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Participation of endogenous neuropeptide Y in the suppression of baroreceptor reflex response by locus coeruleus in the rat
Regulatory Peptides, 48 (1993) 293-300
293
© 1993 Elsevier SciencePublishers B.V. All rights reserved 0167-0115/93/$06.00 REGPEP 01534
Participation of endogenous neuropeptide Y in the suppression of baroreceptor reflex response by locus coeruleus in the rat Julie Y . H . C h a n a, C h e n g - D e a n Shih b and Samuel H . H . C h a n b Department of Medical Research, Veterans General Hospital- Taipei and bInslitllte of Pharmacology, National Yang-Ming Medical College. Taipei (Taiwan. R OC)
(Received 18 February 1993; revised version received 11 May 1993; accepted 14 May 1993) K e y words: Endogenous neuropeptide Y; Locus coeruleus; Baroreceptor reflex response; ~l-Adrenoceptor;
Nucleus tractus solitarii; Rat
Summary We evaluated the potential participation of endogenous brain neuropeptide Y (NPY) in the suppression of baroreceptor reflex (BRR) response by locus coeruleus (LC), using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Bilateral microinjection of an antiserum against NPY (1:20, 20 nl) into the caudal one-third level of the nucleus tractus solitarii (NTS), the terminal site for baroreceptor afferent fibers, significantly reversed the suppressive effect of electrical or chemical activation of the LC on the BRR response. Treatments with NPY (4.65 pmol, 20 nl), normal rabbit serum, aCSF and heat-inactivated NPY or NPY antiserum, on the other hand, were ineffective. The LC-promoted inhibition of the BRR response was also attenuated by the ~l-adrenoceptor antagonist prazosin (50 pmol, 20 nl), either microinjected alone or in combination with NPY antiserum into the bilateral NTS. Mathematical treatment of our data revealed that the depressive effect on the BRR response of NPY or N E released at the NTS following LC activation manifested different time-course and magnitude. The one by endogenous NPY maximized at 40 rain and amounted to no more than 20?0 of, whereas that by NE peaked at 10 min and contributed no less than 30?o to, the suppression. These results suggest that both endogenous NPY and N E may participate in the suppression of BRR response by the LC at the NTS.
Introduction Correspondence to: J.Y.H. Chan, Department of Medical Re-
search, Veterans General Hospital-Taipei,Taipei 11217,Taiwan, ROC.
Our laboratory recently reported [ 1 ] that the locus coeruleus (LC) of the pons exerts an inhibitory mod-
294 ulation on the baroreceptor reflex (BRR) control mechanism. This modulatory effect involves the ~ladrenoceptors located at the nucleus tractus solitarii (NTS), the terminal site for baroreceptor afferent fibers [2,3]. We also showed [4] that the endogenous neuropeptide Y (NPY) in the brain exerts a tonic suppressive effect on the BRR response at the NTS. Apart from its high density of norepinephrine (NE)-containing neurons [5], a substantial number of neuropeptides, including NPY, has been visualized in the rat LC [6-9]. Some of the NE-containing neurons in the LC also show positive immunoreactivity to NPY [6,9]. Neuroanatomically, a high percentage of the NPY-containing perikarya in the LC functions as projecting neurons to various brain regions [8,9]. The physiological significance of such efferent projections of NPY-containing neurons in the LC, however, has not been fully defined. Two interesting corollaries arise from the foregoing observations, and formed the working hypothesis for the present study. First, it is possible that NPY-containing neurons that exert a tonic inhibitory effect on the BRR response at the NTS may arise from the LC. Second, it is likely that activation of the LC may result in the release of at least N E and NPY at the NTS, both of which may influence the BRR response. We demonstrated that activation of the LC may indeed elicit a NPY-related suppression of the BRR response. Furthermore, this suppression exhibited a time-course and magnitude that was different from the inhibition induced by the LC-originated noradrenergic neurotransmission. Materials and Methods
A nimal preparation Experiments were carried out in adult, male Sprague-Dawley rats (250-300 g) anesthetized with pentobarbital sodium (40 mg/kg, i.p., with 10 mg/ kg/h supplements). The right femoral artery and vein were routinely cannulated to measure systemic arterial pressure and administer drugs. Arterial pressure was monitored via a pressure transducer (Statham
P231D), and heart rate was determined using a biotachometer (Gould 20-4615-65) triggered by the arterial pressure pulses. 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 animal was placed in a stereotaxic headholder (Kopf), with the rest of the body placed on a heating pad and elevated to a suitable position. Pulsatile and mean systemic arterial pressure, as well as heart rate, were recorded continuously on a polygraph (Gould RS 3600). All data were collected from animals with a maintained rectal temperature of 37°C and a steady systemic arterial pressure throughout the experiment. Electrical and chemical activation of the locus coeruleus Electrical activation of the LC was delivered by a stainless-steel bipolar concentric electrode (Rhodes Medical SNE-100, tip diameter: 100 #m). The stereotaxic coordinates used were: 1.0-1.2 mm caudal to the lambda, 1.1-1.3 mm lateral to the midline and 5.0-6.0 m m below the cortical surface. The LC was electrically activated by a 10-s train of 1-ms rectangular pulses, at 10-60 /~A 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 10 m m H g upon electrical activation [ 10] was first identified. The stimulus parameters, chiefly intensity, were subsequently adjusted to a condition at which LC stimulation elicited only minimal cardiovascular effects. Neuronal perikarya in the LC were activated, in some experiments, by L-glutamate (1 nmol) stimulation [ 11 ]. This was achieved via a glass micropipette (tip diameter: 50-80/~m), 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 for complete diffusion of the solution. Possible volume effect of microinjection was controlled by injecting the same amount of artificial cerebrospinal fluid (aCSF).
