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Department of Physiology and Institute for Biomedical Research, University of Sydney, New South Wales 2006, Australia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Physiological and anatomic methods were used to determine whether neurons in the rostral ventrolateral medulla (RVLM), nucleus tractus solitarius (NTS), or hypothalamic paraventricular nucleus (PVN) mediate the cardiovascular response evoked from the dorsomedial hypothalamic nucleus (DMH), which is believed to play a key role in mediating responses to stress. In urethane-anesthetized rats, activation of neurons in the DMH by microinjection of bicuculline resulted in a large increase in arterial pressure, heart rate, and renal sympathetic nerve activity. The pressor and sympathoexcitatory responses, but not the tachycardic response, were greatly reduced after bilateral muscimol injections into the RVLM even when baseline arterial pressure was maintained at a constant level. These responses were not reduced by muscimol injections into the PVN or NTS. Retrograde tracing experiments identified many neurons in the DMH that projected directly to the RVLM. The results indicate that the vasomotor and cardiac components of the response evoked from the DMH are mediated by pathways that are dependent and independent, respectively, of neurons in the RVLM.
rostral ventrolateral medulla; nucleus tractus solitarius; paraventricular nucleus; stress
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE HYPOTHALAMUS has
long been recognized as a key brain region involved in the integration
of the physiological responses to stress. In particular, recent studies
indicate that the dorsomedial hypothalamic nucleus (DMH) is an
important component of the central pathways mediating the
cardiovascular response to emotional stress. Activation of DMH
neurons, by microinjection of either excitatory amino acids or the
-aminobutyric acid (GABA) receptor antagonist bicuculline methiodide
results in increases in arterial pressure and heart rate (5, 8,
25, 26) as well as hemodynamic, neuroendocrine,
gastrointestinal, and behavioral changes similar to those evoked by
acute emotional stress (7, 8, 10). Furthermore, inhibition
of neurons in the DMH greatly reduces the pressor and tachycardic
response evoked by air stress in the conscious rat (29).
The increases in arterial pressure and heart rate evoked by activation of DMH neurons have been shown to be sympathetically mediated (8). However, very little is known about the descending pathways that mediate the sympathoexcitatory response evoked from the DMH. Previous anatomic studies indicate that the DMH contains no or very few neurons that project directly to the spinal cord (12, 33, 34), implying that the descending sympathoexcitatory pathway from the DMH must include one or more synaptic connections in other supraspinal nuclei. One possible relay nucleus is the rostral ventrolateral medulla (RVLM), which contains presympathetic neurons that play a pivotal role in cardiovascular regulation (3, 11, 19). However, previous anatomic studies using the method of anterograde transport do not indicate a major direct projection from the DMH to the RVLM (33, 34). On the other hand, it is possible that there are indirect projections from the DMH to the RVLM, because the DMH projects to the midbrain periaqueductal gray and to the nucleus tractus solitarius (NTS) (33, 34), both of which in turn project to the RVLM region containing presympathetic neurons (3).
The descending projections of the DMH to the brain stem, however, are relatively small compared with the intrahypothalamic projections of the DMH (34). One of the major targets of these projections is the hypothalamic paraventricular nucleus (PVN), particularly its parvocellular part (33, 34). Furthermore, the PVN is a major source of direct projections to the spinal sympathetic preganglionic nuclei, RVLM and NTS (3, 30), and, therefore, may also be an important component of the central pathway mediating the sympathoexcitatory response evoked from the DMH.
