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NEUROHUMORAL CONTROL OF CIRCULATION AND HYPERTENSION
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Submitted 10 January 2003 ; accepted in final form 24 February 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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raphe nuclei; conscious rat; heart rate; blood pressure; body temperature
Accompanying the thermoregulatory changes associated with blockade of GABAA receptors in the RP in some but not all of these studies were increases in heart rate and blood pressure. Our recent findings in conscious rats confirmed that microinjection of BMI at this site elevates heart rate (18). In the same study, microinjection of the GABAA receptor agonist and neuronal inhibitor muscimol at the same sites was found to have little effect on heart rate and blood pressure under baseline conditions but to markedly reduce the tachycardia seen in experimental air jet stress. Thus neurons in the same region of the RP might contribute to the increases in sympathetic outflow to specific target organs or systems seen in specific circumstances unrelated to regulation of body temperature. Like those concerned with thermoregulation, these neurons also appear to be under tonic inhibition mediated through GABAA receptors.
On the basis of the above findings, we theorized that tonic activity of neurons in the region of the RP plays a key role in the maintenance of normothermia at room temperature in conscious rats but that neuronal activity in this region contributes little if at all to basal cardiovascular homeostasis. Thus the purpose of this study was to characterize the effect of microinjection of BMI and muscimol into the same region of the RP with respect to core body temperature, heart rate, and blood pressure in conscious freely moving rats under basal laboratory conditions.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Surgical preparation. Rats were anesthetized (80 mg/kg ketamine and 11.5 mg/kg xylazine ip, supplemented as required), and the flexible catheter of a telemetric transmitter (Data Sciences) was inserted into the abdominal aorta through the right femoral artery. The body of the transmitter was placed into the abdominal cavity and sutured to the abdominal wall.
Five days after transmitter implantation, rats were again anesthetized and placed in a stereotaxic apparatus with the incisor bar set 3.3 mm below the interaural line for placement of a microinjection guide cannula into the RP. The skin overlying the dorsal surface of the skull was cut and retracted, and soft tissue was removed from the exposed surface. In eight rats, the guide cannula (26 gauge, Plastics One, Roanoke, VA) was then positioned to target the region of the RP studied previously (coordinates: anterior-posterior -2.8 mm; left-right 0.0 mm; height-depth -1.1 mm; interaural line as reference point) and secured by three stainless steel screws, Vetbond glue, and cranioplastic cement. Dummy wire cannulas were inserted in the guides, and rats were returned to their home cages for recovery. In experiments intended to serve as controls for neuroanatomic specificity, guide cannulas were intentionally placed lateral, anterior, or posterior to the targeted region of the RP. At least 5 days were allowed for recovery.
Experimental procedures. Assessment of placement of the guide cannula relative to the active region of the medullary raphe was then assessed in each rat by noting the cardiovascular response to microinjecting the GABAA receptor antagonist bicuculline methiodide (BMI, 40 pmol/100 nl) in the conscious freely moving subject under baseline conditions. These and all other microinjections were performed in a laboratory where ambient temperature was maintained at 24–25°C as follows. On the day of the experiment, rats were placed in their home cage on the telemetry receiver plate. The dummy cannula was then removed, the microinjector (33 gauge, Plastics One) connected to a 10-µl Hamilton syringe with Teflon tubing (ID 0.12 mm; OD 0.65 mm; Bioanalytic Systems, West Lafayette, IN) was inserted into the guide cannula, and the rat was left undisturbed for an additional 30–40 min. The Hamilton syringe was mounted in an infusion pump (Sage, Boston, MA) that was used to deliver a volume of 100 nl of injectate over 30 s. After the injection of BMI, the injector was left in place for at least 20 min while the animal remained undisturbed in its cage. The microinjection was considered successful if, immediately after removal of the microinjector, flow appeared within 5 s after the pump was reactivated, indicating that the injector was not clogged and that injectate had been successfully delivered.
