receptor agonists caused centrallymediated increases in blood pressure [13]. Several authors have observed differential changes of indices of central dopaminergic activity in the spontaneously hypertensive rat (SHR) (for review, see [14]). Moreover, the development of hypertension in these rats can be inhibited by depletion of brain dopamine, either with the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) or with electrolytic lesions in the substantia nigra [15]. One major central dopaminergic system that has been implicated in cardiovascular regulation is the mesolimbic (A10) system which originates in the midbrain ventral tegmental area (VTA). Electrical stimulation of the VTA in both rabbits and cats has been shown to produce an increase in blood pressure [9,12]. A number of authors
* Corresponding author. Fax: (61) (3) 521 1362. 0304-3940194/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00450-0
have found that dopamine D2 receptor density in brain regions innervated by this system were different between hypertensive and normotensive rats [5,6]. The VTA provides main ascending dopaminergic efferents to the nucleus accumbens, olfactory tubercle, amygdala and habenular nucleus and descending efferents to the locus coeruleus [7,11]. The aim of the present study was to investigate cardiovascular responses to either electrical or chemical stimulation of the VTA in the rat brain. We used the substance P analogue DiMe-C7 for chemical stimulation of the VTA as this brain region is densely innervated by substance P containing neurons, such as those descending from the habenular nucleus and striatum [1]. DiMeC7 is a metabolically stable analogue of substance P which produces prolonged stimulation of A10 dopaminergic cell bodies [3]. Effects of micro-injection of DiMeC7 into the VTA include an increase in locomotor and exploratory behaviour in rats [2] and a stimulation of dopamine turnover in the nucleus accumbens [4]. All experiments followed the guidelines of the Austra-
143
J.L. Cornish, M. van den Buuse/Neuroscience Letters 176 (1994) 142 146
lian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Alfred Hospital/Baker Medical Research Institute Animal Experimentation Committee. We used male SpragueDawley rats (250 350 g). In the case of electrical stimulation of the VTA, the rats were anaesthetised with urethane (1.4 g/kg, i.p.) and implanted with femoral artery catheters for the measurement of blood pressure and heart rate. Jugular vein catheters were implanted for the intravenous administration of drugs. The rats were then placed in a stereotaxic frame (David Kopf) with the nosebar set at zero. A unipolar electrode was inserted unilaterally at an angle of 12° , through a burr hole in the left hand side of the skull, at c°-°rdinates ° f 5 3 mm p°steri°r' 2"5 mm lateral and 8"5 mm ventral of the bregma. The electrical circuit was completed by attaching an earth lead to the trapezius muscle of the animal. Frequency-response curves were then obtained by applying 5 s stimulus trains (5 ms block pulse duration) at a current of 80 #A, using a Grass $88 stimulator via a Grass voltage~zurrent converter (model PSIU6). Ascending frequency-levels (10-120 Hz) were tested at 1 min intervals. Five min after the completion of the first curve, haloperidol (0.5 mg/kg; Serenace, Searle), the dopamine Dl receptor antagonist SCH 23390 (0.1 mg/kg; Research Biochemicals) or vehicle (0.9% saline, 1 ml/kg) was administered i.v., and 15 rain later a second frequency-response curve was obtained. Each animal underwent only one experimental procedure. The animals were killed with an i.v. overdose of pentobarbitone sodium (60 mg/ml, Nembutal, Bomac Laboratories, Australia) and their brains were dissected out and fixed in 4% formalin. For experiments involving chemical stimulation of the VTA, the rats underwent two surgical procedures under anaesthesia induced with a mixture of methohexitone sodium (40 mg/kg, Brietal Sodium, Eli Lilly), pentobarbitone sodium (30 mg/kg) and atropine sulphate (0.5 mg/kg, Sigma). The first procedure involved the implantation of a metal guide cannula (C313-G, Plastic Products Company, Roanoke, VA, USA) into the region of the VTA using the co-ordinates previously mentioned, These cannulas were secured with dental cement and three small screws placed in the skull. The cannulas were kept patent with a chronically placed dummy cannula (C313-DC) cut to the appropriate length. After 5 7 days recovery, the animals underwent surgery for implantation of chronic catheters into the abdominal aorta and jugular vein. These animals were then allowed another 5 7 days recovery before being studied while conscious, We pretreated the rats 15 min before central injections with either haloperidol (0.5 mg/kg, i.v.) or saline (1 ml/ kg, i.v.), and then microinjected 10 nmol of [pGlu s, MePhe8, Sarg]-substance P5 L~ (DiMe-C7, Auspep, Vic., Australia) unilaterally into the VTA. The central injection was in 1.0 #1 of saline which was infused over 1 min
f-------"---'-~. ~
~
/ //,
1
\-
' ~!~'~ F h
VTA ~,~-"x~f~., ( ~___..__
I/
.
