- Email: [email protected]
Cardiovascular Actions of Neuropeptide Y and Social Stress
Peptides, Vol. 19, No. 1, pp. 85–92, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(97)00266-0
Cardiovascular Actions of Neuropeptide Y and Social Stress HARRY KLEMFUSS,1 SCOTT SOUTHERLAND AND KAREN T. BRITTON Department of Psychiatry, Veterans Affairs Medical Center and University of California at San Diego, San Diego, CA 92161 Received 10 April 1997; Accepted 28 August 1997 KLEMFUSS, H., S. SOUTHERLAND AND K. T. BRITTON. Cardiovascular actions of Neuropeptide Y and social stress. PEPTIDES 19(1) 85–92, 1998 —The role of central neuropeptide Y (NPY) in the cardiovascular response to social stress was evaluated in freely moving rats using telemetry. In unstressed rats, intracerebroventricular (ICV) administration of NPY and the selective Y1 receptor agonist [Leu31, Pro34]-NPY decreased blood pressure and heart rate, while the selective Y2 agonist NPY13–36 transiently raised blood pressure. NPY and [Leu31,Pro34]-NPY blunted elevations in blood pressure and pulse rate following exposure to the resident-intruder procedure, an established social stress paradigm. In contrast, the Y2 agonist significantly augmented stressinduced pressor effects. These observations indicate that the hypotensive effects of ICV NPY appear to be mediated by the Y1 receptor subtype and the NPY receptor subtypes may mediate opposing cardiovascular actions in response to stressful stimuli. © 1998 Elsevier Science Inc. Stress Resident-intruder Neuropeptide Y Heart rate Locomotor activity
Y1 receptor
NEUROPEPTIDE Y (NPY), a 36-residue member of the pancreatic polypeptide family, is one of the most abundant peptides in the central nervous system (1,41) and among the most highly conserved peptides phylogenetically (29), implying that it may play an important physiological role. At least two subtypes of NPY receptors, designated Y1 and Y2, have been characterized, although other forms have been proposed (14,40). The substituted analogue [Leu31,Pro34]NPY is an agonist displaying high affinity for the Y1 receptor and low affinity for the Y2 receptor (12,46), while the truncated analogue NPY(13–36) is an agonist with low affinity for the Y1 receptor and high affinity for the Y2 receptor type (33). NPY may be a component of the autonomic and endocrine responses to acute stress. Peripherally, NPY directly mediates vasoconstriction and augments the pressor action of vasoconstrictors such as norepinephrine (15,45,49). Receptors of the Y1 subtype mediate vasoconstriction postsynaptically, while those of the Y2 type act presynaptically in the regulation of catecholamine release (46,47). Physical
Y2 receptor
Blood pressure
stressors such as immobilization, cold, hypoxia, hemorrhage, and handling increase plasma NPY concentrations in animals (9,34,52). Increased plasma levels of immunoreactive NPY are also reported in humans experiencing emotional stress (26) and panic disorder (6). Central administration of NPY causes behavioral changes similar to benzodiazepines in several animal models of anxiety including the conflict test (20,22), elevated plus-maze (8,20), and fear-potentiated startle test (8). Y1 receptors in the amygdala are thought to be a critical site for the anxiolytic action of NPY (8,19,20). Intracerebroventricular (ICV) NPY injection protects against some stress-induced pathology, such as gastric lesions (21,27,36) and fear-induced stimulation of colonic motility (16,44). On the other hand, behavioral signs of anxiety are elicited by an antisense oligodeoxynucleotide to NPY messenger ribonucleic acid injected ICV (48) or directly into the amygdala (18). These findings are generally consistent with a role of NPY in stress reduction. In contrast to systemic treatment, ICV administration of NPY lowers blood pressure and heart rate in anesthetized or
