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Am J Physiol Regul Integr Comp Physiol 294: R1276-R1284, 2008. First published February 20, 2008; doi:10.1152/ajpregu.00437.2007
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Medullary pathways mediating the parasubthalamic nucleus depressor response

John Ciriello, L. Pastor Solano-Flores, M. Patricia Rosas-Arellano, Gilbert J. Kirouac, and Tanja Babic

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

Submitted 21 June 2007 ; accepted in final form 15 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The parasubthalamic nucleus (PSTN) projects extensively to the nucleus of the solitary tract (NTS); however, the function of PSTN in cardiovascular regulation is unknown. Experiments were done in {alpha}-chloralose anesthetized, paralyzed, and artificially ventilated rats to investigate the effect of glutamate (10 nl, 0.25 M) activation of PSTN neurons on mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). Glutamate stimulation of PSTN elicited depressor (–20.4 ± 0.7 mmHg) and bradycardia (–26.0 ± 1.0 beats/min) responses and decreases in RSNA (67 ± 17%). Administration (intravenous) of atropine methyl bromide attenuated the bradycardia response (46%), but had no effect on the MAP response. Subsequent intravenous administration of hexamethonium bromide blocked both the remaining bradycardia and depressor responses. Bilateral microinjection of the synaptic blocker CoCl2 into the caudal NTS region attenuated the PSTN depressor and bradycardia responses by 92% and 94%, respectively. Additionally, prior glutamate activation of neurons in the ipsilateral NTS did not alter the magnitude of the MAP response to stimulation of PSTN, but potentiated HR response by 35%. Finally, PSTN stimulation increased the magnitude of the reflex bradycardia to activation of arterial baroreceptors. These data indicate that activation of neurons in the PSTN elicits a decrease in MAP due to sympathoinhibition and a cardiac slowing that involves both vagal excitation and sympathoinhibition. In addition, these data suggest that the PSTN depressor effects on circulation are mediated in part through activation of NTS neurons involved in baroreflex function.

lateral hypothalamus; blood pressure; bradycardia; baroreceptor reflex; nucleus of the solitary tract

THE PARASUBTHALAMIC NUCLEUS (PSTN) is a cytoarchitectonically distinct cell group at the caudal level of the lateral hypothalamic area (LHA) lying just dorsomedial to the subthalamic nucleus (15, 29). More recently, the PSTN has been characterized as a preautonomic lateral hypothalamic cell group in which neurons express β-preprotachykinin mRNA (15). Additionally, PSTN neurons have been shown to project to both forebrain and brain stem structures previously implicated in feeding behavior and cardiovascular regulation (15). Within the brain stem, the PSTN innervates predominantly the dorsal vagal complex, especially the caudal aspects of the medial subnucleus of the nucleus of the solitary tract (mNTS) (15). The mNTS is known to function as the primary site of termination of baroreceptor afferent inputs (6). In addition, stimulation of this region NTS elicits cardiovascular responses similar to those observed during systemic activation of arterial baroreceptors (11, 17). Although these data suggest that the PSTN may affect cardiovascular function, its role in cardiovascular regulation is not known.

Activation of the caudal LHA in close proximity to the region of the PSTN (1, 24, 25), has been shown to elicit either increases or decreases in arterial pressure (AP) and heart rate (HR) or no changes in HR accompanying the AP changes (1, 24, 25). These cardiovascular responses have been suggested to be mediated by descending pathways to medullary autonomic sites (1). Interestingly, electrical stimulation of the adjacent subthalamic nucleus has been shown to elicit increases in AP, HR, and renal sympathetic nerve activity (RSNA) (3, 4). These observations suggest that the injections made in the study by Pajolla et al. (25) may have spread to the subthalamic nucleus and, in turn, may have masked the bradycardia responses evoked by lateral hypothalamic stimulation. On the other hand, as electrical stimulation does not distinguish between activation of neuronal cell bodies and fibers of passage, it is possible that the responses observed during stimulation of the subthalamic nucleus may have been due to activation of pathways originating in sites remote from those stimulated in the nucleus.