All rats were then subjected to trials in which the effect of microinjection of muscimol (80 pmol/100 nl) and saline vehicle (100 nl) on body temperature, heart rate, and blood pressure was assessed in random order at 2- to 3-day intervals. In four of the rats in which guide cannula placements were determined to be in the active region of the RP (based on the tachycardia in response to microinjection of BMI), responses to additional microinjections were examined. In three of these rats, two additional doses of muscimol (10 and 20 pmol) were also tested, again at 2- to 3-day intervals in random order. In the remaining rat, the effect of three successive microinjections of muscimol (80 pmol/100 nl) at 30-min intervals was studied.
Histology. After the last session, rats were injected with pentobarbital (100 mg/kg), and microinjection sites were marked with 100 nl of 2.5% Alcian Blue dye. Brains were immediately perfused in situ with 30 ml of cold saline (4°C) followed by 30 ml of 4% paraformaldehyde. Brains were then removed and postfixed in 4% paraformaldehyde at least overnight and then saturated with 30% sucrose and cut in 40-µm coronal sections on a freezing microtome. Mounted sections were stained with 1% neutral red. Injection sites were determined according to the atlas of Paxinos and Watson (11) by a blind observer.
Data analysis. A Dataquest telemetry system (Data Sciences) was used for measurement of core body temperature as well as arterial pressure and heart rate.
Results are expressed as means ± SE. Comparisons were made between groups with repeated-measures ANOVA where appropriate. Newman-Keuls was employed for post hoc analysis. Limits of probability considered significant were 5% or less.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Effects of BMI. Microinjection of BMI into the RP in conscious rats resulted in marked increases in heart rate as has been reported for anesthetized animals (Fig. 2). Increases of at least 15 beats/min were detectable within 1 min of injection in every experiment and reached mean maximum increase (change from baseline heart rate just before injection) of +94 ± 13 beats/min (range = 42–130 beats/min) at 6.9 ± 0.8 min after microinjection. Subsequent injection of saline vehicle into the RP (see below) or similar injection of BMI at sites outside the RP (Fig. 2) failed to alter heart rate. Increases in heart rate evoked from the RP were sometimes accompanied by pressor responses (maximum change from baseline = 2–36 mmHg; mean = +20 ± 4 mmHg) that occurred within the first 10 min after injection (mean = 3.7 ± 0.7 min). Owing to the variability of these changes, blood pressure after injection of BMI into the RP was not significantly different from that seen after similar microinjections at surrounding medullary sites (Fig. 2).
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In contrast to the marked effects on heart rate, microinjection of BMI into the RP had little or no effect on body temperature. Twenty minutes after microinjection of BMI, body temperature was not significantly different from either baseline body temperature 5–15 min before injection or from mean body temperature 20 min after injection of BMI at other sites (repeated measures ANOVA, P > 0.05; Fig. 2). However, the ability to detect an effect of blockade of GABAA receptors in the RP on body temperature may have been confounded somewhat by a progressive fall in the parameter that occurred in this group during the baseline period (Fig. 2). As a result, mean body temperature in rats with injection sites in the RP was significantly lower than in rats with injection sites outside this region from 11 min before injection of BMI to 7 min after injection. However, from this lowered baseline core temperature (i.e., temperature just before microinjection), modest increases ranging from 0.16 to 0.34°C (mean = +0.26 ± 0.03°C at 15.7 ± 0.8 min; n = 7) occurred within 20 min after injection of BMI into the RP. In contrast, subsequent microinjection of saline into the RP in these same animals (see below) was followed by maximal increases of only 0.04–0.15°C within 20 min (mean = +0.08 ± 0.02°C at 8.7 ± 1.7 min; n = 7), and injection of BMI at sites outside the RP was followed by small (0.05–0.06°C) increases in temperature above preinjection baseline in only two of five rats tested. Thus microinjection of BMI (40 pmol) into the RP in conscious rats evoked marked changes in heart rate but had little if any effect to increase body temperature.