1
Requests for reprints should be addressed to Harry Klemfuss, Ph.D., Research Service -151, San Diego VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161; E-mail: [email protected]
85
86
awake, catheterized rats (4,13,30,38). Hypotension and bradycardia are also reported following injection into nucleus of the solitary tract, area postrema, and caudal ventrolateral medulla (15,30,42). However, increases in blood pressure have been reported following NPY injection into area postrema (42), nucleus of the solitary tract, or posterior hypothalamus (31,50). It has been reported that intraventricular administration of the Y1 receptor agonist [Leu31,Pro34]-NPY produces hypotension, while the Y2 agonist NPY(13–36) elicits a vasopressor response in awake, catheterized rats (3). The idea that Y1 and Y2 receptors mediate opposing cardiovascular actions is further supported by the observation that low doses of NPY(13–36) antagonize the hypotension produced by NPY itself in catheterized rats (2,3,35,51). The purpose of the present study was to characterize the effects of NPY on the cardiovascular and locomotor responses to a social stressor, using the ‘‘resident-intruder’’ procedure, a social stressor in which a socially naive male ‘‘intruder’’ rat is placed into the home territory of a resident counterpart, resulting in a variety of agonistic interactions (11,25). The intruder rat typically exhibits autonomic changes consistent with a ‘‘stress response’’, i.e. increased heart rate, blood pressure, and renal and mesenteric resistance to blood flow, during the procedure (11,32,39,43). Heart rate may remain elevated for as long as one h after the procedure, but blood pressure tends to return to normal within 10 – 40 min after the intruder is returned to his home cage (32,43). After the preliminary dose-response study, we tested whether central administration of NPY would attenuate the stress-induced increases in heart rate and blood pressure displayed by the intruder rats during and after the resident-intruder procedure. We then tested the Y1 receptor agonist [Leu31,Pro34]-NPY and the Y2 receptor agonist NPY(13–36) to determine possible receptor heterogeneity in the acute cardiovascular and locomotor effects of NPY. METHOD Subjects and Surgical Procedures Male Wistar rats, weighing 200 –250 g at the time of surgery, were implanted with a sterile Data Sciences (St. Paul, MN) PA-C40 device under halothane anesthesia. This device consists of a transmitter capsule and a fluid-filled catheter coated with antithrombogenic film. The tip of the catheter, which is filled with a biocompatible gel, was inserted into the abdominal aorta just cranial to the bifurcation of the aorta and distal to the renal arteries, and secured with tissue adhesive and a fiber patch according to manufacturer’s recommendations (10). The transmitter capsule was sutured to the inside surface of the peritoneum. The device could be turned on and off without disturbing the rat by passing a magnet beneath the cage. Radiofrequency signals from this device were collected by a receiver located under the shoebox cage for 10 s every min. Mean values for systolic pressure, diastolic pressure, heart rate, and locomo-
KLEMFUSS ET AL.
