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Cardiovascular depressor responses to stimulation of substantia nigra and ventral tegmental area GILBERT J. KIROUAC AND JOHN CIRIELLO Department of Physiology, Health Sciences Centre, University of Western Ontario, London, Ontario, Canada N6A 5C1 dopamine; arterial pressure; central cardiovascular pathways with relatively large concentrations and volumes of Glu or kainic acid was reported to elicit responses in the rat similar to those elicited by electrical stimulation (24). As these cardiovascular responses to stimulation of the SN and VTA were blocked by the peripheral and central administration of a DA antagonist, it was suggested that these responses were mediated by mesotelencephalic DA projections (11, 12, 24). However, the location of the neurons within the mesencephalon that may be responsible for these cardiovascular responses has not been clearly defined, as the relatively large microinjections of neuroactive substances that produced cardiovascular responses (11, 12, 24, 29) would have also stimulated neurons in regions outside the SN or VTA. Furthermore, the cardiovascular responses elicited by electrical stimulation of these regions (6, 7, 24) may have been due to activation of fibers that course through these regions (15–17) but that originated in other central sites. Therefore, the present study was done to systematically explore the ventral mesencephalon for cardiovascular-responsive sites in the a-chloralose-anesthetized rat using microinjections of small volumes (10 nl) of the excitatory amino acid Glu, which is known to selectively excite neuronal cell bodies and not fibers of passage (18). Experiments were also done to determine whether these responses were mediated by stimulation of DA neurons in the ventral mesencephalon. Finally, experiments were done to identify the components of the peripheral autonomic nervous system that mediated the cardiovascular responses. METHODS (SN) and the ventral tegmental area (VTA) of the ventral mesencephalon contain the dopamine (DA) neurons that form the mesotelencephalic DA pathway that innervates the striatum, cerebral cortex, and limbic system (15–17). This mesotelencephalic DA pathway is generally thought to function in the regulation of motor and behavioral responses mediated by neuronal mechanisms in the forebrain (1, 22). Recent experimental evidence suggests that the mesotelencephalic DA system may play an important role in the regulation of the cardiovascular system. Stimulation of VTA neurons electrically or with the microinjection of the neurokinin receptor agonist DiMe-C7 or the excitatory amino acid L-glutamate (Glu) has been shown to elicit variable changes in arterial pressure (AP) and heart rate (HR) in the rat and rabbit (11, 12, 23, 29). Similarly, electrical stimulation of the SN in the cat (6, 7) or the rat (24) was also shown to elicit increases in AP and HR. In addition, chemical stimulation of the SN THE SUBSTANTIA NIGRA Experiments were done in 17 male Wistar rats (300–500 g) anesthetized with a-chloralose (60 mg/kg iv and additional doses of 20–30 mg/kg every 1–2 h) after equithesin induction (0.3 ml/100 g ip). All experimental procedures were done in accordance with the guidelines on the use and care of laboratory animals as set out by the Canadian Council on Animal Care and approved by the Animal Care Committee at the University of Western Ontario. Polyethylene catheters (PE-50) were inserted into the femoral artery and vein for the recording of AP and the administration of drugs, respectively. AP was recorded via a Statham pressure transducer (model P23 Db), and a Grass tachograph (model 7P4FG) that was triggered by the AP pulse was used to monitor HR. Both AP and HR were recorded continuously on a Grass polygraph (model 79D). The trachea was cannulated and the animals were artificially ventilated using a small rodent ventilator (model 683, Harvard Apparatus) with a mixture of 5% room air-95% O2. The animals were paralyzed with pancuronium bromide (Pavulon; initial dose 1 mg/kg iv followed by supplementary doses of 0.5 mg/kg every 30 min) to eliminate the possibility that the cardiovascular responses elicited during stimulation 0363-6135/97 $5.00 Copyright r 1997 the American Physiological Society H2549 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017 Kirouac, Gilbert J., and John Ciriello. Cardiovascular depressor responses to stimulation of substantia nigra and ventral tegmental area. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H2549–H2557, 1997.—Experiments were done in a-chloralose-anesthetized, paralyzed, and artificially ventilated rats to investigate the effect of L-glutamate (Glu) stimulation of the substantia nigra (SN) and ventral tegmental area (VTA) on arterial pressure (AP) and heart rate (HR). Glu stimulation of the SN pars compacta (SNC) elicited decreases in both mean AP (MAP; 218.9 6 1.3 mmHg; n 5 52) and HR (226.1 6 1.6 beats/min; n 5 46) at 81% of the sites stimulated. On the other hand, stimulation of the SN pars lateralis or pars reticulata did not elicit cardiovascular responses. Stimulation of the adjacent VTA region elicited similar decreases in MAP (218.0 6 2.6 mmHg; n 5 20) and HR (225.4 6 3.8 beats/min; n 5 17) at ,74% of the sites stimulated. Intravenous administration of the dopamine D2-receptor antagonist raclopride significantly