The main aim of this study was to determine the role of the RVLM, NTS, and PVN in mediating the pressor, sympathoexcitatory, and cardiac components of the cardiovascular response evoked by activation of neurons in the DMH. For this purpose, we tested the effect of inhibition of neurons in the PVN, RVLM, and NTS on the cardiovascular response evoked from the DMH. In addition, in a parallel series of anatomic experiments, retrograde tracing was performed to determine the extent of direct projections from DMH neurons to the RVLM and/or NTS in the medulla.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Physiological Experiments
General procedures. Experiments were performed on male Sprague-Dawley rats (8-12 wk old, 360-500 g, Laboratory Animal Services, University of Sydney, New South Wales, Australia) anesthetized with urethane (1.3-1.5 g/kg ip). All experiments were carried out in accordance with the guidelines for Animal Experimentation of the National Health and Medical Research Council of Australia. Body temperature was monitored with a rectal probe and maintained in the range of 37-38°C with a thermoregulated heating pad. Catheters were placed in a femoral vein and a femoral artery, and the trachea was cannulated. The head was placed in a stereotaxic frame with the tooth bar fixed 19 mm below the interaural line. The renal sympathetic nerve was exposed, and in those experiments in which microinjections were made into the medulla, the dorsal surface of the medulla was also exposed, as previously described in detail (32).
A small craniotomy was made near the bregma to allow for the later insertion of a micropipette into the DMH or PVN. After completion of all surgical procedures, neuromuscular blockade was induced with alcuronium chloride (0.1 mg/kg iv every 1-2 h), and the animals were artificially ventilated at a level that maintained end-tidal carbon dioxide (measured with a Datex Engstrom carbon dioxide monitor) in the range of 4.0-4.5%. The effects of alcuronium chloride were allowed to wear off before each additional dose was administered. The adequacy of anesthesia without neuromuscular blockade was verified by the absence of a withdrawal response to nociceptive stimulation of a hind paw and during neuromuscular blockade by a stable baseline arterial pressure, heart rate, and renal sympathetic nerve activity (RSNA). Supplemental doses of urethane (0.1 g/kg iv) were administered if necessary. The mean arterial pressure (MAP), heart rate, and RSNA were recorded continuously as previously described (32).Microinjections of drugs. Microinjections of the GABA receptor antagonist bicuculline (20 nl of 1 mM solution) were made into sites in the DMH on the left side by use of a micropipette held in a micromanipulator. The tip of the micropipette was first positioned using the coordinates for the DMH, determined according to the atlas of Paxinos and Watson (18) in a track located 3.1-3.3 mm posterior and 0.5- 0.7 mm lateral to the bregma and at a depth of 8.6 mm below the dura. A microinjection of bicuculline was then made into this site. Usually, this resulted in a pressor response of at least 25 mmHg, but if it did not, the micropipette tip was repositioned usually 0.2 mm more rostral or caudal. In all experiments, no more than two sites were tested in this way before a pressor response of at least 25 mmHg was obtained. All subsequent microinjections of bicuculline were then made into this site.
Microinjections of the GABA receptor agonist muscimol were made either unilaterally or bilaterally into the pressor region in the RVLM (100 nl of 10 mM solution), bilaterally into the NTS (40 nl of 10 mM solution), or unilaterally into the PVN (40 nl of 10 mM solution). In the case of the RVLM or NTS, microinjections were made using a micropipette held in a second micromanipulator at an angle of 20° (tip rostral), whereas in the case of the PVN the microinjection was made from a micropipette positioned vertically. Injections into the NTS were made into the sites 0.5 mm rostral to the obex, 0.6 mm lateral to the midline, and 1.5 ventral to the dorsal surface. The pressor region in the RVLM was identified as the site at which a microinjection of sodium glutamate (40-50 nl of 50 mM solution) evoked a pressor response of at least 25 mmHg. Usually, less than three penetrations of the medulla were required to identify the pressor region on each side. The rostrocaudal, mediolateral, and dorsoventral coordinates of this site (determined with respect to the obex, midline, and dorsal surfaces, respectively) were noted, and the subsequent microinjection of muscimol was made into this identified pressor region. In the case of the PVN, the micropipette tip was positioned by use of the coordinates for the PVN according to the atlas of Paxinos and Watson (18) in the track located 1.8 mm posterior and 0.5 mm lateral to the bregma and at a depth of 7.6 mm below the dura. The vehicle solutions were 10 mM phosphate-buffered saline (pH 7.4) for microinjections of glutamate and artificial cerebrospinal fluid (pH 7.4) for bicuculline or muscimol. Microinjections were made by pressure, and the volume injected was measured by determining the displacement of the meniscus in the pipette with respect to a horizontal grid viewed through an operating microscope.Experimental procedures. The general strategy in these experiments was to test the effect on the response evoked by disinhibition of neurons in the DMH of blockade of neurons in the 1) RVLM pressor region on the side ipsilateral to the DMH injection site, 2) RVLM pressor region on both sides, 3) NTS on both sides, and 4) PVN on the ipsilateral side.