tor activity (gross changes in position, as detected by the receiver) were calculated, using algorithms provided by Data Sciences, for each 10 s epoch and were recorded using the Dataquest IVt data collection and analysis system. About three weeks after transmitter implant surgery, each rat was again anesthetized with halothane and stereotaxically implanted with an indwelling stainless steel cannula directed at the lateral ventricle, as previously described (7). Each implanted rat was housed singly in a standard shoebox cage (45 3 22 3 22 cm), in a sound- and light-attenuating isolation enclosure, on a 12:12 light-dark cycle, for at least one week between procedures. Food and water were provided ad lib at all times, and litter was changed twice per week. Study 1: NPY Dose–Response Procedure Six implanted rats were maintained on a reverse light schedule with lights on from 1800 to 0600 h. By 0700 h on test days telemetry devices were magnetically activated, and recording of blood pressure, heart rate, and activity began in the test rats’ home cages. At about 0930 h (2.5 h after lights off) each test rat was removed from the home cage and injected icv with porcine NPY (0.5, 1, 8, or 32 ug/rat 5 0.12, 0.24, 1.9, or 6.5 nmol/rat) or an equal volume (2 ul) of saline vehicle, injected over 60 s. These doses cover the range of concentrations at which ICV NPY has been reported to have anxiolytic actions in the rat. All injections were performed by an experienced technician careful to minimize the potential stress of the injection procedure. After injection, each test rat was returned to the home cage. Rats were left undisturbed for at least 6 h after injection while blood pressure and activity were continuously recorded. During injections and transfers between cages, 1 or 2 data time points were lost. Each rat was injected 5– 6 times at approximately weekly intervals. This separation of treatment times was chosen since even mild stress such as sham injection or social interaction may produce changes in behavior and blood pressure lasting as long as several days (Seifritz et al., submitted; Klemfuss et al., in preparation). There was no indication that hemodynamic parameters changed over the 6 week period. Study 2: NPY versus Social Stress The same animals were subjects in the second study which followed generally the same procedures. Each rat received either NPY (8 ug/rat) or vehicle control. Following each drug treatment, rats were either left undisturbed for 6 h or else subjected to the resident-intruder procedure starting 30 min after injection. Each rat was tested once with vehicle and once with 8 ug NPY under both unstressed and social stress conditions, at approximately weekly intervals.
STRESS, NPY, AND BLOOD PRESSURE
Resident–Intruder Procedure The social stress consisted of a confrontation between the telemetry-implanted ‘‘intruder’’ rat and an unoperated ‘‘resident’’ rat, following procedures previously described (24,37). Residents were adult male Long–Evans rats, housed in a large shoebox cage (45 3 38 3 42 cm) with a female Wistar rat. Residents were used only when pups were between the ages of 5–21 days. Bedding was changed approximately once per week, after a resident-intruder confrontation. One half hour before a confrontation, females and pups were removed from the residence cage and housed separately. Thirty min after injection, each intruder rat was gently removed from the home cage and introduced into the residence cage. At the same time the home cage telemetry receiver was placed under the residence cage. For the next 15 min of confrontation, behavior of the two rats was continuously monitored for attacks and evasive behaviors. In the event of a clear attack, or if no attack had occurred by 15 min after confrontation, the intruder rat was placed in a protective wire mesh cage (30 3 18 3 13 cm) within the residence cage. The resident could sense the intruder, and could threaten him, but could not physically attack. Thirty min after the beginning of the confrontation the intruder was returned to his home cage, and blood pressure, heart rate, and locomotor activity were recorded for at least another 6 h. Study 3: Y1 and Y2 Receptor Agonists This study was identical to Study 2, with the following exceptions: 1) Drug treatments consisted of either 8 ug/rat of the Y1 agonist [Leu31,Pro34]-NPY or 6 ug/rat of the Y2 agonist NPY(13–36), or saline vehicle. The molar concentration of each dose was approximately equal to the 8 ug dose of NPY; 2) Lights were on from 0400 to 1600 h, so