The present study was done to investigate the effect of activation of PSTN neurons in the {alpha}-chloralose anesthetized rat using microinjections (10 nl) of the excitatory amino acid L-glutamic acid (Glu) on AP, HR, and RSNA, and the sensitivity of the baroreceptor reflex. Glu was chosen as it is well known to selectively excite neuronal cell bodies and not fibers of passage (14). In addition, studies were done to determine whether these effects were mediated in part through an obligatory synapse in the NTS using CoCl2 injections into NTS (18).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experiments were done in male Wistar rats (300–450 g; Charles River Canada, St. Constant, Canada; n = 46) anesthetized with {alpha}-chloralose (60–80 mg/kg iv and additional doses of 10–20 mg/kg every 1–2 h) after induction with the short-lasting anesthetic Equithesin (pentobarbital sodium and chloral hydrate mixture; 0.3 ml/100 g ip) (10). 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 (PE)-50 catheters 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), 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 by using a Harvard Apparatus rodent ventilator (model 683) with a mixture of 5% room air and 95% O2. The animals were paralyzed with pancuronium bromide (Pavulon; Organon Canada, Toronto, ON; 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 of the PSTN were secondary to muscular activity or related to respiratory changes (16). All surgical procedures were done prior to the administration of the pancuronium bromide. During the course of the nonsurgical portions of the experiment, the animals were allowed to recover periodically from the pancuronium bromide to determine the depth of anesthesia by examining withdrawal reflexes. As pancuronium bromide is known to have a vagolytic effect, in some cases (n = 3) stimulation of the PSTN was done in nonparalyzed animals. Body temperature was monitored and maintained at 37 ± 0.5°C by a heating pad controlled by a Yellow Springs Instruments temperature controller (model 73).

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 caudal LHA and the ventral mesencephalic region. In addition, in some animals a partial occipital craniotomy was done to expose the dorsal surface of the brain stem overlying the region of the NTS. All exposed nervous tissue was covered with cotton pellets soaked in warm medical fluid (prod. no. 360; Dow Corning, Midland, MI) to prevent drying.

Chemical stimulation of the PSTN. Glu stimulation of the PSTN region was done by using single- or double-barreled glass micropipettes pulled from 5-µl Socorex capillary tubing (Mississauga, ON, Canada) with external tip diameters that ranged between 35 to 50 µm. Micropipette tips were lowered through the region of the caudal lateral hypothalamus according to a stereotaxic atlas of the rat brain (25), with each stimulation site 200–500 µm apart. The PSTN region was explored systematically on a grid pattern from 3.7 to 4.3 mm caudal to bregma, 1.3 to 2.2 mm lateral to the midline, and 7.0 to 8.2 mm ventral to the dorsal surface of the brain for sites that elicited changes in AP and/or HR. Solutions of Glu (0.25 M; 10 nl; Sigma, St. Louis, MO) (16) in 0.9% physiological saline were microinjected by the application of pressurized nitrogen pulses controlled by a picospritzer (General Valve, Fairfield, NJ) (8, 16). The injected volume (10 nl) was measured by direct observation of the fluid meniscus in the micropipette by using a microscope fitted with an ocular micrometer that was calibrated to allow a 2-nl resolution. As the microinjection of excitatory amino acids has been reported to result in a decrease in the excitability of neurons in the vicinity (up to a radius of 500 µm) of an injection site (19) after their initial depolarization, a minimum period ranging between 0.5 to 5 min was allowed between each microinjection of Glu during the mapping of the PSTN region. Control injections (10 nl) 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 (16).

Renal sympathetic nerve recordings. In five animals, following a left flank incision, a retroperitoneal dissection was used to expose the left renal nerve (7). The renal nerves were identified coursing along the renal artery and vein, separated from the blood vessels, and crushed distal to the recording electrode. The isolated nerves were placed on a bipolar platinum-iridium electrode, and the signal was amplified and passed through a band-pass filter (50–3,000 Hz) (5). Background noise was determined at the completion of each experiment after the systemic administration of the ganglionic blocker hexamethonium bromide (20 mg/kg iv). This value was subtracted from the integrated values obtained for each RSNA recording. The raw nerve activity was recorded on an oscilloscope and stored on disk until analyzed using a Powerlab data acquisition system, and integrated nerve activity was recorded on a Grass 79 polygraph.