In the first series of experiments, the control response to a unilateral injection of bicuculline into the DMH was recorded. Then, 60 min after the injection of bicuculline, a microinjection of muscimol (1 nmol) was made into the RVLM pressor region on the side ipsilateral to the injection site in the DMH. Approximately 5-10 min later, when the MAP, heart rate, and RSNA had stabilized, a second microinjection of bicuculline was then made into the DMH. In the second series of experiments, the procedure was the same, except that microinjections of muscimol were made into the RVLM pressor region on both sides. Bilateral microinjections of muscimol into the RVLM resulted in a marked fall in the resting level of MAP, in confirmation of previous findings (14). Therefore, in some experiments, phenylephrine solution (3-5 µg · kg
1 · min1) was
infused continuously at a rate sufficient to maintain MAP at a level
close to the level before muscimol microinjections into the RVLM.
In the third series of experiments, the procedure was similar to the
second series except that muscimol was injected bilaterally into the
NTS. Finally, in the fourth series of experiments, the procedure was
similar to the first series except that muscimol was injected
unilaterally into the PVN on the side ipsilateral to the injection site
in the DMH. A control series of experiments was also performed in which
the procedure was the same as in the fourth series except that the
vehicle solution, instead of muscimol, was injected into the
ipsilateral PVN.
Histology. At the end of each experiment, a microinjection of the vehicle solution containing fast green dye was made into the DMH injection site with use of the same coordinates as used for injections of bicuculline. The animal was then euthanized by an overdose of pentobarbital sodium, and the brain was removed and placed in a solution of 0.1 M phosphate buffer of pH 7.4 containing 4% paraformaldehyde for 24 h. Subsequently, 50-µm-thick coronal sections of the hypothalamus were cut on a freezing microtome. The labeled microinjection sites were identified by examining the sections under a microscope.
Data analysis. The baseline values of MAP, heart rate, and RSNA were measured as the average values of these variables for the 1-min period immediately preceding microinjection of bicuculline into the DMH. Similarly, the peak values of MAP, heart rate, and RSNA after bicuculline microinjection were measured as the average values of these variables over a 1-min period at the time when the evoked increases in each of these variables was maximal (within 5-10 min after microinjection). Comparisons among responses evoked by microinjections of bicuculline into the DMH before and after microinjection of muscimol or vehicle solution into the RVLM, NTS, or PVN were determined by the paired t-test. A value of P < 0.05 was taken to indicate a statistically significant difference. All values are presented as means ± SE.
Anatomic Experiments
In three adult Sprague-Dawley rats (320-400 g body wt), two different types of retrogradely transported tracers (fluorescein-labeled and rhodamine-labeled microspheres) were injected unilaterally into the NTS and the RVLM for the purpose of determining whether neurons in the DMH projected to either or both of these medullarly regions.Surgical procedures. The rats were anesthetized with a mixture of acepromazine (0.5 mg/kg), xylazine (4 mg/kg), and ketamine (80 mg/kg) injected intraperitoneally. After induction of anesthesia, the long-acting opiate agonist buprenorphine (Temgesic, 0.01 mg/kg) was injected subcutaneously to provide preemptive analgesia. An endotracheal tube was inserted and the rat was artificially ventilated. The rat was placed in a stereotaxic frame, and the dorsal medulla was exposed. Microinjections of the microspheres were made from a micropipette positioned such that its tip was located either in the NTS or in the RVLM, using stereotaxic coordinates. In the case of the NTS, the tip was positioned 0.5 mm rostral to the obex, 0.6 mm lateral to the midline, and 1.5 ventral to the dorsal surface. In the case of the RVLM, the tip of the micropipette was positioned using previously determined coordinates, such that it was located within the C1 area in the RVLM, which is known to correspond to the location of presympathetic pressor neurons (19). The locations of the injection sites were subsequently determined histologically (see Histological procedures).