~
QVTA~I~
/
~
/
~I'~_~,~..,Xxr~ r ~
~
~
~
Fig. 1. Typical examples of midbrainsectionsof rats withan electrode (top panel) or guide cannula (bottom panel) placed into the region of the VTA. Right panels show histological sections and left panels represent corresponding diagrams modified from lhe atlas of Paxinos and Watson [8]. Thick arrows and shaded areas show the site of the electrode tip or guide cannula. Animals which showed placement outside of this region were discardedfrom lhe analyses.
with a Hamilton 10 #1 micro-syringe and a Harvard Apparatus syringe infusion pump (Model 22, South Natick, MA, USA). Each of the seven rats used in this part of the study was tested with both treatments in random order on two separate occasions, wilh at least 48 h between the experiments. At the completion of both experiments, the animals were killed and their brains taken as for the acute experiments. In all experiments, pulsatile blood pressure and mean arterial pressure (MAP) were recorded with a Statham P23XL pressure transducer and displayed on a Neomedix Systems Neotrace recorder. Heart rate was derived off the pressure pulse by tachographs. For histological verification of the stimulation and injection sites, 30 #m sections of the midbrain were cut using a Leitz freezing microtome and were stained with Cresyl violet. Electrode and guide cannula placement in the VTA were verified by the comparison of these sections with the brain atlas of Paxinos and Watson [8]. Only results from animals with electrode or cannula placement inside the VTA (Fig. 1) were analysed. Results are expressed as mean values _+ standard error
144
./.L. CmwLvh, '~1 van &'t~ Buu.~c/.%,uroscicm'c Lcm'rs l - 6
luO4, 142 146
blood pressure and heart rate responses to local DiMeC7 infusion were calculated w i t h a graphics computer
30 2o
10 0 .
. ~
30"~ 3E E. 2oa. 5; m. <
.
.
.
.