Peripheral autonomic nervous system blockade. In seven additional animals, to determine which components of the peripheral autonomic nervous system were involved in mediating the cardiovascular responses to PSTN stimulation, cardiovascular responsive sites in the PSTN were retested following the administration of the muscarinic receptor blocker atropine methyl bromide (1–2 mg/kg iv). After 10–15 min, the nicotinic receptor blocker hexamethonium bromide (20 mg/kg iv) was also administered, and the same site in the PSTN was restimulated with Glu (8).

Injections of CoCl2, into the mNTS. In 12 experimental animals, the dorsal surface of the medulla was exposed following a partial occipital craniotomy. The dura was cut and reflected laterally, and the caudal floor of the fourth ventricle was exposed by gently removing the vermis of the cerebellum by suction. The calamus scriptorius was used as the point of reference for all brain stem injections. Micropipettes were lowered stereotaxically into the caudal mNTS bilaterally (stereotaxic coordinates: 0.5 mm rostral to calamus scriptorius, 0.6 mm lateral to the midline, and 0.5 mm ventral to the dorsal surface of the medulla). Cardiovascular responsive sites in the PSTN region were retested following the bilateral injections into the mNTS of the synaptic blocker CoCl2 (5 mM in 0.9% physiological saline; 100 nl; Fisher Scientific, Fair Lawn, NJ) (13, 20, 27, 30). The same stimulation site in the PSTN region identified previously that produced a cardiovascular response was retested at 2, 5, 10, and 20 min postinjection of either the synaptic blocker or vehicle (0.9% physiological saline).

To determine whether the changes in the magnitude of the cardiovascular responses after the injection of CoCl2 into the mNTS were due to local damage of neuronal tissue as a result of the injections into the mNTS or to the repeated stimulation of the PSTN by Glu, control experiments were done in which 100 nl of 0.9% physiological saline was injected into the region of the caudal mNTS bilaterally at similar sites to those used for the injections of CoCl2. The PSTN was restimulated 2–20 min later as described above. The experiments for CoCl2 or the vehicle were done in separate groups of animals.

Activation of PSTN and mNTS with Glu. To determine whether activation of mNTS neurons altered the magnitude of the cardiovascular responses elicited by activation of the PSTN, the effect of microinjection of Glu into the mNTS (10 nl) on the AP and HR responses elicited by Glu injections into the PSTN was tested in a separate series of experiments (n = 9). Injections of Glu were made into PSTN 5 min before (control) and at 0.5, 1.0, and 5.0 min after microinjection of Glu into the mNTS.

Activation of arterial baroreceptors. To determine whether activation of PSTN neurons exerted an effect on the baroreceptor reflex, the effect of microinjection of Glu into the PSTN on the reflex bradycardia elicited by the systemic increase in mean AP (MAP) to an intravenous injection of phenylephrine (Phe; 4 µg/kg; in 0.1 ml of saline) was tested in animals under chloralose anesthesia (n = 5) (10). Control responses for PSTN stimulation and those elicited by injections of Phe were obtained 5 min before the experimental protocol that consisted of injecting Phe 0.5, 2.5, and 5.0 min after microinjection of Glu into the PSTN. In each animal, injections of Phe were made while testing only one site in each side of the PSTN.

Histological localization of stimulation sites. At the completion of an experiment in which single-barreled micropipettes were used, the micropipette was withdrawn from the last site of injection, filled with Pontamine Sky blue in PBS (pH 7.2) without removing it from the stereotaxic holder, and lowered back to the same site at which a 20-nl microinjection of the dye was made to mark the center of the injection site in the PSTN region (8, 10, 16). In some experiments, the second-barrel of a double-barreled micropipette was filled with Pontamine Sky blue. This was injected to mark the center of the stimulation site in the PSTN region (20 nl) or of the injection site in the NTS (100 nl) (8, 13, 16). The injection sites and the resulting micropipette tracks were later identified histologically. The injection of the saline and/or Pontamine Sky blue dye in the PSTN did not elicit cardiovascular responses.