295 Evaluation of the baroreceptor reflex response Similar to the evaluation used in our previous studies [ 12,13 ], the sensitivity of B RR response was evaluated using a modification of the method by Smythe et al. [14]. In essence, we employed the slope of the linear portion of the regression line that relates maximal reflex bradycardia to transient hypertension elicited by three doses of phenylephrine (2.5, 5.0 or 10.0/ag/kg, i.v.) to represent the sensitivity of BRR. The order of different doses of phenylephrine was altered randomly to avoid sequential dependency. Modulation of the BRR response by the LC was assessed by evaluating their interactions. For this purpose, the dorsal pontine nucleus was electrically stimulated simultaneously with the induction of the reflex. In experiments that employed chemical activation of the LC, the sensitivity of BRR response was assessed within 10 rain following microinjection of L-glutamate. Microinjection of chemicals into the bilateral nucleus tractus solitarii Direct, bilateral microinjection of chemicals into the NTS was carried out sequentially with a stereotaxically positioned ( -0.5 to + 0.5 mm from the obex, 0.35 to 0.50 mm lateral to the midline, and 0.35 to 0.90 mm from the surface of the fourth ventricle) glass micropipette (tip diameter: 50-80 /~m), connected to a nanoliter infusion pump. A total volume of 20 nl was delivered into the NTS on each side, with aCSF serving as control. The temporal effect of chemical treatments on the BRR response or the LC-mediated suppression of the BRR response was evaluated before and at 0-10, 15-25, 30-40 or 45-55 min after microinjecting the chemicals into the bilateral NTS. Chemicals used included rat NPY (4.65 pmol, Peninsula Lab.), rabbit NPY antiserum (1:20, Incstar), normal rabbit serum (Jackson Lab.), or heat-inactivated (60 + 5°C for 15-20 min) NPY and NPY antiserum. The NPY antiserum showed no significant cross-reactivity with peptides having similar
molecular structures. All stock solutions were stored frozen, and thawed immediately before use. Histology At the conclusion of each experiment, the brain was removed and fixed in 30~o sucrose in 10~o formaldehyde-saline. 25-/~m frozen sections stained either with Neutral red or Cresyl violet were used to verify the location of stimulation or microinjection sites. 1 ~o Evans blue was added to the injection medium to aid in such verifications. Statistical analysis The effect of chemical treatments on the sensitivity of BRR response or the LC-promoted suppression of the BRR response was statistically assessed using two-way analysis of variance (ANOVA) with repeated measures. This was followed by the Scheffe multiple range test for a posteriori comparison of means at corresponding time intervals. All results were considered statistically significant at P < 0.05. Results
Effect of activation of LC on the B R R response In agreement with our previous findings [ 1 ], electrical activation of the LC (Fig. 1A)reduced the sensitivity of the BRR response (slope: 0.96 to 0.61). We also ascertained with chemical stimulation that such an inhibition was due mainly to activation of the LC perikarya, but not fibers of passage. L-Glutamate (1 nmol) microinjected into the LC similarly depressed the BRR response (Fig. 1B), evaluated within 10 min following application of the excitatory amino acid, by reducing the slope from 0.94 to 0.62. The overall suppression of the BRR response by LC activation amounted to 43.6 + 6.5 ~o (mean + S.E.M., n = 10) (Fig. 2). Effect of N P Y or N P Y antiserum on the LC-induced suppression of B R R response The LC-induced inhibition of BRR response was significantly antagonized in animals pretreated with
296 INCREASE IN SAP ( m m H g )
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Fig. 1. Effects of activation of LC by electrical stimulation (A) (10-s train of 1-ms rectangular pluses, at 10-20 #A, 10-20 Hz) or by L-glutamate (B), (1 nmol, 50 nl) on the sensitivity of BRR response.
microinjection of NPY antiserum (1:20) into the bilateral NTS (Fig. 2). This antagonism followed a
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time-course that peaked at 30-40 min postinjection and endured for at least 55 min. On the other hand, bilateral application of NPY (4.65 pmol) into the same nucleus elicited no discernible effect on the LC-promoted suppression of the BRR response (Fig. 2). We also confirmed that the capability of NPY antiserum to reverse LC-promoted BRR depression was specific to its biologic activity (Fig. 3). When microinjected into the NTS, both heat-inactivated NPY antiserum or NPY produced no appreciable effect on the LC-induced BRR suppression. Control injection of the same amount of normal rabbit serum also elicited no discernible influence on the inhibition of the BRR response by LC activation.