~ ( HALOPERIDOL
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I 020
40 6o 80 100 " 120 Stimulation frequency (Hz)
program (Graphpad Inplot, San Diego, CA. USA). Electrical stimulation of the VTA in anaesthetised rats (Fig. I) consistently produced a frequency-dependent increase in blood pressure which was phase-locked with the onset and offset of the stimulation. Effects on heart rate were variable, with some animals showing moderate increases, while other animals exhibited bradycardic responses or no heart rate changes (data not shown). In all curves obtained before vehicle or antagonist treatment. maximum increases in blood pressure occurred at frequencies of 100 120 Hz (Fig. 2) with similar MAP plateau values and EFs0 values (Table 1). Treatment with haloperidol caused a small, but statistically significant reduction in basal blood pressure (P = 0.005; Table 1). After haloperidol pretreatment, the frequency-response curve was significantly depressed (Fig. 2), with a 35% inhibition of the maximal plateau (P < 0.001' Table 1). Treatment with haloperidol also caused the frequency-response curve to shift to the right, resulting in a significant increase in the EFso. values (P = 0.014: Table 1). The plateau and EFs,, values of the frequency-response curves after treatment with either saline or SCH 23390 were not significantly different from those obtained before these respective treatments (Table l ). Microinjection of DiMe-C7 into the VTA of conscious rats (Fig. 1) produced an increase in blood pressure with little effect on heart rate (Fig. 3). The pressor responses were of 10-15 mmHg in magnitude, were maximal 10-15 min after the infusion of DiMe-C7 (Fig. 3) and remained elevated above baseline for much of the remaining pcriod. Pretreatment with haloperidol did not alter basal MAP; however, it completely blocked the pressor response elicited by chemical stimulation of the VTA
Fig. 2. Changes in blood pressure produced by electrical stimulation of the VTA with increasing frequencies in urethane-anaesthetised rats•
Table 1
The top, middle and b o t t o m panels show the effect of vehicle (n = 6), haloperidol (n = 9) and SCH 23390 (n = 6) pretreatment, respectively, after the initial control curve• Treatment with haloperidol produced a marked reduction in pressor responses. F o r baseline values, see Table l. *P < 0.05 for difference between the plateau values of responses
Frequency response curves to electrical stimulation of the VTA in anaesthetised rats .......................... Treatment Basal M A P AMAP plateau EFs0 (Hz) (mmHg) (mmHg)
before and alter haloperidol treatment.
Control
85.8 _+ 2.0
34.5 _+ 2.7
3916 + 4.0
Saline ( I ml/kg)
87.0 _+ 2.1
29.6 _+ 3.2
41.6 +_ 3.2
Control Haloperidol ~0.5 mg/kg)
83.1 + 2.8 74.2 _+ 2.3*
33.0 _+ 1.7 21.3 + 1•2"
46.2 +_ 3.3 55.3 _+ 4.1'
Control SCH 23390 (0.1 mg/kg)
85.3 + 5.1 84.2 + 3.4
32•0 + 5.0 33.9 _+ 4.7
43.2 _+ 2.9 46.3 _+ 4.0
of the mean (S.E.M.) and were statistically analysed with
analysis of variance (ANOVA) for repeated
measures.
Blood pressure responses to electrical stimulation of the VTA were frequency-dependent and reached a plateau at higher frequencies. Thus, frequency-response curves obtained from the experiments with electrical stimulation were analysed by sigmoidal curve fitting (SIGMOID, Baker Medical Research Institute), which yielded plateau values and effective frequency values that produced 50% of the maximal pressor response (EF50) for each curve. The area under the curve (AUC) of the
d M A P plateau is the maximal increase in M A P seen with electrical stimulation. EFs0 value is the frequency required to induce 50% o f the
maximalpressor response. *P < 0.05 for difference between
treated and control values. The number of animals was 6 (saline group), 9 (haloperidol) and 6 (SCH 23390),
J.L. Cornish, M. van den Buuse/Neuroscience Letters 176 (1994) 142 146
--O--Control + DiMe-C7 ~Haloperidol
+ DiMe-C7
15-
T E E ~" <
1050-5-10- ,..
.
.
.
.
.
.
5o"E" ~ m v
0.