A microinjection of fluorescein-labeled microspheres was first made into the NTS, after which a microinjection of rhodamine-labeled microspheres was made from a second pipette into the RVLM. In one experiment, the tracers were reversed, such that rhodamine-labeled microspheres were injected into the NTS, and fluorescein-labeled microspheres were injected into the RVLM. In all cases, the volume of tracer injected was ~80 nl, determined as described above for the physiological experiments (see Microinjection of drugs). On completion of these procedures, the micropipette was removed, and the dura mater, overlying muscles, and skin were sutured. The animals were monitored closely during the subsequent waiting period of 5-7 days. For 1-2 days after surgery, antibiotic (benzilpenicillin, 100 mg/kg) and an analgesic (Temgesic, 0.01 mg/kg) were administered twice daily.Histological procedures. At the end of the waiting period, the animal was deeply anesthetized with pentobarbitol sodium (60 mg/ml ip) and perfused transcardially with heparinized saline solution containing sodium nitrite (5%), followed by phosphate-buffered saline (0.1 M, pH 7.4) containing 4% paraformaldehyde. The brain was removed and placed in 4% paraformaldehyde in phosphate-buffered saline for 24 h. Subsequently, 50-µm-thick coronal sections of the medulla and hypothalamus were cut on a freezing microtome. In some experiments, adjacent sections of the medulla were processed for tyrosine-hydroxylase immunoreactivity by using the same procedure as previously described in detail (15).
Mapping of labeled neurons. The sections were examined with the use of an Olympus BH-2 microscope. Initially, dark-field optics were used to identify anatomic landmarks, and subsequently, fluorescein- and rhodamine-labeled microspheres were visualized using fluorescence optics with appropriate filters. Neurons that were double-labeled for both tracers were identified by switching between filters. Neurons in medullary sections that were immunoreactive for tyrosine-hydroxylase were identified using bright-field optics. The locations of single- and double-labeled neurons in representative hypothalamic sections through the DMH and of the injection sites and tyrosine-hydroxylase-immunoreactive neurons in the medulla were mapped using the Magellan Image Analysis Program.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Physiological experiments
Cardiovascular response to disinhibition of the DMH.
Unilateral microinjection of bicuculline (20 pmol) into the DMH
resulted in a significant increase in MAP accompanied by a large
increase in heart rate and RSNA (Fig. 1,
Table 1). The MAP, heart rate, and
RSNA typically began to increase within 10-20 sec after the
injection, reached a peak after 5-10 min, and then declined
gradually, returning to baseline levels after ~30 min (Fig. 1).
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Effects of inhibition of RVLM neurons on
cardiovascular response to disinhibition of the DMH.
After unilateral microinjection of muscimol (1 nmol) into the RVLM
pressor region, there was a small decrease in baseline MAP, heart rate,
and RSNA, which was significant only in the case of RSNA (Table
2). After unilateral microinjection of
muscimol into the RVLM, however, the pressor and sympathoexcitatory
responses to disinhibition of the DMH were significantly reduced (by
35-40%), although the tachycardic response was not affected
(Figs. 2 and 3).
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Effects of inhibition of NTS neurons on
cardiovascular response to disinhibition of the DMH.
After bilateral microinjections of muscimol into the NTS, there was a
moderate increase in baseline MAP and a large increase in baseline RSNA
(Table 2) but no significant change in the increase in MAP and heart
rate evoked by disinhibition of the DMH (Fig. 5). In contrast, there was a significant
reduction in the magnitude of the sympathoexcitatory response
(expressed as a percentage of the baseline level) (Fig. 5). It should
be noted, however, that when measured relative to the control level of
RSNA before injections of muscimol in the NTS, the increase in RSNA
evoked by disinhibition of the DMH was not significantly different
before and after injections of muscimol in the NTS (140 ± 29%
vs. 121 ± 4%, n = 5 rats, P > 0.3).