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 caudal lateral hypothalamus and mesencephalon, and the medulla oblongata were cut at 40 µm on a Bright's cryostat and stained with either thionin or Neutral red. The location of the marked sites of stimulation and the resulting micropipette tracks were histologically identified, and the remaining injection sites in any one animal were determined by extrapolation from the marked site along the micropipette tracks. Injection sites were mapped on projection drawings of each brain and later mapped on diagrams of transverse sections of the rat brain modified from a stereotaxic atlas (29).

Data analysis. MAP was calculated by adding one-third of the pulse pressure to the diastolic pressure. Control levels of MAP or HR were calculated 1 min before the injections. A cardiovascular responsive site in the caudal lateral hypothalamus and PSTN was defined as a site at which Glu injections elicited a change in either MAP or HR of > 5 mmHg or > 10 beats/min, respectively. All values were expressed as means ± SE calculated for the magnitude of the cardiovascular changes before and after the injections CoCl2 or saline into the NTS. These values were compared using an ANOVA for repeated measures, followed by the Tukey-Kramer multiple-comparison post hoc test. The effects of the nicotinic and muscarinic blocking agents on the MAP and HR responses were similarly compared statistically. The effect of PSTN activation on the baroreceptor reflex was analyzed by using an ANOVA followed by Dunnett's multiple comparison test. In all comparisons, a P value of <0.05 was taken to indicate statistical significance (GraphPad Prism; GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The resting MAP and HR in the {alpha}-chloralose anesthetized, paralyzed, and artificially ventilated rat were found to be 112 ± 5 mmHg and 403 ± 5 beats/min, respectively. Glu microinjections into histologically verified sites in the PSTN regions (Figs. 1, 2A and 3A) elicited decreases in MAP (–20 ± 1 mmHg; n = 50; Figs. 2B and 3B). Additionally, all depressor responses were accompanied by decreases in HR (–20 ± 1 beats/min; Figs. 2C and 3B). These data are summarized in Figure 2E. Furthermore, at all sites tested (n = 15), PSTN stimulation also decreased RSNA (67 ± 17%) (Fig. 2D). Glu injections into the PSTN of three additional nonparalyzed animals elicited qualitatively and quantitatively similar MAP (–18 ± 5 mmHg; n = 8) and HR (–22 ± 7 beats/min; n = 8) cardiovascular responses to those observed within the paralyzed animals. In addition, a few sites (n = 4) were found in the adjacent subthalamic nucleus in the nonparalyzed animals that only elicited a small increase in MAP (9 ± 4 mmHg).


Figure 1
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  		Fig. 1. A series of transverse sections of the rat brain taken through the region of the caudal lateral hypothalamic area (extending from –3.7 to –4.45 mm caudal to bregma) showing the location of histologically identified sites that elicited decreases in arterial pressure (AP) and heart rate (HR). {blacktriangledown}, sites that elicited decreases in mean AP (MAP) and HR of >15 mmHg or beats/min; bullet, sites that elicited decreases in MAP and HR of 5–15 mmHg or beats/min; {circ}, sites that elicited MAP and HR responses of <5 mmHg or beats/min, respectively, or no responses; {blacktriangleup}, sites that elicited increases in MAP. ZI, zona incerta; STN, subthalamic nucleus; cpd, cerebral peduncle; PST, preparasubthalamic nucleus; LHA, lateral hypothalamic area; mtt, mammillothalamic tract; PH, posterior hypothalamic nucleus; fx, fornix; DMH, dorsomedial hypothalamic nucleus; FF, fields of Forel; LHAp, posterior lateral hypothalamic area; PSTN, parasubthalamic nucleus; pm, principal mammillary tract; PMd, dorsal premammillary nucleus; AL, anterior lobe, pituitary gland; PMv, ventral premammillary nucleus; PVp, periventricular hypothalamic area; 3V, third ventricle; ml, medial lemniscus; fr, fasciculus retroflexus; VTA, ventral tegmental area; LM, lateral mammillary nucleus; pm, principal mammillary tract; MM, medial mammillary nucleus. All sections drawn have been modified from a stereotaxic atlas (29).

 

Figure 2
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  		Fig. 2. A: brightfield photomicrograph of a transverse section of the forebrain (stained with thionin) taken through the region of the LHAp showing and a glutamate injection site (*) centered in the PSTN. Stimulation of this site elicited a decrease in MAP (B), HR (C), and renal sympathetic nerve activity (RSNA; D). Arrow in D indicates the time of injection in (B–D). E: summary of the changes in MAP and HR following stimulation of the sites in the PSTN. Numbers in brackets in E are the number of PSTN sites stimulated with glutamate. bpm, Beats/min.