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Fig. 2. Time-course changes in the suppressive effect of electrical stimulation of the LC on the sensitivity of BRR response (open bar) and its influence by microinjection into the bilateral NTS of NPY (4.65 pmol, hatched bar) or N P Y antiserum (NPYAb, 1:20, crossed bar). Values presented are mean + S.E.M., n = 6-10 animals per group. Significant difference ( P < 0.05) exists between the 4 groups in the A N O V A analysis, and * P < 0 . 0 5 vs. BRR; ~ 7 P < 0 . 0 5 vs. L C + B R R in the Scheffe multiple range test.
Effect of prazosin and~or NPY antiserum on the LCinduced suppression of BRR response
We next compared the roles played by %adrenoceptors and NPY at the NTS on the LCpromoted suppression of the BRR response. Similar to our previous findings [1], microinjection of prazosin into the bilateral NTS, at a dose (50 pmol, 20 nl) that did not by itself change the basal BRR response, antagonized the depressive effect of the LC
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Fig. 3. Time-course changes in the suppressive effect of electrical stimulation of the LC on the sensitivity of BRR responses and its influence by microinjection into the bilateral N T S of heatinactivated N P Y ([NPY], 4.65 pmol, open bar), N P Y antiserum ([NPYAb], 1:20, crossed bar) or normal rabbit serum (NRS, 1:20, hatched bar). Values presented are mean +_S.E.M., n = 5-8 animals per group. Significant difference (P < 0.05) exists between the 4 groups in the A N O V A analysis, and * P < 0 . 0 5 vs. BRR in the Scheffe multiple range test.
Fig. 4. Time-course changes in the suppressive effect of electrical stimulation of the LC on the sensitivity of BRR responses and its influence by microinjection into the bilateral N T S of prazosin (PRA, 50 pmol, hatched bar), and N P Y antiserum (NPYAb) plus PRA (1:20 + 50 pmol, crossed bar). Data on (LC + BRR, filled bar) and (NPYAb + LC + BRR, open bar) from Fig. 2 were redrawn for comparison. Values presented are m e a n + S.E.M., n = 5-10 animals per group. Significant difference ( P < 0.05) exists between the 4 groups in the A N O V A analysis, and * P < 0 . 0 5 vs. LC + BRR in the Scheffe multiple range test.
on the BRR sensitivity (Fig. 4). This antagonism was significant for approx. 25 min. Simultaneous application of prazosin (50 pmol) and NPY antiserum (1:20) into the bilateral NTS also appreciably blunted the LC-induced inhibition of the BRR response (Fig. 4). This action was similar in time-course and magnitude to that produced by NPY antiserum pretreatment alone.
on the LC-induced BRR depression further includes the additional effect of phasically released NPY or NE at the NTS during LC stimulation. It follows that a subtraction of corresponding results by NPY antiserum or prazosin should logically reveal the specific BRR suppressive action of NPY or NE released at the NTS following LC activation. Fig. 5 demonstrates the outcome of such a mathematical treatment. It revealed that the suppression of the BRR response by endogenous NPY or NE, released at the NTS during LC activation, exhibited different time course and magnitude. The respective peak effect of NPY and NE was approx. 40 and 10 min. Furthermore, LC-induced noradrenergic neurotransmission at the NTS contributed to no less than 30~/o of the suppressive effect on the BRR response. On the other hand, even at its peak, the contribution by NPY from the LC was no more than 20%.
Participation of endogenous NE and NPY in the LCinduced suppression of BRR response The results produced by administration into the NTS of NPY antiserum (cf. Ref. 4) or prazosin (Fig. 4), we reasoned, represent the tonic, intrinsic influence of endogenous NPY or NE that originates from various brain nuclei, including those from the LC, on the sensitivity of BRR response at the NTS. Superimposed upon the above actions, the antagonism by NPY antiserum (Fig. 2) or prazosin (Fig. 4)
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-60 Fig. 5. Time-course effects of endogenous NPY ('NPY', filled bar) or norepinephrine ('NE', open bar), released at the NTS upon LC activation, on the sensitivity of BRR response. Values are normalized to percent of preinjection control, and are presented as the mean.