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Effects of inhibition of PVN neurons on
cardiovascular response to disinhibition of the DMH.
After microinjection of muscimol into the ipsilateral PVN, there was no
significant change in the baseline MAP, heart rate, and RSNA or in the
magnitudes of the increases in these variables evoked by disinhibition
of the DMH (Table 3). However, the
durations of the responses were significantly reduced compared with the control responses (Table 3). In a control series of experiments, injection of the vehicle solution into the ipsilateral PVN had no
effect on the magnitudes or durations of the responses (Table 3).
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Anatomic experiments
Injection sites. In the NTS, the injection sites extended from ~1.0 mm rostral to the obex to 0.75 mm caudal. The injection sites were mostly restricted to the NTS, although they did extend ventrally into the hypoglossal nucleus. In the RVLM, the injection sites extended from the caudal pole of the facial nucleus to ~1.5 mm more caudal and were ventromedial to the compact formation of the nucleus ambiguus. This region corresponds to the C1 area of the medulla (19), which was confirmed by the demonstration that the unilateral injection sites corresponded closely to the location of the group of tyrosine-hydroxylase neurons that could be visualized on the side contralateral to the injection site.
Distribution of retrogradely labeled neurons.
As previously reported, neurons retrogradely labeled from both the NTS
and RVLM were located in the PVN and lateral hypothalamus (22,
35, 36). In addition, within the region corresponding to the
DMH, including the compact and diffuse portions of this nucleus as
defined by the atlas of Paxinos and Watson (18), there
were also, in all experiments, numerous cells retrogradely labeled from
either the NTS (30-100 cells/section) or the RVLM (150-500
cells/section) and, in addition, a significant number of neurons
retrogradely labeled from both the NTS and RVLM (30-50 cells/section) (Figs. 6 and
7). The number of such double-labeled cells represented ~40% and 15% of all cells retrogradely labeled from the NTS and RVLM, respectively. The projections from the DMH to
the RVLM and to the NTS were bilateral in all cases. In two rats, there
were similar numbers of retrogradely labeled cells in the DMH on each
side after unilateral injections of microspheres into the RVLM or NTS.
In the third animal, there were significantly more cells retrogradely
labeled from the RVLM and NTS (~100% and 50% more, respectively) in
the DMH on the side ipsilateral to the injection site compared with the
contralateral DMH (Fig. 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The results of our study confirm previous findings (5, 6, 8) that disinhibition of the DMH evokes an increase in MAP and a large increase in heart rate, which is mediated mainly by an increase in cardiac sympathetic activity, and show for the first time that this is accompanied by a large increase in RSNA. The main new finding of this study, however, is that the increase in MAP and RSNA evoked from activation of DMH neurons is greatly reduced by inhibition of neurons in the RVLM, whereas the evoked tachycardia is unaffected. Thus the results indicate that RVLM neurons are an essential component of the central pathways mediating the increased sympathetic vasomotor activity in response to activation of DMH neurons. In contrast, the descending pathways mediating the tachycardia are independent of the RVLM.