 

Figure 3
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  		Fig. 3. A: brightfield photomicrograph of a transverse section of the forebrain (stained with Neutral red) taken through the region of the LHAp showing a series of micropipette tracks through the region of PSTN (track 1) and adjacent LHAp (tracks 2–3). B: cardiovascular responses elicited by stimulation of the sites marked (1a-3) in the photomicrograph. Note that as the pipette is lowered through the region of PSTN, decreases in AP and HR are elicited, whereas injections of glutamate above (1a) or below (1d) either elicited small or no cardiovascular responses. Additionally note that as sites are stimulated medially to the PSTN (tracks 2–3) the magnitude of the responses decreases the further away the site of injection is from PSTN. Arrows in B indicate time of the glutamate injections (10 nl, 0.25 M), and the calibration bar in 3 (1 min) applies to all tracings. LHAvl, lateral hypothalamic area, ventrolateral zone.

 
Figure 3 shows a representative experiment in which the micropipette was lowered through the region of the PSTN and the adjacent lateral hypothalamic region (Fig. 3A). As the micropipette was moved medially from within the PSTN (sites marked 1a–1d) to within the LHA (sites marked 2–3), the magnitude of the depressor and bradycardia responses decreased (Fig. 3B). Additionally, as the micropipette was lowered through the region of the PSTN (sites marked 1a–1d), the magnitude of the cardiovascular responses became progressively larger as the injection site approached the PSTN (Fig. 3, A–B, sites marked 1b and 1c). The largest responses were elicited from sites within the PSTN (sites marked 1b–1c). Stimulation of sites (n = 34) dorsal or ventral to PSTN in the adjacent LHA or lateral to the PSTN in the subthalamic nucleus elicited either small decreases or increases in MAP or no cardiovascular responses (Fig. 1).

Figure 4 shows the effect of decreasing the volume of the Glu microinjected into the PSTN on the magnitude of the cardiovascular responses. Note that stimulation of the same site in PSTN elicited qualitatively similar responses but of smaller magnitude with the smaller volume. Reproducibility of the initial cardiovascular responses can be seen after increasing again the volume to the initial 10 nl volume. This last injection elicited responses not different from those observed after the first injection of the same volume of Glu indicating that the smaller responses elicited with the smaller volumes were not due to mechanical damage as a result of the multiple injections, but most likely were due to the smaller number of neurons activated within the injection site.


Figure 4
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  		Fig. 4. Representative experiment showing the effect of microinjections of different volumes (2–10 nl) of 0.25 M glutamate into the same site within PSTN on AP and HR. Injections were made at 5-min intervals beginning with the largest volume (10 nl) and ending with the same 10 nl volume. Arrows indicate time of microinjections.

 
Single- or multiple-control injections of the vehicle (10 nl) into the same PSTN sites did not evoke any cardiovascular response. In contrast, repeated injections of Glu (up to 6 tested at 0.5- to 5.0-min intervals) at any one responsive site were found to elicit both qualitatively and quantitatively similar cardiovascular responses as the initial Glu injection (Fig. 4).

Effect of systemic muscarinic and nicotinic receptors blockers on the cardiovascular responses to PSTN stimulation. To investigate which peripheral component of the autonomic nervous system contributed to the cardiovascular responses elicited by activation of PSTN neurons, the muscarinic receptor blocker atropine methyl bromide and/or the nicotinic receptor blocker hexamethonium were administered intravenously (n = 7). Atropine methyl bromide did not alter the magnitude of the MAP responses (Fig. 5A) while significantly attenuating (46%) the HR responses (Fig. 5B). Following injections of hexamethonium bromide, both the MAP responses and the remaining HR responses to Glu injections into PSTN were abolished (depressor and bradycardia responses were reduced by 92% and 94%, respectively; Fig. 5).