-50-
-loo
. 0
. . . . 16 20 30 40 50 Time after DiMe-C7infusion (rain)
60
Fig. 3. Changes in mean arterial pressure ( M A R top panel) and heart rate (bottom panel) after local infusion of DiMe-C7 into the VTA of conscious rats. Baseline blood pressures were 100.3 + 3.0 and 98.8 _+ 2.1 m m Hg, and baseline heart rates were 381.0 + 12.8 and 381.9 +_ 11.5 beats/min for vehicle (n = 7) and haloperidol (n = 7) pretreated animals, respectively. Pretreatment with haloperidol caused a complete inhibition of the pressor response produced with DiMe-C7 infusion and also revealed a marked bradycardia,
(P = 0.001). After this pretreatment, a marked bradycardia was observed with a maximum of approximately 65 beats/rain at 20 min after injection (Fig. 3). Heart rate had returned to near resting levels by the end of the 60 min monitoring period. There was a statistically significant overall difference between heart rate values obtained with or without haloperidol pretreatment (P < 0.001). The AUC values for the blood pressure responses to DiMe-C7 infusion were 602+125 units for the saline-treated rats, which was significantly different from - 8 6 + 134 units calculated for the haloperidol treated animals (P < 0.001). Similarly, the A U C value for the heart rate responses to DiMe-C7 infusion of the salinetreated animals (1522+ 517 units) was significantly greater than that calculated for the haloperidol treated rats (-2743 + 595 units, P = 0.001). In the present study, we observed that both electrical and chemical stimulation of the VTA caused an increase
145
in blood pressure. In both cases the involvement of dopamine D 2 receptors was suggested by the finding that pretreatment with haloperidol produced a 35% inhibition of electrical stimulation and a complete blockade of the pressor response produced after DiMe-C7 infusion into the VTA. The pressor response to electrical stimulation of the VTA began immediately upon stimulation and lasted only as long as the pulse duration. This suggests that the effects elicited by this stimulation were most likely sympathetic in origin. Indeed, in preliminary experiments we observed that pretreating the animals with the ganglion blocking agent, pentolinium, cornpletely inhibited any pressor effect of electrical stimulation (data not shown). Approximately 35% of the response elicited with electrical stimulation appears to be produced by stimulation of dopamine D2 receptors, which could be mediated by dopaminergic projections from the VTA to brain regions such as the nucleus accumbens or the amygdala, or by descending projections to regions such as the locus coeruleus. The latter possibility may be considered as lesions of the posterior hypothalamus had little effect on the pressor response elicited by electrical stimulation of the cat VTA [9]. Since this lesion was anterior to the VTA this suggests the involvement of lower brainstem regions, such as the locus coeruleus, in the pressor response elicited. The antagonist results from this study imply that approximately 65% of the pressor response produced by electrical stimulation of the VTA is not mediated by either dopamine D Lor D2 receptors. This response m a y be caused by stimulation of non-dopaminergic efferents of the VTA as approximately one third of the n e u r o n s in this brain area are non-dopaminergic cells which a r e intermixed with dopaminergic cell bodies [11]. There is also the possibility that the electrical stimulation technique has affected fibres of passage in the ventral midbrain, such as ascending noradrenergic neurones, to increase blood pressure. The metabolically stable substance P analogue DiMe-C7 is thought to directly stimulate dopaminergic neuronal cell bodies by actiw~ting substance P receptors in this area [2,3,4]. In addition, we used conscious rats for this part of the study, avoiding any influence of anaesthesia on the results. Chemical stimulation of the rabbit VTA with glutamate produced pressor and bradycardic responses [12]. In the present study, chemical stimulation of the rat VTA with DiMe-C7 also induced a long-lasting pressor response but failed to produce a decrease in heart rate. The complete inhibition of the pressor response by haloperidol suggests that dopamine De receptors are the major mediators of this effect. In recent preliminary experiments, we observed that the pressor effect of intra-VTA injections of DiMe-C7 could also be blocked by pretreatment with the selective dopamine De receptor antagonist raclopride (unpublished data). The bradycardia revealed