Role of the RVLM in Mediating the Sympathoexcitatory Component of Response
Before discussing the physiological significance of our observations, some methodological issues need to be considered. Although bilateral inhibition of the RVLM (by injections of muscimol) greatly reduced the increase in RSNA evoked by disinhibition of the DMH, bilateral inhibition also resulted in a large decrease in MAP. However, bilateral inhibition of the RVLM virtually abolished the sympathoexcitatory response to DMH disinhibition even when MAP was maintained with phenylephrine infusion, showing that this reduction in the response was not simply a consequence of reduced perfusion pressure. On the other hand, the baseline RSNA was also significantly reduced (by ~40%) even when MAP was maintained constant. Therefore, it could be argued that, after inhibition of the RVLM, the resulting large reduction in excitatory input to spinal sympathetic preganglionic neurons would render them less responsive to inputs from other sources, regardless of whether or not they are mediated by the RVLM. However, even after unilateral inhibition of the RVLM block, which reduced baseline RSNA only slightly (by 15%), the magnitude of both the pressor and sympathoexcitatory responses were reduced to a far greater degree (by 35-40%). In contrast, we have recently shown (31) that the sympathoexcitatory response evoked by disinhibition of the PVN on one side is not reduced by inhibition of the contralateral RVLM, presumably because the descending pathway from the PVN to the RVLM is almost entirely ipsilateral (22). This previous finding (31) therefore indicates that unilateral inhibition of the RVLM does not affect a response evoked by activation of a hypothalamic nucleus unless the descending pathway mediating the response includes a synapse in the RVLM. Therefore, the finding in the present study that unilateral inhibition of the RVLM significantly reduces the renal sympathoexcitatory response, and that bilateral inhibition virtually abolishes it, indicates that the descending sympathoexcitatory pathway from the DMH is bilateral and includes essential synapses in the RVLM.At the same time, the possibility cannot be ruled out that there is also a descending pathway from the DMH to the spinal sympathetic vasomotor outflow that is independent of the RVLM. Even if such a pathway exists, however, our results indicate that it is not capable of significantly activating renal sympathetic activity under conditions where the normal tonic excitatory input to the spinal sympathetic outflow from the RVLM is absent.
Previous studies (33, 34) using the method of anterograde transport of Phaseolous vulgaris leucoagglutinin have demonstrated a projection from the DMH to the RVLM, although in those studies relatively few labeled fibers were observed in the RVLM, suggesting that the projection was relatively minor. In contrast, we observed many neurons retrogradely labeled from the RVLM, indicating that there was a significant direct projection from the DMH to the RVLM. The reason for this apparent inconsistency between our results and previous studies using anterograde tracing is not clear, although it is conceivable that DMH neurons projecting to the RVLM do not take up or transport P. vulgaris leucoagglutinin as readily as the fluorescent microspheres used in the present study.
In any case, our results together with previous studies (33, 34) indicate that a direct projection from the DMH to the RVLM exists, although the magnitude of this projection and its precise site of termination in the RVLM is not established. It has been shown previously that microspheres are not taken up by undamaged fibers (13), and in our experiments care was taken to minimize the damage to tissue resulting from the microinjection procedure. Therefore, even though we cannot rule out the possibility that the neurons in the DMH were retrogradely labeled by microspheres taken up by fibers of passage rather than nerve terminals, our anatomic observations are consistent with the possibility that at least a part of the sympathoexcitatory response evoked by disinhibition of the DMH is mediated by a direct projection from the DMH to RVLM presympathetic neurons.
Our anatomic study also demonstrated neurons outside the DMH that were retrogradely labeled from the RVLM. Some of these neurons may also project to RVLM presympathetic neurons, such as neurons in the perifornical region believed to have a cardiovascular function (23). It is therefore conceivable that part of the cardiovascular response evoked by microinjection of bicuculline into the DMH is due to spread of the bicuculline to adjacent structures such as the perifornical area. However, previous detailed mapping studies (5, 26) have demonstrated that, when microinjections of bicuculline are made into sites within and surrounding the DMH, the largest and shortest latency responses are evoked from sites within the DMH, whereas microinjections into sites immediately surrounding the DMH resulted in much smaller or no increases in arterial pressure and heart rate. Consistent with these previous studies, we found in the present study that microinjections of bicuculline into the DMH evoked large sympathoexcitatory responses of short onset latency. We therefore conclude that activation of neurons within the DMH rather than surrounding regions are primarily responsible for the evoked sympathoexcitatory responses.
A significant proportion (~15%) of DMH cells retrogradely labeled from the RVLM were also retrogradely labeled from the NTS. Conversely, ~40% of DMH cells retrogradely labeled from the NTS were also retrogradely labeled from the RVLM. This observation is interesting, in view of previous studies showing that electrical stimulation of the "hypothalamic defense area," the location of which appears to overlap at least partly with that of the DMH (8), leads to an increase in arterial pressure, heart rate, and renal sympathetic activity (4) together with inhibition of the baroreceptor reflex, via a modulatory action on NTS neurons (27). Our anatomic observations therefore raise the possibility that some DMH neurons provide excitatory inputs to RVLM presympathetic neurons as well as having a modulatory influence on NTS neurons subserving the baroreceptor reflex.