attenuated both the MAP (70%) and the HR (54%) responses elicited by stimulation of the transitional region where the SNC merges with the lateral VTA (SNC-VTA region). Intravenous administration of the muscarinic receptor blocker atropine methyl bromide had no effect on the magnitude of the MAP and HR responses to stimulation of the SNC-VTA region, whereas administration of the nicotinic receptor blocker hexamethonium bromide significantly attenuated both the depressor and the bradycardic responses. These data suggest that dopaminergic neurons in the SNC-VTA region activate a central pathway that exerts cardiovascular depressor effects that are mediated by the inhibition of sympathetic vasoconstrictor fibers to the vasculature and cardioacceleratory fibers to the heart. H2550 SN AND VTA DEPRESSOR RESPONSES the nicotinic receptor blocker hexamethonium bromide (20 mg/kg iv) was also administered and the same site in the SN-VTA region was restimulated with Glu. The a-receptor agonist phenylephrine (5–10 mg · kg21 · min21 iv) was administered using a Harvard infusion pump (model 22) to maintain a stable and normal level of AP after the precipitous decrease in AP caused by the administration of hexamethonium. Histological localization of stimulation sites. At the completion of most experiments, a 20-nl microinjection of Pontamine Sky blue in phosphate-buffered saline (pH 7.2) was made through the second barrel of the two-barreled micropipettes to mark the center of the injection site (Fig. 1). The injection site and the resulting micropipette tracts were later identified histologically. The injection of the phosphate-buffered saline and/or Pontamine Sky blue dye did not elicit cardiovascular responses. The animals were perfused transcardially with 100 ml of 0.9% saline followed by 100 ml of 10% Formalin. The brains were removed and stored in 10% Formalin for at least 24 h. Frozen serial transverse sections of the mesencephalon were cut at 40 µm on a Bright’s cryostat and stained with Neutral red. The location of the marked sites of stimulation and/or micropipette tracts was verified, and the remaining injection sites in any one animal were determined by extrapolation from the marked site along the micropipette tract. Injection sites were mapped on diagrams of transverse sections of the rat brain modified from a stereotaxic atlas (26). In addition, injection sites were grouped into SN pars compacta (SNC), SN pars reticulata (SNR), SN pars lateralis (SNL), VTA, retrorubral nucleus, red nucleus, and reticular formation above the medial lemniscus according to the stereotaxic atlas of the rat brain by Paxinos and Watson (26). Data analysis. A cardiovascular-responsive site was defined as a site at which Glu injections elicited a change in either mean AP (MAP) or HR of .5 mmHg or .10 beats/min, respectively. Means 6 SE were calculated for the magnitude of the cardiovascular changes and for the onset latency, latency to the peak response, and duration of the MAP and HR responses. These values were compared using analysis of variance (ANOVA). In addition, the effects of the nicotinic, muscarinic, and DA blocking agents on the MAP and HR responses were compared using an ANOVA for repeated measures. A P value of ,0.05 was taken to indicate statistical significance. RESULTS In the initial series of experiments, 278 Glu injections were made at sites in the region of the ventral mesencephalon (Fig. 2). The baseline MAP was 120.6 6 1.3 mmHg and the baseline HR was 350.5 6 3.2 beats/min in the a-chloralose-anesthetized rat. Of the 100 injection sites histologically verified in the SN, 52 (52%) elicited cardiovascular responses (Fig. 2). These responsive sites were localized to the SNC or regions of the SNL and SNR immediately adjacent to the SNC (Fig. 2). Most (91%) Glu microinjections in the SNC elicited decreases in MAP that ranged between 25 and 245 mmHg. The majority (n 5 46; 81%) of these depressor responses were accompanied by decreases in HR that ranged between 210 and 250 beats/min (Fig. 2; Table 1). Only one site was found in the SN that elicited bradycardia without a concomitant decrease in MAP. A representative experiment showing the effect of stimulation of the SNC region is shown in Fig. 3. Note Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017 of brain tissue were secondary to muscular activity or related to respiratory changes. All surgical procedures were done before the administration of the paralyzing agent. During the course of the nonsurgical portions of the experiment, the animals were allowed to recover periodically from the paralyzing agent to determine the depth of anesthesia by examining withdrawal reflexes. Body temperature was monitored and maintained at 37 6 1.0°C by a heating pad controlled by a temperature controller (model 73; Yellow Springs Instruments). The animal was placed in a Kopf stereotaxic frame, and a hole was drilled through the parietal bone to expose the brain tissue overlying the ventral mesencephalon. Chemical stimulation of the ventral mesencephalon. Glu stimulation of the mesencephalon was done by using doublebarreled glass micropipettes pulled from 5-µl Socorex capillary tubing (Mississauga, Canada) with tip diameters that ranged between 35 and 50 µm. Solutions of Glu (0.25 M; Sigma Chemical, St. Louis, MO) in phosphate-buffered saline (pH 7.2) were microinjected (10 nl) by the