Figure 5
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  		Fig. 5. Summary of effect of intravenous administration of muscarinic and nicotinic receptor blockers on the MAP and HR responses to activation of PSTN neurons. Note that atropine significantly attenuates only the HR responses, while injections of the ganglionic blocker hexamethonium bromide abolished the remaining HR response and the MAP response. *Significantly different (P < 0.05) from control; **significantly different (P < 0.05) from control and atropine effects.

 
Effect of blockade of synaptic transmission in the mNTS on the cardiovascular responses to PSTN stimulation. Microinjection of CoCl2 into the caudal region of the mNTS (n = 6; Fig. 6) bilaterally resulted in an attenuation of the depressor (Fig. 6A) and bradycardia (Fig. 6B) responses elicited by the injection of Glu into the PSTN. Effects of CoCl2 on the PSTN responses were observed to occur within approximately the first 2 min after the injection into the mNTS and were observed to be maximal at ~10 min following the mNTS injection. The magnitude of the cardiovascular responses to the injection of Glu into the PSTN returned to control values by ~20 min after the injection into the mNTS (Fig. 6). The microinjection of CoCl2 into the caudal regions of the mNTS did not alter the resting level of AP or HR in the chloralose-anesthetized animal as previously seen in urethane anesthetized or conscious rats following lesions of NTS (28).


Figure 6
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  		Fig. 6. Summary of effect of CoCl2 or saline bilateral injections (100 nl) into the caudal nucleus of the solitary tract (NTS) on the AP and HR responses to stimulation of PSTN. Note that saline injections into the NTS did not alter the magnitude of either the MAP or HR responses. On the other hand, injections into the NTS of CoCl2 significantly (*P < 0.05) attenuated both the MAP and HR responses at about 5–10 min after the NTS injections.

 
Microinjection of 0.9% physiological saline into the similar sites in the mNTS, bilaterally (n = 6; Fig. 6) had no effect on the magnitude of the depressor and bradycardia responses elicited by Glu injections into the PSTN or on resting levels of AP and HR.

Effect of activation of mNTS neurons on the cardiovascular responses to PSTN stimulation. To determine the effect of prior injection of Glu into the mNTS on the magnitude of the cardiovascular responses to stimulation of the PSTN, PSTN was stimulated at 0.5–5.0 min after injections of Glu into the ipsilateral mNTS. As summarized in Fig. 7, stimulation of both the mNTS and PSTN were found to elicit qualitatively similar cardiovascular responses. Stimulation of the PSTN, 0.5–1.0 min after the mNTS injection, elicited MAP responses that were not quantitatively different from either the original PSTN or mNTS responses. On the other hand, the HR responses elicited by stimulation of the PSTN at 0.5–1.0 min after mNTS injections was significantly larger than the original PSTN response, although not different in magnitude from the mNTS response. Both the MAP and HR responses to PSTN stimulation returned to their original pre-mNTS injection levels after about 5.0 min.


Figure 7
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  		Fig. 7. Bar chart summarizing the effect of ipsilateral injections of glutamate (20 nl; 0.25 M) into the NTS on the magnitude of the MAP and HR responses to activation of the PSTN (10 nl, 0.25 M). Note that the algebraic summation of the two independent responses (PSTN and NTS) is greater than the responses to PSTN alone after the injection into the NTS, suggesting that stimulation of PSTN and NTS activate the same neuronal pools. a,b,cAny two bars with the same letter are not significantly different, whereas, any 2 bars with different letters are significantly different (P < 0.05).

 
Effect of PSTN stimulation on the baroreceptor reflex. To investigate whether activation of PSTN neurons by Glu altered the vagal HR component of the baroreflex, changes in systemic blood pressure were used to activate arterial baroreceptors before an injection of Glu into the PSTN. As summarized in Fig. 8, activation of PSTN neurons increased the magnitude of the reflex decrease in HR resulting from the activation of arterial baroreceptors. Glu injection into PSTN potentiated the HR response during activation of the baroreflex between 0.5 and 2.0 min (Fig. 8, B-C) after activation of the baroreflex. The potentiated reflex HR response returned to control values when the baroreflex was activated 15 min after the Glu injection into the PSTN (Fig. 8, B-C). During this same corresponding period of time, the gain of the baroreflex was increased (Fig. 8C) as the magnitude of the systemic AP rise to Phe injections was found to be similar throughout the course of experiment (Fig. 8A), while the magnitude of the reflex HR responses increased (Fig. 8B).


