Inhibition by locus coeruleus on the baroreceptor reflex response in the rat

Neuroscience Letters, 144 (1992) 225-228 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Inhibition by locus coeruleus on the baroreceptor reflex response in the rat Julie Y.H. Chan ax, Shiu-Fang Jang b and Samuel H.H. Chan b aInstitute of Neuroscience and blnstitute of Pharmacology, National Yang-Ming Medical College, and 'Department of Medical Research, Veterans General Hospital-Taipei, Taipei (Taiwan, ROC) (Received 12 May 1992; Revised version received 26 June 1992; Accepted 26 June 1992)

Key words: Locus coeruleus; Baroreceptor reflex; Nucleus tractus solitarii; Noradrenergic neurotransmission; ~-Adrenoceptor; Rat We evaluated the modulation of baroreceptor reflex (BRR) response by locus coeruleus (LC) in adult, male Sprague-Dawley rats anesthetized with urethane (1.5 g/kg, i.p.). Under an electrical stimulation condition that did not appreciably alter the basal systemic arterial pressure and heart rate, the LC significantly suppressed the BRR response. Microinjection of L-glutamate (1 nmol, 50 nl) into the LC essentially duplicated this depressant effect. Intracerebroventricular (i.c.v.) administration of the ~-adrenoceptor antagonist, prazosin (6.5 nmol), appreciably blunted the inhibition by LC on the BRR response. Yohimbine (6.5 nmol), the ~2-adrenoceptor blocker, however, was ineffective. Direct microinjection of prazosin (50 pmol), but not yohimbine (50 pmol), into the terminal site of baroreceptor afferents at the nucleus tractus solitarii (NTS) also significantly blunted the suppressive effect of LC on the BRR response. These results suggest that the LC may produce an inhibition on the BRR response by a process that involves the ~-adrenoceptors located in the NTS.

The locus coeruleus (LC), a pontine nucleus with high density of norepinephrine-containing neurons [5], is evident to play an important role in central neural control of cardiovascular functions. Anatomic studies [11] demonstrated the distribution of axons from the LC to many brainstem areas that are involved in circulatory regulation, including dorsal motor nucleus of the vagus nerve and the nucleus tractus solitarii. When electrically [17] or chemically [18] activated, LC elicits changes in arterial pressure and heart rate. Biochemically, the turnover of catechol [2, 19] and indole [2] neurotransmitters in the LC is affected by changes in arterial blood pressure. Pathophysiologically, several forms of experimental hypertension exhibit alterations in the morphology and activity of LC neurons [7, 15]. Baroreceptor reflex (BRR) is one of the fundamental mechanisms by which the central nervous system regulates the peripheral cardiovascular performance [16]. Apart from operating directly via the BRR, central neural control of circulation may also function indirectly through modulation of this fundamental feedback loop by other neural substrate. Thus, modulation of the BRR response by the hypothalamus [14] or nucleus reticularis gigantocellularis of the medulla [3] has been reported. Corre.q~omh,m'e.-S.H.H. Chan, Institute of Pharmacology. National Yang-Ming Medical College. Taipci 11221, Taiwan. Republic of China, Fax: 886-2-8264372.

Despite our knowledge regarding its participation in many facets of cardiovascular control, the role played by the LC and its noradrenergic efferents in the modulation of BRR response is still not forthcoming. The present study demonstrated that the LC may exert an inhibitory modulation on the BRR response, in a process that is likely to involve the cq-adrenoceptors located in the nucleus tractus solitarii (NTS), the terminal site of the baroreceptor afferents [4]. Experiments were carried out in adult, male SpragueDawley rats (250-300 g) anesthetized with urethane (1.5 g/kg, i.p.). The right femoral artery and vein were routinely cannulated to measure arterial pressure and administer drugs. The arterial pressure was monitored via a pressure transducer (Statham P231D), and heart rate was determined using a biotachometer (Gould 20-461565) triggered by the arterial pressure pulses. Pulsatile and mean systemic arterial pressure, as well as heart rate, were recorded continuously on a polygraph (Gould RS 3600). The trachea was intubated to maintain its patency and to facilitate artificial ventilation using a rodent respirator (Harvard 683). Following the completion of surgery, the head of the rat was placed in a stereotaxic headholder (Kopf), with the rest of the body placed on a heating pad and elevated to a suitable position. All data were collected from animals with a maintained rectal temperature of 37°C and a steady systemic arterial pressure throughout the experiment.