application of pressurized nitrogen pulses controlled by a pneumatic pump (Medical Systems, Great Neck, NY). The injected volumes were measured by direct observation of the fluid meniscus in the micropipettes by using a microscope fitted with an ocular micrometer. As the microinjection of excitatory amino acids using similar volumes has been reported to decrease the excitability of neurons in the vicinity (up to 500 µm) of an injection site (25), a minimum period of 5 min was allowed between each microinjection of Glu. Control injections of the vehicle were also made at similar sites to determine whether the observed cardiovascular responses during Glu injections were due to the vehicle or mechanical stimulation of the neuronal tissue. In addition, in two animals the cardiovascular-responsive region of the SN and VTA was explored for cardiovascular responses elicited by the microinjection of the vehicle only. This was done to eliminate the possibility that the observed cardiovascular effects during Glu injection were due to a distortion of neuronal tissue as a result of the multiple injections in the region. Micropipette tips were lowered into the ventral mesencephalon according to a stereotaxic atlas of the rat brain (26). The SN and VTA and the regions that are located dorsally to the SN-VTA region were explored systematically on a grid pattern from 2.5 to 4.0 mm rostral to the intra-aural line, from midline to 3.0 mm lateral to the midline, and from 5.5 to 8.5 mm ventral to the surface of the brain. The micropipette was gradually lowered through the region of the ventral mesencephalon with each stimulation site 300–500 µm apart. Approximately 15–18 injections were made into the SN-VTA region in any one animal. Effect of pretreatment with DA antagonists. Cardiovascularresponsive sites in the SN-VTA region were retested after the intravenous administration (0.1 ml/100 g) of the D1 DAreceptor antagonist Sch-23390 hydrochloride (0.1–0.4 mg/kg; Research Biochemicals, Natick, MA), the D2 DA-receptor antagonist raclopride L-tartrate (2 mg/kg; Research Biochemicals), or saline vehicle. Similar doses of these DA-receptor antagonists have previously been shown to produce changes in motor activity in the rat (20, 21). The same stimulation site in the SN-VTA region previously shown to produce a cardiovascular response was retested at 30 min and 3 h after infusion of the antagonists or vehicle. The experiments for each antagonist or the vehicle were done in separate groups of animals. Effect of autonomic blockade. In five additional rats, cardiovascular-responsive sites in the SN-VTA region were retested 5 min after the administration of the muscarinic receptor blocker atropine methyl bromide (1 mg/kg iv). After 10 min, SN AND VTA DEPRESSOR RESPONSES H2551 that, as the micropipette was lowered through the region of the SNC, the magnitude of the cardiovascular responses became progressively larger. Stimulation of sites deep within the SNR did not elicit cardiovascular responses (Figs. 2 and 3). Stimulation of 39 of 109 (36%) sites in the reticular formation just dorsal to the medial lemniscus elicited significantly smaller depressor responses (Table 1). In all experimental cases, the largest decreases in MAP and HR were observed when the injection site was either in or immediately adjacent to the SNC. The onset latency of the MAP response to stimulation of the SNC (6.9 6 0.4 s) was significantly shorter than the onset latency of the MAP response elicited by stimulation of the reticular formation dorsal to the medial lemniscus (9.1 6 0.7 s). No statistical differences were found between the onset latency of the HR responses to stimulation of the two regions. Of 27 injection sites histologically verified within the VTA region (Fig. 2), 20 sites (74%) elicited decreases in MAP (218.0 6 2.6 mmHg) and 17 sites (63%) elicited decreases in HR (225.4 6 3.8 beats/min). A larger number and a greater magnitude of the cardiovascular responses were elicited by stimulation of the region of the VTA that is located lateral to the fasciculus retroflexus and medial to the medial lemniscus (Figs. 1 and 2). In addition, stimulation sites in the adjacent red nucleus and the prerubral field elicited a few smaller depressor responses (Table 1; Figs. 1 and 2). Figure 1 shows a representative experiment involving a microin- jection tract through the prerubral field and the lateral region of the VTA. Note that decreases in MAP and HR become progressively larger as the stimulation sites involve the prerubral fields closest to the VTA and the large magnitude of the depressor responses elicited from the lateral portion of the VTA (Fig. 1). Stimulation of most of the sites tested within the retrorubral field, adjacent to the VTA, also elicited decreases in both MAP and HR (Fig. 2, Table 1). No statistical differences were observed between the onset latency of the MAP responses after stimulation of the SNC (6.9 6 0.4 s) and VTA (6.9 6 0.9 s). In addition, the onset latency of the HR responses to stimulation of the SNC (16.2 6 0.7 s) and the VTA (15.4 6 1.4 s) occurred significantly later than the onset latency of the MAP responses to stimulation of these areas. Figure 4 shows the effect of decreasing the volume of the Glu microinjection into the SNC. Note that stimulation of this region with different volumes (10, 4, and 2 nl; n 5 5) of 0.25 M Glu elicited depressor responses of approximately equal magnitude (Fig. 4; Table 2). Control injections of the vehicle (10 nl) into the same sites did not evoke any cardiovascular response. Similarly, multiple injections of the vehicle into this cardiovascular-responsive region did not elicit cardiovascular responses. In contrast, repeated injections of Glu at any one responsive site elicited cardiovascular responses that were both qualitatively and quantitatively similar to those elicited by the initial Glu injection. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017 Fig. 1. A and B: photomicrographs showing a deposit of Pontamine Sky blue dye that marks the injection site in the ventral tegmental area (VTA) (C). B is an increased magnification of the inset in A, showing neurons in the VTA that have incorporated the dye and may represent the neurons activated by the injection at that site. SNC, substantia nigra pars compacta; PR, prerubral field; ml, medial lemniscus. Calibration marks in A and B, 100 µm. C: representative micropipette tract through the PR and VTA ,3.8 mm rostral to the intra-aural line, showing the arterial pressure (AP) and heart rate (HR) responses elicited during microinjection of L-glutamate (Glu) at different dorsoventral sites (filled circles). Note that the magnitude of the cardiovascular depressor responses elicited from stimulation of the PR increases as the micropipette tract approaches the VTA, at which point the largest cardiovascular responses are elicited. Arrows, time of Glu injections. SNR, substantia nigra pars reticulata. Transverse section of mesencephalon is modified from stereotaxic atlas of rat brain (Ref. 26). H2552 SN AND VTA DEPRESSOR RESPONSES DISCUSSION Fig. 2. Transverse sections of rat brain taken through region of the VTA extending from 2.6 to 4.2 mm rostral to the intra-aural line (A) and showing location of histologically identified sites tested for AP (B) and HR responses (C). cp, cerebral peduncle; CG, central gray matter; DpMe, deep mesencephalic nucleus; dtgx, dorsal tegmental decussation; fr, fasciculus retroflexus; IP, interpeduncular nucleus; MB, mammillary body; MT, medial terminal accessory nucleus of the optic tract; R, red nucleus; RRF, retrorubral field; SNL, substantia nigra pars lateralis; tfp, transverse fibers of the pons; ZI, zona incerta. Open inverted triangles, sites that elicited decreases in mean AP (MAP) or HR of ,15 mmHg or beats/min, respectively; small filled circles, sites that elicited decreases in MAP or HR of 15–30 mmHg or beats/min, respectively; large filled circles, sites that elicited decreases in MAP or HR of .30 mmHg or beats/min, respectively; open circles, sites that did not elicit cardiovascular responses. Transverse sections are modified from stereotaxic atlas of rat brain (26). To determine whether activation of DA-containing neurons may be involved in the mediation of the cardiovascular responses to Glu stimulation of the transitional region where the SNC merges with the lateral VTA (SNC-VTA region), we administered the D2 DA-receptor antagonist raclopride and the D1 DAreceptor antagonist Sch-23390 intravenously after a cardiovascular-responsive site was identified in the SNC-VTA region. Restimulation of the same site 30 min after the intravenous administration of raclopride resulted in a significant attenuation of the decreases in both MAP and HR (Figs. 5 and 6). Restimulation of the same site within the SNC-VTA region 3 h after the administration of raclopride elicited depressor responses similar in magnitude to those elicited by microinjection before the raclopride treatment. Intravenous administration of the vehicle (Fig. 6) or Sch-23390 (0.1 and 0.4 mg/kg; n 5 3) did not alter the magnitude of This study has demonstrated that chemical stimulation of the ventral mesencephalon with Glu elicits decreases in MAP that were often accompanied by decreases in HR. Stimulation of the SNC and VTA consistently elicited the most marked cardiovascular responses. Stimulation of regions immediately adjacent to the SNC and VTA evoked smaller responses. The cardiovascular responses to stimulation of the SNC-VTA region were not secondary to changes in muscular tone or respiration inasmuch as they were elicited in paralyzed and artificially ventilated animals. However, these cardiovascular responses were due to inhibition of sympathetic activity to the vasculature and the heart. This conclusion is based on the finding that the responses were not affected by the systemic administration of the muscarinic receptor antagonist atropine methyl bromide but were abolished after administration of the nicotinic receptor antagonist hexamethonium bromide. The observation that the onset latency of the HR response was greater than that Table 1. MAP and HR responses to stimulation of the different regions of the ventral mesencephalon with 10 nl of 0.25 M Glu MAP, mmHg HR, beats/min SNC SNL SNR 218.9 6 1.3a 226.1 6 1.6a (46/57) 0b (0/16) 0b (0/27) RF VTA R RRF 211.7 6 1.3c (39/109) 218.0 6 2.6a (20/27) 213.0 6 2.6a,c (7/13) 217.8 6 2.7a,c (10/10) 220.0 6 1.7a (22/109) 225.4 6 3.8a (17/27) 215.3 6 1.7c (6/13) 224.5 6 3.0a (8/10) (52/57) 0b (0/16) 0b (0/27) Values are means 6 SE. Values in parentheses are no. of responsive sites/no. of sites tested. MAP, mean arterial pressure; HR, heart rate; Glu, L-glutamate; SNC, substantia nigra pars compacta; SNL, substantia nigra pars lateralis; SNR, substantia nigra pars reticulata; RF, reticular formation; VTA, ventral tegmental area; R, red nucleus; RRF, retrorubral field. Within any column, any 2 superscript letters that are different indicate statistically different values (P , 0.05), whereas any 2 superscript letters that are the same indicate no significant difference between values. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017 the cardiovascular depressor responses elicited by stimulation of the SNC-VTA region. Intravenous injections of either saline, raclopride, or Sch-23390 did not alter basal blood pressure levels. To investigate the contribution of different components of the autonomic nervous system to the cardiovascular responses elicited by activation of the neurons in the SNC-VTA region, we first administered the muscarinic receptor blocker atropine methyl bromide intravenously. The magnitude of the response of the MAP or HR responses elicited by stimulation of the same SNC-VTA sites was not significantly altered after the administration of atropine (Fig. 7). However, after the intravenous injection of the nicotinic receptor blocker hexamethonium bromide, the MAP response was abolished, whereas the HR responses to stimulation of the same SNC-VTA sites were significantly attenuated (Fig. 7). These data are summarized in Table 3. SN AND VTA DEPRESSOR RESPONSES H2553 of the MAP response suggests that the MAP responses elicited during stimulation of the SNC-VTA region were likely not secondary to the concomitant cardiac slowing. This observation and the finding of sites that elicited bradycardic responses in the absence of a change in MAP suggest that these sympathoinhibitory effects are mediated by different central pathways. Stimulation of the SNC and the lateral region of the VTA where DA neurons have been described to form a continuous layer of cells that merges indistinctly with the DA neurons of the SNC (8) elicited cardiovascular depressor responses in most (93%) of the sites stimulated. In addition, stimulation of the adjacent retrorubral field, which contains DA neurons that are continuous with the caudal extension of the SNC (8), produced depressor responses in almost all the sites tested. The observation that the location of the most responsive site was found in the same region that DA neurons have been described to be located suggests that DA neurons may be involved in mediating the cardiovascular responses to stimulation of the SNC-VTA region. This possibility was tested by observing the effect of the intravenous administration of DA-receptor antagonists on the magnitude of the depressor responses to stimulation of the SNC-VTA region. Administration of the D2 DA-receptor antagonist raclopride abolished the decrease in MAP and attenuated the HR responses elicited by stimulation of the SNC-VTA region. On the other hand, administration of the D1 DA-receptor antagonist Sch-23390 had no effect on the responses. The observation that administration of neither of the two compounds altered resting AP suggests that the AP depressor responses elicited by stimulation of the SNCVTA region are mediated by central DA mechanisms Fig. 4. Representative experiment showing effect of microinjections of different volumes (10, 4, and 2 nl) of 0.25 M Glu into same site within the SNC on AP and HR. Injections were made at 5-min intervals beginning with the largest volumes. Arrows, time of Glu microinjections. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017 Fig. 3. Representative micropipette tract through the region of ventral mesencephalon ,3.8 mm rostral to intra-aural line showing AP and HR responses elicited during microinjection of Glu at different dorsoventral sites along the tract (filled circles). Note that the largest cardiovascular responses were elicited from region of the SNC, whereas no responses were elicited by stimulation of SNR or reticular formation of dorsal tegmentum. Arrows, time of Glu injection. Transverse section of the mesencephalon is modified from stereotaxic atlas of rat brain (26). H2554 SN AND VTA DEPRESSOR RESPONSES Table 2. MAP and HR responses to stimulation of the SNC region with different volumes of 0.25 M Glu Glu (0.25 M) 10 nl 4 nl 2 nl MAP, mmHg HR, beats/min 232.0 6 4.1 223.8 6 2.6 223.0 6 3.7 231.0 6 4.9 224.0 6 5.1 223.8 6 5.4 Values are means 6 SE. For each volume 5 sites were stimulated in different animals. Fig. 5. Representative experiment showing effects of administration of raclopride (2 mg/kg iv) on magnitude of AP and HR responses during stimulation of SNC-VTA region before (A) and 30 min (B) and 3 h after administration of raclopride (C). Note that cardiovascular depressor responses to stimulation of the same site (A) were attenuated at 30 min (B) but returned to control values 3 h after raclopride administration (C). Arrows, time of Glu injections. Fig. 6. Bar charts showing magnitude of MAP (A) and HR (B) changes elicited by Glu stimulation of the same site within SNC-VTA region before (baseline) and after administration of raclopride (2 mg/kg iv, n 5 5) or saline (n 5 5). * Significantly different (P , 0.05) from control responses. proposed to be mediated by the release of vasopressin in the systemic circulation by means of central DA mechanisms (12), whereas the responses to stimulation of the SNC were proposed to be mediated by central DA release in the dorsal