It should be mentioned that histologically verified microinjection sites indicated that all chemicals were administered primarily into the caudal one-third portion of the NTS. As such, they correspond to the major site of termination of the baroreceptor afterents in this nucleus [2,3], and where NPY or N E nerve terminals [ 5,15,16 ] as well as receptor binding sites for N PY or ~1-adrenoceptors [ 17-19] have been demonstrated. Discussion
We demonstrated in the present study that the endogenous NPY may participate in the LCpromoted suppression of the BRR response via an action that may at least take place at the NTS. Our data further revealed a differential involvement, both in time course and magnitude, of NPY and NE at the NTS, in this modulatory process of LC on the BRR sensitivity. Neurons that contain NPY are present in the LC [6-9]. NPY-containing nerve terminals [ 15] and receptor binding sites for NPY [ 17,18] are localized in
the NTS. Microinjection of NPY into the bilateral NTS produces a suppression of the BRR response [4]. The LC-induced inhibition of BRR sensitivity was antagonized by microinjection of NPY antiserum into the NTS (cf. Fig. 2). Together, these observations suggest strongly that activation of LC results in the release of NPY at the NTS that exerts the inhibitory action on BRR response. This suggestion is supported by our observed lack of significant augmentation of LC-induced BRR suppression in animals pretreated with microinjection of NPY to the bilateral NTS (cf. Fig. 2). We reasoned that occupation of a majority of the NPY binding sites at the NTS by the exogenous NPY may render the subsequently released NPY upon LC activation ineffective. It is likely that activation of LC resulted in the release of both NPY and NE at the NTS. Based on the logic of a mathematical treatment we previously developed to study the action of endogenous neurotensin [ 13], the present study also revealed that NPY and ~-adrenoceptors at the NTS may participate differentially in the LC-promoted BRR suppression. Under similar stimulus conditions for LC, the former evoked an action that manifested a process of slower maximization and lesser magnitude. One possible explanation for this differential effect is that NPY, like other neuropeptides, exhibits a slow time course of action [20]. Another possibility is the difference in the quantity of LC neurons that contain NPY or NE as their chemical substances. Whereas most of the LC neurons show positive immunoreactivity for dopamine-/~-hydroxylase [5], only around 20~o of nerve cells in the LC contain NPY [21]. A wealth of evidence demonstrates an interaction between NPY and NE in the central nervous system [22-26]. Thus, it is possible that NPY may participate in the LC-promoted BRR suppression by modulating the activity of the cq-adrenoceptors at the NTS. This possibility, however, is unlikely to take place since simultaneous administration of NPY antiserum and prazosin into the NTS elicited a blunting action on the LC-induced BRR depression that
299 was similar in time course and magnitude to pretreatment with N P Y antiserum alone (cf. Fig. 4). The binding characteristics o f ~l-adrenoceptors in the medulla oblongata are not significantly influenced by N P Y [22]. Furthermore, different signal transduction mechanism(s) are engaged by N P Y and a l-adrenoceptors. N P Y inhibits cyclic A M P accumulation in slices that contain the N T S regions o f the rat [27]. In addition, pertussis toxin-sensitive guanine nucleotide-binding proteins (e.g., Gi or G o ) are involved in N P Y - i n d u c e d cardiovascular effects [28]. On the other hand, inositol triphosphate liberation [29,30], mobilization o f cytosolic Ca 2+ and activation of a calmodulin-dependent system [ 30] are proposed to be involved in the cellular responses to central ~ - a d r e n o c e p t o r s activation. As we pointed out previously [ 1 ], whether the LC exerts a direct action on the N T S neurons is not immediately clear. Earlier studies [31,32] suggest the existence of a direct innervation from the LC to the NTS, although the chemical property o f this pathway is currently unknown. Whereas a small portion of the NPY-containing neurons form intrinsic circuits within the LC [33], the majority send their efferent fibers to different brain regions and nuclei [8,9,33]. However, whether the NPY-containing nerve terminals at the N T S take origin from the LC is also not clear. Ongoing experiments from our laboratory have yet to identify LC neurons with positive N P Y immunoreactivity to be simultaneously labeled by retrogradely transported horseradish peroxidase, upon being microinjected into the caudal level of the NTS. Alternatively, an indirect link between the L C and the N T S may be involved in the L C - p r o m o t e d B R R suppression [ 1 ], and the hypothalamus may serve as a possible intermediate. A direct projection of N P Y containing LC neurons to the paraventricular nucleus o f the hypothalamus is present [8,9]. This hypothalamic nucleus is at least partly responsible for the depression of the B R R response [34]. However, the participation of a hypothalamic NPYergic pathway in the LC-elicited suppression of the B R R response remains to be delineated.
We previously demonstrated that the endogenous brain N P Y exerts a tonic inhibitory action on the B R R [4]. The present study further reveals a novel physiologic function for N P Y by demonstrating its participation in the LC-elicited suppression of the B R R response. Thus, the endogenous brain N P Y may participate in central neural control o f cardiovascular function at two operating levels: to directly modulate the sensitivity of the B R R and to take part in the suppression of this reflex mechanism by the LC.
Acknowledgement This study was supported in part by research grant N S C 8 2 - 0 4 1 2 - B 0 7 5 - 0 5 8 from the National Science Council, and V G H - 1 6 0 from the Veterans General Hospital-Taipei, Taiwan, Republic of China to J.Y.H.C.