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Electrical activation of the LC was delivered by a stainless-steel bipolar concentric electrode (Rhodes Medical SNE- 100, 100 Hm tip diameter). The stereotaxic coordinates used were: 1.0 1.2 mm caudal to lambda, 1.1 1.3 mm lateral to the midline and 5.0-6.0 mm below the cortical surface. The LC was electrically activated by a 10-s train of 1-ms rectangular pulses, at 10--60 HA and 10 20 Hz, using a Grass $88 stimulator equipped with a constant-current isolation unit (Grass PSIU6). A locus in the LC capable of increasing the mean systemic arterial pressure by l0 mmHg upon electrical activation [17] was first identified. The stimulus parameters, chiefly intensity, were subsequently adjusted to a condition at which LC stimulation elicited only minimal cardiovascular effects. Similar to our previous studies [12, 13], arterial baroreceptors were stimulated by a transient hypertension induced by a bolus injection of phenylephrine (5 /lg/kg, i.v.). The quotient that represents the unit reflex decrease in heart rate per unit increase in mean systemic arterial pressure (beats/min per mmHg) was used as the index for the BRR response [12, 13]. Modulation of the BRR response by the LC was assessed by evaluating their interactions when this dorsal pontine nucleus was electrically activated simultaneously with the induction of the reflex. Individual quotients were further normalized to a percentage of prestimulation control to compensate for variations between animals. The LC was activated, in some experiments, by L-glutamate to stimulate the neuronal perikarya [10]. This was achieved via a glass micropipette (tip diameter: 50-80 Hm), which was connected to a nanoliter infusion pump (WPI A1400). A total volume of 50 nl was delivered unilaterally into the LC over at least 1 min to allow tbr complete diffusion of the solution. The effect on the BRR response was evaluated immediately following application of L-glutamate into the LC. Possible volume effect of microinjection was controlled by injecting the same amount of aCSF. Equimolar concentration of 0t-adrenoceptor antagonists, prazosin (Pfizer) or yohimbine (Lilly), was used to investigate the participation of ~-adrenoceptors in the LC-mediated inhibition of BRR response, lntracerebroventricular (i.c.v,) administration of the chemicals was carried out as described in our previous studies [12, 13]. A total volume of 2.5 H1 was delivered over at least 1 min to allow for full diffusion of the solution. Direct, bilateral microinjection of 0t-adrenoceptor antagonists into the NTS, at a volume of 20 nl, was carried out as described for application of L-glutamate into the LC. The coordinates used were: -0.80 to +0.50 mm from and 0.35 to 0.50 mm lateral to the obex, and 0.35-0.80 mm below the surface of the brainstem. The effect of each pretreat-

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Fig. 1. Representative tracing showing the effect of activating the locus coeruleus (LC) with electrical stimulation (Bar: 10-s train of 1-ms rectangular pulses at 20 HA and 10 Hz) (upper panels) or microinjection of t,-glutamate (L-GLU: 1 nmol, 50 nl) (lower panels) on the baroreceptor reflex (BRR) response elicited by phenylephrine (PE, 5/tg/kg, i.v.). HR, heart rate; SAP and MSAE pulsatile and mean systemic arterial pressure.

ment on the BRR response or the LC-modulated BRR response was evaluated immediately following its application. At the conclusion of each experiment, the brain was removed and fixed in 30% sucrose in 10% formaldehydesaline. Thirty-Hm frozen sections stained either with Neutral red or Cresyl violet were used to verify the location of stimulation and i.c.v, or microinjection sites. Evans blue 1% was added to the injection medium to aid in such verifications. The results were statistically assessed using the analysis of variance (ANOVA), followed by the Student-Newman-Keuls test for a posteriori comparison of means as appropriate. All results were considered statistically significant at P < 0.05. Under an electrical stimulation condition that produced no discernible effect on basal systemic arterial pressure and heart rate, the LC significantly suppressed the BRR response induced by an intravenous injection of phenylephrine (Fig. 1, upper panels). We estimated that the degree of this inhibition amounted to 47.3 + 3.4% (mean + S.E.M., n = 30). Such a suppressive effect of LC on the BRR response was due mainly to the activation of neuronal perikarya, but not axons of passage, because

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Fig. 2. Effect of i.c.v, pretreatment with equimolar concentration (6.5 nmol) of prazosin (A) or yohimbine (B) on the baroreceptor reflex response (PRA-BRR or YOH-BRR) or locus coeruleus (LC)-induced BRR suppression (PRA-LC-BRR or YOH-LC-BRR). The control BRR and its inhibition by electrical activation of the LC (LC-BRR) were included for comparison. Values are mean _+ S.E.M., n = 6 animals per group. *P < 0.05 vs. BRR response in the Student NewmanKeuls test.