146
J.L. ('ornish, M. van den Buuse / Neur oscicnce Letters 176 ~ 1994 j 142 146
after haloperidol treatment could be either dopamine D I receptor-mediated or non-dopaminergic in origin. The latter effect may be mediated through non-dopaminergic neurones that originate in the VTA and project to structures involved in the regulation of heart rate, such as the locus coeruleus [ 10,11 ]. H e a r t r a t e in rats injected with DiMe-C7 is obviously under the influence of a number of mechanisms: on the one hand, the increase in blood pressure observed in
these animals would normally produce a baroreflexmediated bradycardia, and central non-dopaminergic mechanisms (see above) also induce a decrease in heart
rate, which becomes apparent when dopamine receptors are blocked with haloperidol. On the other hand, there could be a cardio-acceleration concomittant with the increase in blood pressure, possibly mediated by an increased sympathetic outflow, which is largely compensated for by the other two mechanisms, In conclusion, the present study shows that electrical or chemical stimulation of the A 10 dopaminergic cells in the VTA elicit a pressor response. The effects are most
likely mediated by dopamine D2 receptors. These results suggest that the A10 dopaminergic system is involved in cardiovascular control and that the projections from midbrain dopaminergic neurons may be associated with cardiovascular homeostasis, [1] Brownstein, M.J., Mroz, E., Kizer, J.S., Palkovits, M. and Leeman, S.E., Regional distribution of substance P in the brain of the rat, Brain Res., 116 (1976) 299-305. [2] Eison, A.S., Eison, M.S. and Iversen, S.D., The behavioural effects of a novel substance P analogue following infusion into the ventral tegmental area or substantia nigra of rat brain, Brain Res., 238 (1982) 137-152. [3] Eison, A.S. and Iversen, S.D., Substance P analogue, DiMe-CV: evidence for stability in rat brain and prolonged central actions, Science, 215 (1982) 188-190.
[4] Elliott, P.J., Alpert, J.E., Bannon, M.J. and lverscn. S.D., Selectiw activation of mesolimbic and mesocortical dopamine metabolism in rat brain by infusion of a stable substance P analogue into the ventral tegmental area, Brain Res., 363 (1986) t45 147. [5] Kirouac, G.J. and Ganguly, P.K., Up-regulation of dopamme receptors in the brain of the spontaneously hypertensive rat: an autoradiographic analysis, Neuroscience, 52 (1993) 135-141. [6] Le Fur, G., Guilloux, F., Kabouche, M., Mihani. N., Ferris, O. and Uzan, U., Central dopaminergic neurons during the development of genetic and DOCA-salt hypertension in the rat, Brain Res., 227 (1981)153-163. [7] Oades, R.D. and Halliday, G.M., Ventral tegrnental (AI0) system: neurobiology. 1. Anatomy and connectivity, Brain Res. Rev., 12 (1987) 117 165. [81 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1982.
[9] Spring. A. and Winkelmiiller, W., Ventral midbrain stimulation.
blood pressure responses and their relation to the dopaminergic nigro-striatal pathways, Pflfigers Arch., 358 (1975) 339--348. [10] Sved, A.F. and Felsten, G., Stimulation of the locus coeruleus decreases arterial pressure, Brain Res., 414 (1987) 119 132. [111 Swanson, L.W., The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat, Brain Res. Bull., 9 (1982)
321 353. [12] Tan, E., Goodchild, A.K. and Dampney, R.A.L., Intense vasocon-
striction and bradycardia evokedby stimulation of neurons within the midbrain and the ventral tegmentum of the rabbit, Clin. Exp. Pharmacol. Physiol., 10 (1983) 305--309. [13l Van den Buuse, M., Central effects ofquinpirole on blood pressure of spontaneously hypertensive rats, J. Pharmacol. Exp. Ther., 262 (1992) 303-311. [14] Van den Buuse, M. and De Jong, W., Pharmacology of central dopaminergic modulation in hypertension, In H. Saito, M. Minami and S.H. Parvez (Eds.), Progress in Hypertension, Vol. 2; Antihypertensive Drugs Today, VSP, Utrecht, 1991, pp. 157 188. [15] Van den Buuse, M., Versteeg, D.G.H. and De Jong, W., Brain dopamine depletion by lesions of the substantia nigra attenuates the development of hypertension in the spontaneously hypertensive rat, Brain Res., 368 (1986) 69-78.