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© 1993 Elsevier SciencePublishers B.V. All rights reserved 0167-0115/93/$06.00 REGPEP 01534
Participation of endogenous neuropeptide Y in the suppression of baroreceptor reflex response by locus coeruleus in the rat Julie Y . H . C h a n a, C h e n g - D e a n Shih b and Samuel H . H . C h a n b Department of Medical Research, Veterans General Hospital- Taipei and bInslitllte of Pharmacology, National Yang-Ming Medical College. Taipei (Taiwan. R OC)
(Received 18 February 1993; revised version received 11 May 1993; accepted 14 May 1993) K e y words: Endogenous neuropeptide Y; Locus coeruleus; Baroreceptor reflex response; ~l-Adrenoceptor;
Nucleus tractus solitarii; Rat
Summary We evaluated the potential participation of endogenous brain neuropeptide Y (NPY) in the suppression of baroreceptor reflex (BRR) response by locus coeruleus (LC), using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Bilateral microinjection of an antiserum against NPY (1:20, 20 nl) into the caudal one-third level of the nucleus tractus solitarii (NTS), the terminal site for baroreceptor afferent fibers, significantly reversed the suppressive effect of electrical or chemical activation of the LC on the BRR response. Treatments with NPY (4.65 pmol, 20 nl), normal rabbit serum, aCSF and heat-inactivated NPY or NPY antiserum, on the other hand, were ineffective. The LC-promoted inhibition of the BRR response was also attenuated by the ~l-adrenoceptor antagonist prazosin (50 pmol, 20 nl), either microinjected alone or in combination with NPY antiserum into the bilateral NTS. Mathematical treatment of our data revealed that the depressive effect on the BRR response of NPY or N E released at the NTS following LC activation manifested different time-course and magnitude. The one by endogenous NPY maximized at 40 rain and amounted to no more than 20?0 of, whereas that by NE peaked at 10 min and contributed no less than 30?o to, the suppression. These results suggest that both endogenous NPY and N E may participate in the suppression of BRR response by the LC at the NTS.
Introduction Correspondence to: J.Y.H. Chan, Department of Medical Re-
search, Veterans General Hospital-Taipei,Taipei 11217,Taiwan, ROC.
Our laboratory recently reported [ 1 ] that the locus coeruleus (LC) of the pons exerts an inhibitory mod-
294 ulation on the baroreceptor reflex (BRR) control mechanism. This modulatory effect involves the ~ladrenoceptors located at the nucleus tractus solitarii (NTS), the terminal site for baroreceptor afferent fibers [2,3]. We also showed [4] that the endogenous neuropeptide Y (NPY) in the brain exerts a tonic suppressive effect on the BRR response at the NTS. Apart from its high density of norepinephrine (NE)-containing neurons [5], a substantial number of neuropeptides, including NPY, has been visualized in the rat LC [6-9]. Some of the NE-containing neurons in the LC also show positive immunoreactivity to NPY [6,9]. Neuroanatomically, a high percentage of the NPY-containing perikarya in the LC functions as projecting neurons to various brain regions [8,9]. The physiological significance of such efferent projections of NPY-containing neurons in the LC, however, has not been fully defined. Two interesting corollaries arise from the foregoing observations, and formed the working hypothesis for the present study. First, it is possible that NPY-containing neurons that exert a tonic inhibitory effect on the BRR response at the NTS may arise from the LC. Second, it is likely that activation of the LC may result in the release of at least N E and NPY at the NTS, both of which may influence the BRR response. We demonstrated that activation of the LC may indeed elicit a NPY-related suppression of the BRR response. Furthermore, this suppression exhibited a time-course and magnitude that was different from the inhibition induced by the LC-originated noradrenergic neurotransmission. Materials and Methods
A nimal preparation Experiments were carried out in adult, male Sprague-Dawley rats (250-300 g) anesthetized with pentobarbital sodium (40 mg/kg, i.p., with 10 mg/ kg/h supplements). The right femoral artery and vein were routinely cannulated to measure systemic arterial pressure and administer drugs. Arterial pressure was monitored via a pressure transducer (Statham
P231D), and heart rate was determined using a biotachometer (Gould 20-4615-65) triggered by the arterial pressure pulses. 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 animal was placed in a stereotaxic headholder (Kopf), with the rest of the body placed on a heating pad and elevated to a suitable position. Pulsatile and mean systemic arterial pressure, as well as heart rate, were recorded continuously on a polygraph (Gould RS 3600). All data were collected from animals with a maintained rectal temperature of 37°C and a steady systemic arterial pressure throughout the experiment. Electrical and chemical activation of the locus coeruleus Electrical activation of the LC was delivered by a stainless-steel bipolar concentric electrode (Rhodes Medical SNE-100, tip diameter: 100 #m). The stereotaxic coordinates used were: 1.0-1.2 mm caudal to the lambda, 1.1-1.3 mm lateral to the midline and 5.0-6.0 m m below the cortical surface. The LC was electrically activated by a 10-s train of 1-ms rectangular pulses, at 10-60 /~A 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 10 m m H g upon electrical activation [ 10] was first identified. The stimulus parameters, chiefly intensity, were subsequently adjusted to a condition at which LC stimulation elicited only minimal cardiovascular effects. Neuronal perikarya in the LC were activated, in some experiments, by L-glutamate (1 nmol) stimulation [ 11 ]. This was achieved via a glass micropipette (tip diameter: 50-80/~m), 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 for complete diffusion of the solution. Possible volume effect of microinjection was controlled by injecting the same amount of artificial cerebrospinal fluid (aCSF).