rats were injected 5.5 h after lights on, and 3) the protective cage for intruders was a perforated plastic container (diameter 16 cm 3 10 cm high). Data Analysis Systolic and diastolic pressure, heart rate, and motor activity means were collapsed into 30 min bins using Dataquestt software and analyzed further using Quattro Pro and SPSS. Pulse pressure (systolic– diastolic) was calculated and analyzed as well. Locomotor activity means were log transformed to normalize variance. Unless otherwise stated, data are expressed as a percent of each rat’s baseline during the 120 min prior to confrontation, to reduce within-group variance. Drugs Porcine NPY (MW 5 4252), the Y1 agonist [Leu31,Pro34]NPY (MW 5 4221), and the Y2 agonist NPY(13–36) (MW 5 2862) were generously supplied by Dr. Jean Rivier of the
87
Salk Institute. All drugs were dissolved in saline immediately before injection. Statistics Analysis of variance (ANOVA) was used to test for effects of NPY dose on pressure, heart rate, and locomotor activity. Repeated measures ANOVA was used to evaluate effects of NPY or NPY receptor agonists in relation to exposure to social stress and time post-injection as within-subjects variables. Bonferroni-corrected ANOVA, with planned comparisons (Tukey HSD) when appropriate, were used to localize effects. Significance is defined as p , 0.05. All results in the text are expressed as mean 6 SD. RESULTS Control Rats Freely moving rats had a baseline systolic pressure of 131 6 9 mmHg and diastolic pressure of 95 6 8 mmHg (n 5 60 observations in 6 rats). Baseline heart rate was 325 6 32 beats/min. Immediately following ICV injection of either drug or saline all rats showed a transient increase in blood pressure and heart rate, which returned toward baseline levels within 15 min. Study 1: NPY Dose Response in Unstressed Rats All four doses of NPY (0.5 to 32 ug/rat) significantly lowered systolic, diastolic and pulse pressure, and heart rate, averaged across 3 h beginning 30 min after injection to avoid injection artifacts. Averaged locomotor activity was not significantly affected by NPY treatment. Figure 1 shows that pulse pressure was significantly decreased at all timepoints, after all doses, compared to control (F(4, 24) . 2.8; p , 0.05). Systolic and diastolic pressure were significantly decreased at the first and third timepoint at all doses, and at the last two time points by the 8 and 32 ug doses. Heart rate was decreased at all time points by the 8 and 32 ug doses. Locomotor activity was statistically decreased at the last three time points only by the lowest (0.5 ug) dose of NPY (p , 0.05, planned comparison to vehicle). Social Stress Following Saline Injection (Study 2) During exposure to threat in the resident-intruder confrontation, blood pressure was not significantly increased relative to baseline in saline-pretreated intruders (Fig. 2; F(1, 20) , 2.5). Both heart rate and locomotor activity rose significantly during the confrontation (F(1, 20) . 8.0). Although most residents attacked the intruder within a few minutes of confrontation, clear defeats (defined by submissive posture of the intruder following attack) occurred in fewer than 10% of confrontations. Defeat did not make any apparent difference in acute or chronic pressure or heart rate response, so data from defeated and undefeated intruders are combined.
88
KLEMFUSS ET AL.
2 (left side), and also decreased gross activity 90 –150 min after injection. Graphs on the right side of Figure 2 show comparable data
FIG. 1. Time course of blood pressure, heart rate, and locomotor activity following graded doses of NPY. All data are presented as percent of each animal’s baseline 6 SEM. Nine rats were injected at time 0 with saline vehicle (E), and 5 rats were given each dose of NPY: 0.5 ug/rat (E), 1.0 ug/rat (h), 8 ug/rat (Œ), and 32 ug/rat (■).
Effect of NPY Pretreatment on Responses to Social Stress The effects of the 8 ug/rat dose were generally similar in unstressed rats in Study 2 as in Study 1: NPY produced significant reductions in mean systolic pressure, diastolic pressure, pulse pressure, and heart rate, as shown in Figure
FIG. 2. Effects of NPY and social stress on blood pressure, heart rate, and activity. Rats were injected at time 0 with saline E or 8 ug NPY Œ. Graphs on the left show data from unstressed animals in Study 2. Graphs on the right show comparable data from rats exposed to social stress 30 – 60 min after injection (shaded bar). *Indicates a significant difference from vehicle control (p , 0.05, planned comparisons).