Fig. 3. Effect of microinjection of equimolar concentration (50 pmol) of prazosin (A) or yohimbine (B) on the baroreceptor reflex response (PRA-BRR or YOH-BRR) or locus coeruleus (LC)-induced BRR suppression (PRA-LC-BRR or YOH-LC-BRR). The control BRR and its inhibition by electrical activation of the LC (LC-BRR) were included for comparison. Values are mean _+S.E.M., n = 6 animals per group. *P < 0.05 vs. BRR response in the Student-Newman-Keuls test.

direct microinjection of L-glutamate (1 nmol) into the LC also inhibited the BRR response (Fig. l, lower panels). In contrast, direct application of aCSF into the LC, or chemical activation of sites adjacent to the LC (e.g. lateral dorsal tegmental nucleus, mesencephalic nucleus of the trigeminal nerve), had no discernible effect. I.c.v. administration, at an equimolar concentration (6.5 nmol), of prazosin (Fig. 2A) or yohimbine (Fig. 2B), did not by itself appreciably alter the BRR response. Nonetheless, the specific cfl-adrenoceptor antagonist, prazosin, significantly blunted the inhibition of BRR response by LC (Fig. 2A). On the other hand, the selective ~2-adrenoceptor blocker, yohimbine, was relatively ineffective (Fig. 2B). Similar to the results obtained from i.c.v, pretreatments, bilateral NTS application of equimolar concentration (50 pmol) of prazosin or yohimbine elicited no significant effect on the BRR response (Fig. 3). In addition, prazosin also reversed the inhibitory action of LC on the BRR response, whereas yohimbine produced minimal effect (Fig. 3). Histologically, the distribution of

verified injection sites concentrated primarily at the caudal one-third level of the NTS, where most of the baroreceptor afferents terminate [4]. The present study demonstrated that activation of neuronal perikarya in the LC produced an inhibitory effect on the BRR response. This suppressive action was exerted under a condition in which LC did not significantly affect the basal arterial pressure and heart rate. Thus, apart from the general notion that activation of the LC alters basal hemodynamics [17, 18], our data suggest that this pontine nucleus may additionally modulate the BRR. More importantly, it is possible that the latter mode of action has a lower threshold of activation. Both ~l- and ~2-adrenoceptor binding sites are present in the NTS [6, 20]. Our results demonstrated that at least the former receptors may participate actively in the LCinduced BRR suppression. However, it is not likely that this suppressive action is exerted tonically, since pretreatment with prazosin did not significantly alter the basal reflex response. The lack of effect by yohimbine implies that the ct2-adrenoceptors at the NTS may not

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play a critical role, either phasically upon LC activation or tonically, in the modulation of the BRR response. Whether the LC exerts a direct, noradrenergic action on the ~l-adrenoceptors in the NTS to elicit inhibition on the BRR response is not immediately clear. Earlier studies [11] suggest the existence of a direct innervation from the LC to the NTS. Preliminary results from our laboratory demonstrated that neurons, albeit relatively few, in the LC with positive dopamine-fl-hydroxylase immunoreactivity were simultaneously labeled with retrogradely transported horseradish peroxidase, upon being microinjected into the caudal level of the NTS. Although the characteristic divergent axonal outflows from the LC [8] may offer a partial answer, the mechanism via which significant inhibition on the BRR response was achieved by relatively few LC-noradrenergic neurons that impinge directly on the NTS require further elucidation. It was recently shown [9] that the LC supplies noradrenergic axons primarily to sensory and association nuclei of the brain stem. On the other hand, bulbar motor nuclei and autonomic areas receive their noradrenergic innervations from non-LC neurons. Thus, the link between the LC and the NTS may be indirect, and the hypothalamus may serve as a possible intermediate. Baroreceptor-induced depression of supraoptic neurons is mediated by a direct noradrenergic input from the LC [1]. The supraoptic nucleus and paraventricular nucleus, in turn, are at least partly responsible for the suppression of the BRR [14]. Nonetheless, the participation of a hypothalamic noradrenergic pathway in the LC-promoted BRR suppression at the NTS must await further investigation. Supported in part by Research Grant NSC-81-0412B075-04 to J.Y.H.C. from the National Science Council, Taiwan, Republic of China. 1 Banks, D. and Harris, M.C., Lesions of the locus coeruleus abolish baroreceptor-induced depression of supraoptic neurones in the rat, J. Physiol., 355 (1984) 383 398. 2 Bhaskaran, D. and Freed, C.R., Changes in neurotransmitter turnover in locus coeruleus produced by changes in arterial blood pressure, Brain Res. Bull., 21 (1988) 191 199. 3 Chan, S.H.H., Kuo, J.S., Chen, Y.H. and Hwa, J.Y., Modulatory actions of the gigantocellular reticular nucleus on baroreceptor reflexes in the cat, Brain Res., 196 (1980) 1-9.

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