295 Evaluation of the baroreceptor reflex response Similar to the evaluation used in our previous studies [ 12,13 ], the sensitivity of B RR response was evaluated using a modification of the method by Smythe et al. [14]. In essence, we employed the slope of the linear portion of the regression line that relates maximal reflex bradycardia to transient hypertension elicited by three doses of phenylephrine (2.5, 5.0 or 10.0/ag/kg, i.v.) to represent the sensitivity of BRR. The order of different doses of phenylephrine was altered randomly to avoid sequential dependency. Modulation of the BRR response by the LC was assessed by evaluating their interactions. For this purpose, the dorsal pontine nucleus was electrically stimulated simultaneously with the induction of the reflex. In experiments that employed chemical activation of the LC, the sensitivity of BRR response was assessed within 10 rain following microinjection of L-glutamate. Microinjection of chemicals into the bilateral nucleus tractus solitarii Direct, bilateral microinjection of chemicals into the NTS was carried out sequentially with a stereotaxically positioned ( -0.5 to + 0.5 mm from the obex, 0.35 to 0.50 mm lateral to the midline, and 0.35 to 0.90 mm from the surface of the fourth ventricle) glass micropipette (tip diameter: 50-80 /~m), connected to a nanoliter infusion pump. A total volume of 20 nl was delivered into the NTS on each side, with aCSF serving as control. The temporal effect of chemical treatments on the BRR response or the LC-mediated suppression of the BRR response was evaluated before and at 0-10, 15-25, 30-40 or 45-55 min after microinjecting the chemicals into the bilateral NTS. Chemicals used included rat NPY (4.65 pmol, Peninsula Lab.), rabbit NPY antiserum (1:20, Incstar), normal rabbit serum (Jackson Lab.), or heat-inactivated (60 + 5°C for 15-20 min) NPY and NPY antiserum. The NPY antiserum showed no significant cross-reactivity with peptides having similar
molecular structures. All stock solutions were stored frozen, and thawed immediately before use. Histology At the conclusion of each experiment, the brain was removed and fixed in 30~o sucrose in 10~o formaldehyde-saline. 25-/~m frozen sections stained either with Neutral red or Cresyl violet were used to verify the location of stimulation or microinjection sites. 1 ~o Evans blue was added to the injection medium to aid in such verifications. Statistical analysis The effect of chemical treatments on the sensitivity of BRR response or the LC-promoted suppression of the BRR response was statistically assessed using two-way analysis of variance (ANOVA) with repeated measures. This was followed by the Scheffe multiple range test for a posteriori comparison of means at corresponding time intervals. All results were considered statistically significant at P < 0.05. Results
Effect of activation of LC on the B R R response In agreement with our previous findings [ 1 ], electrical activation of the LC (Fig. 1A)reduced the sensitivity of the BRR response (slope: 0.96 to 0.61). We also ascertained with chemical stimulation that such an inhibition was due mainly to activation of the LC perikarya, but not fibers of passage. L-Glutamate (1 nmol) microinjected into the LC similarly depressed the BRR response (Fig. 1B), evaluated within 10 min following application of the excitatory amino acid, by reducing the slope from 0.94 to 0.62. The overall suppression of the BRR response by LC activation amounted to 43.6 + 6.5 ~o (mean + S.E.M., n = 10) (Fig. 2). Effect of N P Y or N P Y antiserum on the LC-induced suppression of B R R response The LC-induced inhibition of BRR response was significantly antagonized in animals pretreated with
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Fig. 1. Effects of activation of LC by electrical stimulation (A) (10-s train of 1-ms rectangular pluses, at 10-20 #A, 10-20 Hz) or by L-glutamate (B), (1 nmol, 50 nl) on the sensitivity of BRR response.
microinjection of NPY antiserum (1:20) into the bilateral NTS (Fig. 2). This antagonism followed a
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time-course that peaked at 30-40 min postinjection and endured for at least 55 min. On the other hand, bilateral application of NPY (4.65 pmol) into the same nucleus elicited no discernible effect on the LC-promoted suppression of the BRR response (Fig. 2). We also confirmed that the capability of NPY antiserum to reverse LC-promoted BRR depression was specific to its biologic activity (Fig. 3). When microinjected into the NTS, both heat-inactivated NPY antiserum or NPY produced no appreciable effect on the LC-induced BRR suppression. Control injection of the same amount of normal rabbit serum also elicited no discernible influence on the inhibition of the BRR response by LC activation.