striatum (24). These earlier observations appear contradictory to those reported in this study. However, this is likely due to experimental approaches used in these studies. First, studies in which electrical stimulation was used to elicit cardiovascular responses are difficult to interpret because of the possibility of activation of fibers passing through the area where the stimulus is applied Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017 involving D2 receptors. In addition, that the HR response was only partially attenuated after the administration of the D2-receptor antagonist suggests that central pathways other than those involving DA neurons mediate a component of the HR responses. It may be argued that the effect observed after raclopride was due to damage of neuronal tissue at the site of injection as a result of the multiple injections of Glu. This possibility is considered unlikely because repeated injections of Glu into the SNC-VTA region after the administration of the D1-receptor antagonist or the saline did alter the magnitude of the cardiovascular responses. In addition, the response to stimulation of the SNC-VTA region returned to control levels 3 h after the administration of the raclopride. It has been previously shown that repeated injections of a low concentration of Glu do not appear to have any significant neurotoxic effects (18, 25). The region of the ventral mesencephalon that contains DA neurons has previously been implicated in the regulation of the circulatory system. First, electrical stimulation of the SNC region in anesthetized and awake cats has been shown to produce increases in MAP and HR, in addition to somatomotor and respiratory responses (6, 7). Second, electrical stimulation and chemical stimulation of the SNC region with the excitatory amino acids kainic acid and Glu in anesthetized rats was shown to produce increases in MAP and HR (24). Third, electrical stimulation and chemical stimulation of the VTA with Glu and the substance P analog DiMe-C7 in awake rabbits and rats was found to also elicit cardiovascular pressor responses (11, 12, 29). The pressor responses to stimulation of the VTA were SN AND VTA DEPRESSOR RESPONSES H2555 Fig. 7. Representative experiment showing control responses before drug administration (A) and effects of muscarinic (atropine methyl bromide; B) and nicotinic (hexamethonium bromide; C) receptor antagonists on magnitude of AP and HR responses during stimulation of SNC-VTA region. Arrows, time of Glu injections. Table 3. Changes in the MAP and HR responses to stimulation of the SNC region after the administration of muscarinic and nicotinic receptor antagonists Control Atropine methyl bromide Hexamethonium bromide MAP, mmHg HR, beats/min 233.8 6 5.1 227.6 6 5.0 0.0 6 0.0* 231.6 6 4.8 230.4 6 5.5 26.6 6 2.9* Values are means 6 SE of 5 sites stimulated in different animals. Atropine methyl bromide and hexamethonium bromide are muscarinic and nicotinic receptor antagonists, respectively. Control responses were obtained just before the administration of atropine and/or hexamethonium. * Significantly different (P , 0.005) from control responses and from responses after atropine treatment. use of different anesthetics in the previous studies accounts for the variable changes in AP and HR reported during stimulation of the SN. It has previously been shown that stimulation of the similar central sites under either urethan or a-chloralose elicited cardiovascular responses that were opposite in direction (9). Stimulation of sites within the medial lemniscus, SNR, SNL, reticular formation, red nucleus, and preand retrorubral fields adjacent to the SNC-VTA region elicited smaller responses. This observation suggests that Glu microinjected into these areas may have diffused a sufficient distance to activate SNC-VTA neurons and to elicit these weaker cardiovascular responses. From Fig. 2, it can be estimated that the diffusion distance for Glu to exert the effect was ,150 µm. Sites in either the medial lemniscus or the SNR .150 µm from the SNC-VTA region were found not to elicit cardiovascular responses. This suggestion is also supported by the finding that the onset latency for the MAP produced by stimulation of the SNC-VTA region was shorter than the onset latency of the responses elicited by stimulation of these extra-SNC-VTA sites. In addition, the finding of cardiovascular-responsive sites within the SNR adjacent to the SNC may have been due to activation of SNC neurons that have dendrites oriented ventrally deep within the SNR (8). However, the possibility cannot completely be excluded that neurons within regions of the reticular formation of the mesencephalon, red nucleus, or rubral fields may also play a role in the regulation of the circulation. Perspectives. Although there is increasing evidence to support the possibility that DA neurons in the ventral mesencephalon are part of a central neuronal network involved in cardiovascular regulation, the function of the mesotelencephalic DA systems in the regulation of the circulation remains speculative. Recent evidence has been obtained suggesting that the activity of DA neurons in the SNC-VTA region may be regulated by inputs from arterial baroreceptors (2, 3, 18, 29). Denervation of the arterial baroreceptors has been shown to produce a decrease in striatal DA content and release and a decrease in