Download Cardiovascular depressor responses to stimulation of substantia

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
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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
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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.
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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).
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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.
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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.
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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
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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-
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(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.
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