Figure 8
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  		Fig. 8. Summary of effect of PSTN stimulation on the magnitude of the reflex decrease in HR evoked by systemic baroreceptor activation using intravenous injections of phenylephrine (Phe). Note that the magnitude of the HR responses and to baroreceptor activation and the gain of the baroreceptor reflex is significantly potentiated for ~0.5–2 min after PSTN stimulation.*Significantly different (P < 0.05) initial control Phe response.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study has demonstrated that chemical activation of a discrete neuronal cell group in the caudal LHA recently defined as the PSTN (15) elicited decreases in MAP, HR, and RSNA that are mediated by a descending neuronal pathway with an obligatory synapse in the mNTS region. In addition, activation of PSTN neurons was found to facilitate the cardiac component of the baroreceptor reflex.

The MAP responses were found to be due to sympathoinhibition as activation of histologically verified sites throughout the PSTN elicited not only decreases in RSNA, but the MAP response was found to be also abolished following the systemic administration of the ganglionic receptor blocker hexamethonium bromide. On the other hand, the decrease in HR to PSTN stimulation was due to both vagal excitation and sympathoinhibition as the bradycardia response was attenuated after systemic injections of the muscarinic receptor blocker atropine methyl bromide and then abolished with the subsequent administration of the nicotinic receptor blocker hexamethonium bromide. It is unlikely that the cardiovascular responses to activation of the PSTN were secondary to alterations in muscular activity or respiratory changes as they were elicited in paralyzed and artificially ventilated animals.

The finding that Glu stimulation of the PSTN in the {alpha}-chloralose anesthetized rat elicited decreases in MAP, HR, and RSNA is a novel observation as little is known about the function of this nucleus within the caudal LHA. The finding that activation of the caudal lateral hypothalamus elicited cardiovascular responses is consistent with earlier observations in both awake and anesthetized animals showing that Glu, DL-homocysteate, or NMDA injections into the posterior hypothalamic area, which may have included the region of the PSTN, elicited decreases in AP (1, 24, 25) and HR (1). The lack of a HR responses in the studies by Pajolla et al. (24, 25) may be the result of the relatively large injection volumes (50–500 nl) used, thus making it difficult to conclude with any certainty in those studies that the injected chemical activated only neurons within that aspect of the lateral hypothalamus. In fact, as electrical stimulation of the adjacent subthalamic nucleus has been shown to elicit both pressor and tachycardia responses (3, 4), the possibility exists that in the study by Pajolla et al. (24, 25) the injection volumes may have been sufficiently large to have activated these subthalamic neurons, which, in turn, may have overridden bradycardia responses evoked by lateral hypothalamic stimulation. Interestingly, in this study we found a few sites within the subthalamic nucleus that elicited only an increase in MAP in nonparalyzed animals suggesting that these responses may have been mediated by changes in muscular activity.

The finding in this study that blockade of synaptic transmission in the caudal mNTS attenuated the responses to stimulation of the PSTN is supported by the earlier observation that the neurons in the PSTN provide an extensive bilateral innervation of this brain stem cardiovascular area (15). This region of the NTS is known to serve as one of the primary sites of termination of baroreceptor afferent fibers (6). These observations suggest that PSTN neurons not only exert an effect on NTS circuits involved in mediating cardiovascular responses, but also indicate that the mNTS region contains an obligatory synapse in the descending pathway from the PSTN that mediates depressor and bradycardia responses. Furthermore, these data also suggest that the PSTN exerts an effect on neurons within mNTS involved in mediating the cardiovascular responses to activation of arterial baroreceptors (11, 17). In support of this suggestion, the present study has demonstrated that stimulation of PSTN not only elicits cardiovascular responses that are qualitatively similar to those evoked during baroreceptor reflex activation (11, 17), but PSTN also facilitates the reflex bradycardia to increases in systemic AP. Additionally, stimulation of PSTN during and immediately after stimulation of mNTS evoked cardiovascular responses that were significantly smaller than the algebraic summation of the individual responses to stimulation of either structure independently. This latter observation can be interpreted to suggest that the PSTN and the mNTS region shared a common neuronal pool involved in mediating the cardiovascular responses and that the PSTN likely stimulated the same neurons within the mNTS that were activated when Glu was injected into the mNTS. Taken together, these data indicate that the NTS is an important relay for information from the PSTN involved in cardiovascular regulation.