the activity of the DA biosynthetic enzyme tyrosine hydroxylase in the striatum (2, 3). In addition, striatal DA release was found to be enhanced after increasing AP by the intravenous administration of phenylephrine and at- Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017 (18, 25). The region of the mesencephalon contains a large number of fiber tracts including the medial lemniscus, fasciculus retroflexus, cerebral peduncle, and fibers forming parts of the medial forebrain bundle. In the present study Glu was used to selectively excite cell bodies without activating neuronal fibers. Second, microinjections of a large volume of neuroactive substances (0.25–1.0 µl) and a large concentration of excitatory amino acids (1–2 µg) in the ventral mesencephalon to elicit cardiovascular responses (11, 12, 24, 29) prevent the precise localization of the neurons that may be involved in mediating the cardiovascular responses. It has been previously shown that microinjections of large volumes and concentrations of Glu may cause a short-lasting excitation followed by a longerlasting inhibition or a decrease in excitability of neurons in the immediate area of the injection (25). Small volumes (2–10 nl) and low concentration (0.4 µg) of Glu were used in this study to define regions of the ventral mesencephalon that produced cardiovascular responses. Third, in previous studies, either awake or anesthetized nonparalyzed or artificially ventilated animals were used (6, 7, 11, 12, 24). Therefore, the possibility exists that changes in respiratory and somatomotor function may have been responsible for the pressor responses observed in these studies during stimulation of the SN-VTA region. This suggestion is supported by the observation that electrical stimulation of the SNC in cats resulted in changes in both respiratory and somatomotor variables (6, 7). In all experiments done in this study the animals were paralyzed and artificially ventilated. Finally, the possibility exists that the H2556 SN AND VTA DEPRESSOR RESPONSES 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. This research was supported by the Heart and Stroke Foundation of Ontario. G. J. Kirouac is the recipient of a Heart and Stroke Foundation of Canada Research Fellowship. Address reprint requests to J. Ciriello. 19. Received 23 January 1997; accepted in final form 23 July 1997. 20. REFERENCES 1. Alexander, G. E., M. D. Crutcher, and M. R. De Long. Basal ganglia-thalamocrotical circuits: parallel substrates for motor, oculomotor, ‘‘prefrontal’’ and ‘‘limbic’’ functions. Prog. Brain Res. 85: 119–146, 1990. 2. Alexander, N., Y. Hirata, and T. Nagatsu. Reduced tyrosine hydroxylase activity in nigrostriatal system of sinoaorticdenervated rats. Brain Res. 299: 380–382, 1984. 3. Alexander, N., D. Nakahara, N. Ozaki, N. Kaneda, T. Sasaoka, N. Iwata, and T. Nagatsu. Striatal dopamine release and metabolism in sinoaortic-denervated rats by in vivo microdialy- 21. 22. 23. sis. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R396–R399, 1988. Alheid, G. F., C. Beltramino, A. Braun, R. R. Miselis, C. François, and J. de Olmos. Transition areas of the striatopallidal system with the extended amygdala in the rat and primate: observations from histochemistry and experiments with monoand transsynaptic tracer. In: The Basal Ganglia IV, Advances in Behavioral Biology, edited by G. Percheron, J. S. McKenzie, and J. Féger. New York: Plenum, 1994, vol. 41, p. 95–107. Alheid, G. F., J. S. de Olmos, and C. A. Beltramino. Amygdala and extended amygdala. In: The Rat Central Nervous System, edited by G. Paxinos. Sydney: Academic, 1995, p. 495– 578. Ångyán, L. Role of the substantia nigra in the behavioralcardiovascular integration in the cat. Acta Physiol. Scand. 74: 175–187, 1989. Ångyán, L. Substantia nigra stimulation and blood pressure effects of locally applied kainic acid. Neuroreport 2: 785–788, 1991. Björklund, A., and O. Lindvall. Dopamine-containing systems in the CNS. In: Handbook of Chemical Neuroanatomy. Classical Transmitters in the CNS, edited by A. Björklund and T. Hökfelt. New York: Elsevier, 1984, vol. 2, pt. 1, p. 55–122. Calaresu, F. R., and G. J. Mogenson. Cardiovascular responses to electrical stimulation of the septum in the rat. Am. J. Physiol. 223: 777–782, 1972. Ciriello, J., and S. A. Janssen. Effect of glutamate stimulation of bed nucleus of the stria terminalis on arterial pressure and heart rate. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H1516– H1522, 1993. Cornish, J. L., and M. van den Buuse. Pressor responses to electrical and chemical stimulation of the rat brain A10 dopaminergic system. Neurosci. Lett. 176: 142–146, 1994. Cornish, J. L., and M. van den Buuse. Stimulation of the rat mesolimbic dopaminergic system produces a pressor response which is mediated by dopamine D-1 and D-2 receptor activation and the release of vasopressin. Brain Res. 701: 28–38, 1995. Dahlström, A., and K. Fuxe. Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol. Scand. 