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Fig. 2. Time-course changes in the suppressive effect of electrical stimulation of the LC on the sensitivity of BRR response (open bar) and its influence by microinjection into the bilateral NTS of NPY (4.65 pmol, hatched bar) or N P Y antiserum (NPYAb, 1:20, crossed bar). Values presented are mean + S.E.M., n = 6-10 animals per group. Significant difference ( P < 0.05) exists between the 4 groups in the A N O V A analysis, and * P < 0 . 0 5 vs. BRR; ~ 7 P < 0 . 0 5 vs. L C + B R R in the Scheffe multiple range test.
Effect of prazosin and~or NPY antiserum on the LCinduced suppression of BRR response
We next compared the roles played by %adrenoceptors and NPY at the NTS on the LCpromoted suppression of the BRR response. Similar to our previous findings [1], microinjection of prazosin into the bilateral NTS, at a dose (50 pmol, 20 nl) that did not by itself change the basal BRR response, antagonized the depressive effect of the LC
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Fig. 3. Time-course changes in the suppressive effect of electrical stimulation of the LC on the sensitivity of BRR responses and its influence by microinjection into the bilateral N T S of heatinactivated N P Y ([NPY], 4.65 pmol, open bar), N P Y antiserum ([NPYAb], 1:20, crossed bar) or normal rabbit serum (NRS, 1:20, hatched bar). Values presented are mean +_S.E.M., n = 5-8 animals per group. Significant difference (P < 0.05) exists between the 4 groups in the A N O V A analysis, and * P < 0 . 0 5 vs. BRR in the Scheffe multiple range test.
Fig. 4. Time-course changes in the suppressive effect of electrical stimulation of the LC on the sensitivity of BRR responses and its influence by microinjection into the bilateral N T S of prazosin (PRA, 50 pmol, hatched bar), and N P Y antiserum (NPYAb) plus PRA (1:20 + 50 pmol, crossed bar). Data on (LC + BRR, filled bar) and (NPYAb + LC + BRR, open bar) from Fig. 2 were redrawn for comparison. Values presented are m e a n + S.E.M., n = 5-10 animals per group. Significant difference ( P < 0.05) exists between the 4 groups in the A N O V A analysis, and * P < 0 . 0 5 vs. LC + BRR in the Scheffe multiple range test.
on the BRR sensitivity (Fig. 4). This antagonism was significant for approx. 25 min. Simultaneous application of prazosin (50 pmol) and NPY antiserum (1:20) into the bilateral NTS also appreciably blunted the LC-induced inhibition of the BRR response (Fig. 4). This action was similar in time-course and magnitude to that produced by NPY antiserum pretreatment alone.
on the LC-induced BRR depression further includes the additional effect of phasically released NPY or NE at the NTS during LC stimulation. It follows that a subtraction of corresponding results by NPY antiserum or prazosin should logically reveal the specific BRR suppressive action of NPY or NE released at the NTS following LC activation. Fig. 5 demonstrates the outcome of such a mathematical treatment. It revealed that the suppression of the BRR response by endogenous NPY or NE, released at the NTS during LC activation, exhibited different time course and magnitude. The respective peak effect of NPY and NE was approx. 40 and 10 min. Furthermore, LC-induced noradrenergic neurotransmission at the NTS contributed to no less than 30~/o of the suppressive effect on the BRR response. On the other hand, even at its peak, the contribution by NPY from the LC was no more than 20%.
Participation of endogenous NE and NPY in the LCinduced suppression of BRR response The results produced by administration into the NTS of NPY antiserum (cf. Ref. 4) or prazosin (Fig. 4), we reasoned, represent the tonic, intrinsic influence of endogenous NPY or NE that originates from various brain nuclei, including those from the LC, on the sensitivity of BRR response at the NTS. Superimposed upon the above actions, the antagonism by NPY antiserum (Fig. 2) or prazosin (Fig. 4)
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-60 Fig. 5. Time-course effects of endogenous NPY ('NPY', filled bar) or norepinephrine ('NE', open bar), released at the NTS upon LC activation, on the sensitivity of BRR response. Values are normalized to percent of preinjection control, and are presented as the mean.