It may be argued that the injections of CoCl2 into the mNTS region exerted an effect on other brain stem sites that may have been involved in mediating the PSTN responses. This suggestion is considered unlikely as injections into the nervous system that ranged from 200 to 300 nl have previously been shown to only diffuse within a radius of ~0.5 mm (2, 13, 27), well within the region of the mNTS (29). It may also be argued that the effect observed after the CoCl2 injections into the mNTS was due to damage of neuronal tissue at the site of injection in the PSTN as a result of a neurotoxic effect resulting from the multiple injections of Glu. However, this possibility is also considered unlikely as repeated injections of Glu into the same PSTN site after the administration of saline into the mNTS did not alter the magnitude of the cardiovascular responses to stimulation of that PSTN site. In addition, the cardiovascular responses to stimulation of the PSTN returned to control levels ~20 min after the administration of CoCl2 into mNTS. Furthermore, repetitive injections of Glu at the same PSTN site was found in this study to elicit cardiovascular responses that were both qualitatively and quantitatively similar, an observation that has been observed in previous studies following multiple Glu injections (8, 13, 16, 27). It has also been previously reported that repeated injections of a low concentration of Glu, as used in this study, do not appear to have any significant neurotoxic affects (14, 19). Therefore, it likely that the CoCl2 disrupted neuronal transmission by altering the synaptic release of neurotransmitter in pathways that mediated the cardiovascular responses to activation of PSTN neurons that terminated in the mNTS, consistent with the observation of direct projections from PSTN neurons to this NTS region (15). CoCl2 injections into brain stem nuclei have previously been shown to disrupt neuronal transmission (2, 13, 27). CoCl2 injections have also been used to block transmission in cardiovascular pathways with a similar time course to that observed in this study (2, 13, 20, 26, 27, 30). This interruption of neuronal transmission with CoCl2 has been shown to be due to a reversible inactivation of calcium channels on the presynaptic terminals (18, 22, 23).

In summary, taken together these data suggest that selective stimulation of the PSTN elicits a cardiac slowing resulting from an activation of the parasympathetic nervous system and inhibition of the sympathetic nervous system, and a decrease in AP and RSNA due to sympathoinhibition. In addition, these responses are mediated in part by activation of neurons within the NTS as injections of a synaptic blocker into the NTS attenuated the MAP and HR responses, while activation of PSTN in conjunction with NTS elicited responses that were less than the algebraic summation of the two individual responses. This latter observation suggests that PSTN and NTS likely share a similar neuronal pool that, when activated, elicits decrease in MAP and HR. Finally, stimulation of the PSTN potentiates the reflex decrease in HR to activation of systemic arterial baroreceptors, suggesting that PSTN is able to modify the baroreceptor reflex response during a blood pressure challenge and likely plays an important role as a hypothalamic preautonomic nucleus in cardiovascular homeostasis.

Perspectives and Significance

The observation that the location of the responsive sites in the PSTN were found in the same region in which neurons express β-preprotachykinin mRNA (15) suggests that these neurons may be involved in mediating the cardiovascular responses to stimulation of the PSTN. However, whether these neurons project to the NTS is not known. Interestingly, the PSTN has been shown to project to a number of forebrain structures that have also been previously implicated in cardiovascular regulation. Activation of neurons in both the bed nucleus of the stria terminalis and central nucleus of the amygdala, forebrain structures that also receive dense projections from the PSTN (15) elicit cardiovascular responses similar to those elicited in this study from the PSTN (8, 13, 27). These observations suggest the possibility that these forebrain areas may also be involved in mediating components of the cardiovascular responses observed in this study from the PSTN, especially as both of these structures have been shown to project to the NTS (9, 12, 21).


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This work was supported by Grant-in-Aid T5847 from the Heart and Stroke Foundation of Ontario.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Ciriello, Dept. of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Univ. of Western Ontario, London, ON, Canada N6A 5C1 (e-mail: john.ciriello{at}schulich.uwo.ca)

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.


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