Effects of muscimol. In contrast to the failure of microinjection of BMI into the RP to evoke a detectable effect on temperature, injection of muscimol (80 pmol) at the same sites was followed by sharp declines (Fig. 3). Decreases in core temperature were seen in every experiment with maximum declines from preinjection baselines ranging from -0.74 to -2.89°C (mean = -2.22 ± 0.25°C) at 60–75 min after injection (mean = +73.9 ± 5.5 min). Increase toward original baseline body temperature was noted by the end of the 90-min observation period in each rat; however, animals were not followed to full recovery. The mean maximal decreases from baseline body temperature observed after injection either of saline vehicle into the RP or of muscimol (80 pmol) into other medullary sites around the RP were -0.41 ± 0.07 and -0.81 ± 0.29°C, respectively. The trend for injections outside the RP to decrease body temperature was a consequence of two experiments in which microinjections of muscimol lowered body temperature by >1°C. In one experiment, injection of muscimol lateral to the RP (site shown in Fig. 1C) lowered body temperature by a maximum of 1.62°Cat +73 min. In another, injection of muscimol at a site in the RP anterior to the most active region (site shown in Fig. 1A) reduced body temperature by 1.34°C at +53 min. After injection of muscimol into the RP, heart rate and blood pressure were no different from that seen after injection of saline vehicle (Fig. 3). Thus injection of muscimol (80 pmol) into the RP markedly lowered body temperature without significantly altering heart rate or arterial pressure.
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The effect of microinjection of 10 and 20 pmol of muscimol into the RP on body temperature was also examined in three of the rats discussed above and compared with the response to injection of muscimol (80 pmol) and saline vehicle in these same animals (Fig. 4). Regression analysis of the data revealed that maximal changes from baseline temperature were significantly correlated with the dose of muscimol injected (r = 0.764; P = 0.004). Thus the effect of microinjection of muscimol into the RP on body temperature was clearly dose related.
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In a single exploratory experiment in another rat from the RP group, we examined the response to three injections of muscimol (80 pmol) repeated at 30-min intervals in the same session and compared this response to the effect of a single injection in this animal. While temperature fell by a maximum of
2.89°C at 
77 min after a single injection of muscimol in this rat,
repeated injections resulted in a profound fall by a maximum of 4.77°C at
102 min after the initial injection.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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These findings extend previous results that pointed to a role for increased neuronal activity in this region in thermoregulatory activity under extreme or pathophysiological conditions. Increased Fos expression in the RP has been reported after exposure to a cold environment (7, 8) and in experimental fever (9). Microinjection of muscimol into the RP in anesthetized rats prevented the increase in core temperature and temperature of interscapular brown fat caused by central injection of prostaglandin E2 into the preoptic area (9). Because the generation of prostaglandin E2 in this brain region is thought to play an obligatory role in fever (13), this observation supports a role for activity of neurons in the RP in the increased body temperature that characterizes this pathophysiological state. Microinjection of BMI into the RP prevented the increase in surface temperature of the tail caused by local heating of the preoptic area to 42°C (16). However, these experiments were performed in anesthetized rats in which basal body temperature had to be artificially supported to maintain basal physiological levels. Thus no conclusions could be drawn regarding the role of the region in normal thermoregulation from these results. Perfusion of the region of the RP with tetrodotoxin using microdialysis was reported to markedly lower body temperature in conscious rats at normal ambient temperature (23°C) (5). Because tetrodotoxin inhibits neuronal activity by blocking most fast sodium channels, its effect in this study might have resulted from blockade of propagation of action potentials in axons passing through the RP rather than from inhibition of neuronal somata in the region. Furthermore, no data were provided that would establish the anatomic specificity of the effect noted. Our results are the first to establish a key role for neurons located in a specific region of the RP in maintenance of normal body temperature in conscious rats under standard laboratory conditions. The present findings indicate that neurons in the RP whose activation has been shown previously to increase the production of heat by metabolic activation of brown fat and/or reduce the loss of body heat by vasoconstriction in the tail are likely to contribute to physiological thermoregulatory activity.