Role of the NTS and PVN in Mediating the Sympathoexcitatory Response
Apart from the RVLM, it is well established that the DMH projects to other regions involved in cardiovascular control, including the NTS, PVN, and midbrain periaqueductal gray (33, 34). All of these regions contain neurons that project directly to the RVLM (3), and it is therefore possible that they may also be relay nuclei in the descending sympathoexcitatory pathway from the DMH. With regard to the NTS, our results indicate that bilateral inhibition of this nucleus has little effect on the magnitude of the evoked pressor and tachycardic response evoked from the DMH. Although the magnitude of the evoked renal sympathoexcitatory response was reduced when expressed as a percentage of the baseline level, this largely reflects the fact that the baseline level of RSNA was greatly increased after bilateral inhibition of the NTS. When measured relative to the control level of RSNA before injections of muscimol in the NTS, the increase in RSNA evoked by disinhibition of the DMH was not significantly changed. These results therefore indicate that the NTS is not a major component of the descending pathways mediating the cardiovascular response to disinhibition of the DMH.The PVN, particularly its parvocellular portion, receives a dense innervation from the ipsilateral DMH (33, 34). The parvocellular PVN in turn has major projections to the NTS, RVLM, and spinal cord (22, 30). Our results showed that unilateral injection of muscimol in the PVN did not reduce the magnitude of the sympathoexcitatory response evoked from the ipsilateral DMH. It could be argued that the projection from the DMH to the contralateral PVN was sufficient to maintain the full response, even after blockade of synaptic transmission in the ipsilateral PVN. This seems unlikely, however, given that the projection from the DMH to the contralateral PVN is much less dense than that to the ipsilateral PVN (34).
Second, it could be argued that the amount of muscimol injected (400 pmol in 40 nl) caused incomplete blockade of the PVN. Muscimol inhibits neurons by acting as an agonist at GABAA receptors, which virtually all neurons possess (2), so that muscimol is thought to be effectively a universal inhibitor of neurons. We injected a high concentration of muscimol (10 mM) to maximize its inhibitory effect. At the same time, the volume injected (40 nl) was chosen in an attempt to minimize the spread of muscimol to the DMH itself, which is located only 1.0-1.5 mm from the PVN. In a previous study, it was shown that bilateral injection of a much lower dose of muscimol (80 pmol) into the PVN was sufficient to greatly reduce the increase in adrenocorticotropic hormone levels evoked by air-jet stress (28). Furthermore, we have recently shown that a dose of 400 pmol of muscimol, when injected bilaterally into the PVN, causes a significant (~30%) reduction in baseline arterial pressure and RSNA (T. Tagawa, M. A. P. Fontes, and R. A. L. Dampney, unpublished observations). Although this effect is not observed when muscimol is injected unilaterally into the PVN (as in the present study), these previous observations indicate that the dose of muscimol used has a significant inhibitory effect on sympathoexcitatory neurons in the PVN.
De Novellis et al. (5) found that the cardiovascular response evoked by bicuculline microinjection into the DMH has a greater magnitude and shorter latency than that evoked from the PVN. Furthermore, it has been shown that inhibition of the DMH, but not of the PVN, blocks the cardiovascular response to air-jet stress (29). Therefore, these previous observations are consistent with the finding of the present study that injection of a large dose of muscimol in the PVN did not significantly reduce the peak sympathoexcitatory response evoked by DMH activation. Taken together, the results of the present and previous studies (5, 29) suggest that the sympathoexcitatory response evoked from the DMH is not dependent on a synapse in the PVN.
Although muscimol injection in the PVN did not reduce the magnitude of the peak response evoked from the DMH, it did significantly reduce the duration of the response from ~30 min to 15-20 min. One possible explanation for this is that the muscimol injected into the PVN diffuses by 15-20 min postinjection to the DMH (a distance of ~1.0-1.5 mm), thus inhibiting sympathoexcitatory neurons initially activated by bicuculline injection.