232: 1–55, 1964. Dampney, R. A. L. Functional organization of central pathways regulating the cardiovascular system. Physiol. Rev. 74: 323–364, 1994. Domesick, V. B. Neuroanatomical organization of dopamine neurons in the ventral tegmental area. Ann. NY Acad. Sci. 537: 10–26, 1988. Fallon, J. H. Topographical organization of ascending dopaminergic projections. Ann. NY Acad. Sci. 537: 1–9, 1988. Fallon, J. H., and S. E. Loughlin. Substantia nigra. In: The Rat Central Nervous System, edited by G. Paxinos. Sydney: Academic, 1995, p. 215–237. Goodchild, A. K., R. A. L. Dampney, and R. Bandler. A method for evoking physiological responses by stimulation of cell bodies, but not axons of passage, within localized regions of the central nervous system. J. Neurosci. Methods 6: 351–363, 1982. Kirouac, G. J., and J. Ciriello. Cardiovascular afferent inputs to ventral tegmental area. Am. J. Physiol. 272 (Regulatory Integrative Comp. Physiol. 41): R1998–R2003, 1997. Kohler, C., H. Hall, S.-O. Ogren, and L. Gawell. Specific in vitro and in vivo binding of 3H-raclopride. A potent substituted benzamide drug with high affinity for dopamine D-2 receptors in the rat brain. Biochem. Pharmacol. 34: 2251–2259, 1985. Lappalainen, J., J. Hietala, B. Sjoholm, and E. Syvalahti. Effects of chronic SCH 23390 treatment on dopamine autoreceptor function in rat brain. Eur. J. Pharmacol. 179: 315–321, 1990. Le Moal, M., and H. Simon. Mesocorticolimbic dopaminergic network: functional and regulatory roles. Physiol. Rev. 71: 155– 234, 1990. Lin, M.-T., B.-L. Tsay, and F. F. Chen. Activation of dopaminergic receptors within the caudate-putamen complex facilitates reflex bradycardia in the rat. Jpn. J. Physiol. 32: 431–442, 1982. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017 tenuated by decreasing carotid AP after carotid artery occlusion (31). Finally, selective activation of arterial baroreceptors has been shown to alter the discharge rate of putative DA neurons (19). These studies suggest that DA neurons of the mesencephalon may be part of a central long-loop baroreceptor reflex pathway controlling AP. The DA neurons of the SNC (the A9 cell group), VTA (the A10 cell group), and retrorubral field (the A8 cell group), which form the three monoaminergic cell groups originally defined by Dahlström and Fuxe (13), have widespread projections to many areas of the cortex, striatum, and limbic system (8, 15, 17). These DA projections are organized with only a crude topography with much overlap between the terminal fields originating from the different groups of DA neurons (8). The regions of the medial SNC and lateral VTA were found in this study to elicit the largest cardiovascular responses. DA neurons of the medial SNC and lateral VTA have been shown to project to an area that has been termed the extended amygdala, which includes structures such as the shell region of the nucleus accumbens, bed nucleus of the stria terminalis, and central amygdala, and the transitional areas between these structures and the remainder of the striatum (4, 5). The extended amygdala is strongly interconnected with forebrain and brainstem centers (4, 5) known to be involved in cardiovascular regulation (14, 28). Stimulation of the bed nucleus of the stria terminalis and the central nucleus of the amygdala have been shown to elicit cardiovascular depressor responses similar to those elicited in this study (10, 27). Therefore, it is possible that stimulation of DA neurons that subserve cardiovascular regulation may activate mechanisms in structures of the extended amygdala to produce cardiovascular depressor responses. This suggestion is supported by several studies showing that stimulation of forebrain DA receptors has depressor effects on the circulation (30). In summary, the DA neurons within the SN-VTA region have been suggested to be involved in the preparation, organization, and initiation of goaldirected behaviors (6, 22). The observation in this study that stimulation of these neurons elicits changes in HR and AP suggests that the SN-VTA region is likely involved in controlling the cardiovascular responses that accompany goal-directed behavior (6, 19). SN AND VTA DEPRESSOR RESPONSES 24. Lin, M.-T., and J.-J. Yang. Stimulation of the nigrostriatal dopamine system produces hypertension and tachycardia in rats. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H2489–H2496, 1994. 25. Lipski, J., M. C. Bellingham, M. J. West, and P. Pilowsky. Limitations of the technique of pressure microinjection of excitatory amino acids for evoking responses from localized regions of the CNS. J. Neurosci. Methods 26: 169–179, 1988. 26. Paxinos, G., and C. Watson. The Rat Brain in Stereotaxic Coordinates (2nd ed.). New York: Academic, 1986. 27. Roder, S., and J. Ciriello. Contribution of bed nucleus of the stria terminalis to the cardiovascular responses elicited by stimulation of the amygdala. J. Auton. Nerv. Syst. 45: 61–75, 1993. H2557 28. Sun, M. K. Central neural organization and control of sympathetic nervous system in mammals. Prog. Neurobiol. 47: 157– 233, 1995. 29. Tan, E., A. K. Goodchild, and R. A. L. Dampney. Intense vasoconstriction and bradycardia evoked by stimulation of neurones within the midbrain ventral tegmentum of the rabbit. Clin. Exp. Pharmacol. Physiol. 10: 305–309, 1983. 30. Van den Buuse, M., and W. De Jong. Pharmacology of central dopaminergic modulation in hypertension. In: Antihypertensive Drugs Today, edited by H. Saito, M. Minami, and S. H. Parvez. Utrecht, The Netherlands: VSP, 1992, vol. 2, p. 157–188. 31. Yang, J.-J., and M.-T. Lin. Arterial baroreceptor information affects striatal dopamine release measured by voltammetry in rats. Neurosci. Lett. 157: 21–24, 1993. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on May 2, 2017