While stimulation of GABAA receptors in the RP clearly lowered body temperature, an opposite effect of blocking GABAA receptors by local microinjection of BMI at the same sites was not as readily apparent in this study. Morrison and colleagues (8) reported that microinjection of BMI into the RP evoked marked increases in the activity of sympathetic nerves innervating interscapular brown fat in anesthetized rats. In the present study, maximal increases from baseline body temperature were significantly greater in the 20 min after injection of BMI into the RP than after either similar microinjection of BMI outside the RP or of saline at the same sites in the RP. However, this effect was relatively small compared with the decrease in body temperature caused by microinjection of muscimol at the same site and was further confounded by the fact that body temperature had fallen by the same small increment during the baseline observation period just before the microinjection of BMI (see Fig. 2). Thus 20 min after microinjection of BMI, mean body temperature was not significantly different from levels seen 5–15 min before injection. One factor that may have contributed to the difference in the effects of BMI vs. muscimol with respect to body temperature may relate to the pharmacokinetic profile of these agents when given by local microinjection at these doses. Microinjection of BMI into the RP in this and other studies (12, 18) or into the dorsomedial hypothalamus (DMH) (1, 4, 15, 17) produces maximal effects on heart rate within 10 min and on core body temperature within 20 min. However, after injection of muscimol into the RP, body temperature continued to fall well beyond these times with nadirs occurring at least 1 h after injection. Thus the greater magnitude of the effect of microinjection of muscimol relative to that of BMI probably reflects at least in part its more prolonged duration of local action. This notion is supported by one experiment in which we attempted to extend muscimol-induced inhibition of local neuronal activity even further by repetitive microinjections of this agent. The result was a continued fall in body temperature to sharply lower levels.
Another factor that may have interacted with the contrast in the duration of local action of BMI and muscimol to contribute to the difference in the magnitude of the effects of the two drugs on body temperature relates to the nature of the systems involved. Microinjection of BMI into the DMH in anesthetized rats markedly and rapidly increased both heart rate and temperature of interscapular brown fat but elevated core body temperature only modestly and with a longer latency to maximal effect (17). Microinjection of BMI into the RP in rats increased heart rate within seconds in the present study and has been reported previously to increase sympathetic nerve activity to brown fat and to decrease tail blood flow with a similarly rapid time course (3). Thus while effects of microinjected BMI on brown fat or cutaneous vasoconstriction may be immediate, consequent increases in body temperature may appear only slowly and thus may never achieve prominence in the absence of a sustained action of the drug.
In contrast to effects on body temperature, stimulation of GABAA receptors in the region of the RP in conscious rats has little effect on baseline heart rate while blockade of GABAA receptors at the same site elicits marked tachycardia. Our findings are in agreement with the results of previous reports that microinjection of BMI into the RP increases basal heart rate in anesthetized rats (3, 8, 12). In contrast, Tanaka and colleagues (16) found that while microinjection of either BMI or of the excitatory amino acid D,L-homocysteate into the RP profoundly affected cutaneous vasoconstrictor activity to the tail, neither treatment elevated heart rate. However, baseline heart rates in their study were often in excess of 500 beats/min, which likely occluded the tachycardia that was seen here in conscious animals and in other studies in anesthetized rats where basal heart rates were lower.
While microinjection of BMI into the RP elicited marked tachycardia, microinjection of muscimol, an effective inhibitor of practically all adult mammalian neurons (6), into the same region had little effect on basal heart rate, confirming previous results in both conscious and anesthetized animals (12, 18). We have also shown that identical treatment with this agent virtually abolishes the dramatic increases in heart rate associated with experimental air stress (18). Therefore, although activity of thermoregulatory neurons in the RP plays a key role in the maintenance of basal body temperature, activity of cardiac sympathetic premotor neurons found in the same region does not contribute significantly to resting heart rate under similar conditions. Instead, these latter neurons are recruited in stress and mediate the tachycardia seen in this circumstance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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, Injection sites within 500 µm of the
targeted region of the raphe pallidus (RP; n = 8); 
, injection
sites judged to be at least 700 µm outside this region at other sites in
the medulla (n = 5). Numbers indicate distance from bregma for each
section.