Descending Pathway Mediating Tachycardia
As previously reported (5, 8), tachycardia is one of the most striking features of the cardiovascular response evoked by activation of the DMH. In confirmation of a previous study (6), the tachycardia was abolished by propranolol and is thus sympathetically mediated. In contrast to the pressor and renal sympathoexcitatory components of the response, however, the tachycardia was entirely unaffected by either unilateral or bilateral inhibition of the RVLM. It was also unaffected by bilateral inhibition of the NTS and unilateral inhibition of the PVN. These observations indicate that the descending pathway from the DMH mediating the cardiac component of the response is separate from that mediating the vasomotor component, and is quite independent of the RVLM, NTS, and PVN. At the same time, previous studies using both retrograde and anterograde labeling techniques (12, 34) indicate that the DMH does not project directly to the spinal cord, so that the descending pathway mediating the tachycardia must include a synaptic relay in one or more supraspinal nuclei. One interesting possibility is the midbrain periaqueductal gray, especially its lateral and ventrolateral portions, which is innervated by the DMH (33, 34). In a recent study using retrograde transynaptic transport of viruses, Farkas et al. (9) found that the predominant descending pathway from the periaqueductal gray to cardiac-related sympathetic neurons included a synaptic relay within the ventromedial medulla, especially the raphe magnus and adjacent gigantocellular reticular nucleus pars alpha. This pathway, which is independent of the NTS and RVLM, could therefore mediate tachycardia evoked by activation of the DMH.Comparison of Pathways Mediating Cardiovascular Responses from the DMH and PVN
Disinhibition of the PVN, like the DMH, also evokes an increase in arterial pressure, heart rate, and RSNA (16, 17, 31). However, the results of the physiological experiments in the present study indicate that the descending sympathoexcitatory pathway from the DMH is bilateral, whereas that from the PVN is mainly ipsilateral (31). These observations are consistent with anatomic observations indicating that the PVN projects almost entirely ipsilaterally to the RVLM (22), whereas the DMH projects bilaterally to the RVLM (present study). An additional difference is that the tachycardia evoked by disinhibition of the PVN is partly mediated by the RVLM (30), whereas the present results show that the tachycardia evoked from the DMH is independent of the RVLM.Perspectives
As reviewed by DiMicco and colleagues (7, 8), there is a substantial body of evidence indicating that DMH is a key nucleus in integrating cardiovascular, behavioral, gastrointestinal, and neuroendocrine responses to stress. The DMH receives inputs from a number of forebrain regions believed to play a role in mediating responses to stress, including the lateral septum, amygdala, and bed nucleus of the stria terminalis (1). Of these regions, the amygdala is thought to be of particular importance (7). For example, activation of the basolateral nucleus of the amygdala generates cardiovascular and behavioral changes similar to that evoked by stress (20, 21), and it has recently been shown (24) that the cardiovascular response evoked from the basolateral nucleus of the amygdala is dependent on synaptic transmission in the DMH.Given the crucial role of the DMH in integrating the physiological responses to stress, it is also of fundamental importance to elucidate the output pathways from the DMH that mediate these responses. The present study provides new information about the output pathways that mediate the cardiovascular components of the response, but further studies are needed to provide a complete description of the neural pathways that subserve all components of the response, including the neuroendocrine and behavioral components.
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ACKNOWLEDGEMENTS |
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This study was supported by the National Health and Medical Research Council of Australia. Dr. M. A. P. Fontes was supported by a Fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil.
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FOOTNOTES |
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Current address of M. Fontes: Departamento de Fisiologia e Biofisica, ICB-UFMG, Belo Horizonte, MG, Brazil (E-mail: peliky{at}mono.icb.ufmg.br).
Address for reprint requests and other correspondence: R. A. L. Dampney, Dept. of Physiology, F13, Univ. of Sydney, Sydney, NSW 2006, Australia (E-mail: rogerd{at}physiol.usyd.edu.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 October 2000; accepted in final form 25 January 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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