It should be mentioned that histologically verified microinjection sites indicated that all chemicals were administered primarily into the caudal one-third portion of the NTS. As such, they correspond to the major site of termination of the baroreceptor afterents in this nucleus [2,3], and where NPY or N E nerve terminals [ 5,15,16 ] as well as receptor binding sites for N PY or ~1-adrenoceptors [ 17-19] have been demonstrated. Discussion
We demonstrated in the present study that the endogenous NPY may participate in the LCpromoted suppression of the BRR response via an action that may at least take place at the NTS. Our data further revealed a differential involvement, both in time course and magnitude, of NPY and NE at the NTS, in this modulatory process of LC on the BRR sensitivity. Neurons that contain NPY are present in the LC [6-9]. NPY-containing nerve terminals [ 15] and receptor binding sites for NPY [ 17,18] are localized in
the NTS. Microinjection of NPY into the bilateral NTS produces a suppression of the BRR response [4]. The LC-induced inhibition of BRR sensitivity was antagonized by microinjection of NPY antiserum into the NTS (cf. Fig. 2). Together, these observations suggest strongly that activation of LC results in the release of NPY at the NTS that exerts the inhibitory action on BRR response. This suggestion is supported by our observed lack of significant augmentation of LC-induced BRR suppression in animals pretreated with microinjection of NPY to the bilateral NTS (cf. Fig. 2). We reasoned that occupation of a majority of the NPY binding sites at the NTS by the exogenous NPY may render the subsequently released NPY upon LC activation ineffective. It is likely that activation of LC resulted in the release of both NPY and NE at the NTS. Based on the logic of a mathematical treatment we previously developed to study the action of endogenous neurotensin [ 13], the present study also revealed that NPY and ~-adrenoceptors at the NTS may participate differentially in the LC-promoted BRR suppression. Under similar stimulus conditions for LC, the former evoked an action that manifested a process of slower maximization and lesser magnitude. One possible explanation for this differential effect is that NPY, like other neuropeptides, exhibits a slow time course of action [20]. Another possibility is the difference in the quantity of LC neurons that contain NPY or NE as their chemical substances. Whereas most of the LC neurons show positive immunoreactivity for dopamine-/~-hydroxylase [5], only around 20~o of nerve cells in the LC contain NPY [21]. A wealth of evidence demonstrates an interaction between NPY and NE in the central nervous system [22-26]. Thus, it is possible that NPY may participate in the LC-promoted BRR suppression by modulating the activity of the cq-adrenoceptors at the NTS. This possibility, however, is unlikely to take place since simultaneous administration of NPY antiserum and prazosin into the NTS elicited a blunting action on the LC-induced BRR depression that
299 was similar in time course and magnitude to pretreatment with N P Y antiserum alone (cf. Fig. 4). The binding characteristics o f ~l-adrenoceptors in the medulla oblongata are not significantly influenced by N P Y [22]. Furthermore, different signal transduction mechanism(s) are engaged by N P Y and a l-adrenoceptors. N P Y inhibits cyclic A M P accumulation in slices that contain the N T S regions o f the rat [27]. In addition, pertussis toxin-sensitive guanine nucleotide-binding proteins (e.g., Gi or G o ) are involved in N P Y - i n d u c e d cardiovascular effects [28]. On the other hand, inositol triphosphate liberation [29,30], mobilization o f cytosolic Ca 2+ and activation of a calmodulin-dependent system [ 30] are proposed to be involved in the cellular responses to central ~ - a d r e n o c e p t o r s activation. As we pointed out previously [ 1 ], whether the LC exerts a direct action on the N T S neurons is not immediately clear. Earlier studies [31,32] suggest the existence of a direct innervation from the LC to the NTS, although the chemical property o f this pathway is currently unknown. Whereas a small portion of the NPY-containing neurons form intrinsic circuits within the LC [33], the majority send their efferent fibers to different brain regions and nuclei [8,9,33]. However, whether the NPY-containing nerve terminals at the N T S take origin from the LC is also not clear. Ongoing experiments from our laboratory have yet to identify LC neurons with positive N P Y immunoreactivity to be simultaneously labeled by retrogradely transported horseradish peroxidase, upon being microinjected into the caudal level of the NTS. Alternatively, an indirect link between the L C and the N T S may be involved in the L C - p r o m o t e d B R R suppression [ 1 ], and the hypothalamus may serve as a possible intermediate. A direct projection of N P Y containing LC neurons to the paraventricular nucleus o f the hypothalamus is present [8,9]. This hypothalamic nucleus is at least partly responsible for the depression of the B R R response [34]. However, the participation of a hypothalamic NPYergic pathway in the LC-elicited suppression of the B R R response remains to be delineated.
We previously demonstrated that the endogenous brain N P Y exerts a tonic inhibitory action on the B R R [4]. The present study further reveals a novel physiologic function for N P Y by demonstrating its participation in the LC-elicited suppression of the B R R response. Thus, the endogenous brain N P Y may participate in central neural control o f cardiovascular function at two operating levels: to directly modulate the sensitivity of the B R R and to take part in the suppression of this reflex mechanism by the LC.
Acknowledgement This study was supported in part by research grant N S C 8 2 - 0 4 1 2 - B 0 7 5 - 0 5 8 from the National Science Council, and V G H - 1 6 0 from the Veterans General Hospital-Taipei, Taiwan, Republic of China to J.Y.H.C.
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