STRESS, NPY, AND BLOOD PRESSURE
89
from rats exposed to social stress 30–60 min after injection (shaded bar). During the 30 min confrontation, intruders showed transient increases in systolic pressure (p , 0.05), heart rate (p , 0.01) and activity (p , 0.001) compared to unstressed animals, without significant interaction between social stress and drug treatment during the confrontation. During the 2 h interval following social stress, the hypotensive effects of NPY were attenuated, but still significantly lower than controls (p , 0.05, Tukey’s HSD). NPY effects on other measures were also decreased, and no longer reached statistical significance. The dose vs. social stress interaction term was significant for systolic pressure (F(1, 5) 5 6.72) and pulse pressure (F(1, 5) 5 7.1 by repeated measures ANOVA (p , 0.05). Diastolic pressure (F(1, 5) 5 6.2), heart rate (F(1, 5) 5 0.1), and gross activity (F(1, 5) 5 4.53) fail to show significant interactions between NPY treatment and social stress. Y1 and Y2 Receptor Agonists in Unstressed Rats As shown in Fig. 3 (left side), the Y1 receptor agonist [Leu31,Pro34]-NPY significantly lowered systolic, diastolic, and pulse pressures by 5–10% compared to saline control immediately following injection and for at least 3 h after injection (p , 0.05 at all time points). These actions were comparable in magnitude and duration to the effects of an equimolar dose of NPY. Unlike NPY, the Y1 agonist had no significant effect on heart rate (p . 0.3). In contrast to both NPY and [Leu31,Pro34]-NPY, the Y2 agonist NPY(13–36) did not alter lower systolic pressure or heart rate in unstressed animals, but transiently raised systolic pressure (p , 0.05) compared to vehicle. Neither agonist produced a significant change in gross motor activity. Y1 and Y2 Receptor Agonists in Socially Stressed Rats During the confrontation, intruders showed increases in blood pressure parameters and heart rate (p , 0.001), but not in locomotor activity (p 5 0.17). There were no significant interactions between social stress and drug treatment during the confrontation (Fig. 3 right side). The Y1 receptor agonist [Leu31,Pro34]-NPY blunted the pressor response and tachycardia observed following social stress. In contrast, the systolic pressure of NPY(13– 36)-treated rats remained significantly elevated for at least 90 min after the end of the confrontation, while the systolic pressure of saline controls had returned to baseline levels by 30 min after confrontation. In stressed rats, mean systolic pressure was significantly lower in Y1 agonist-treated animals compared to Y2-agonist-treated rats, during the 2 h after return to the home cage (p , 0.05, Newman–Keuls). The interaction between agonist treatment and social stress on systolic pressure was statistically significant by repeated measures ANOVA (F(2, 10) 5 5.56; p , 0.05). The Y1 receptor agonist [Leu31,Pro34]-NPY also attenu-
FIG. 3. Effects of Y1 and Y2 agonists on responses to social stress. Rats were injected at time 0 with saline E, 8 ug(leu,pro)NPY Œ or 6 ug NPY(13–36)■. Graphs on the left show data from unstressed animals in Study 3. *Indicates a significant difference from vehicle control (p , 0.05, planned comparisons). Graphs on the right side show comparable data from rats exposed to social stress 30 – 60 min after injection (shaded bar).
ated stress-induced tachycardia and blood pressure changes during the first 60 min following stress. No other significant main effects of stress or drug treatment, or interactions
90
KLEMFUSS ET AL.
between agonist treatment and social stress, were found in stressed rats (F(2, 10) , 2.3; p . 0.15). DISCUSSION The results of the present study confirm that central administration of NPY to freely-moving rats produces a marked and prolonged vasodepressor and bradycardic action (4,13,17) at doses that have potent anxiolytic actions in other behavioral paradigms (8,22). These actions appear to be mediated by the Y1 receptor subtype since equimolar doses of NPY and the selective Y1 agonist [Leu31,Pro34]NPY lowered blood pressure to a similar degree and with a similar time course, while the selective Y2 agonist NPY(13– 36) was inactive. Thus, the predominant cardiovascular action of centrally administered NPY is to lower blood pressure via Y1 receptor activation. The resident-intruder stress paradigm is an ethologically relevant procedure that produces behavioral and physiological changes consistent with increased stress, including tachycardia, increased blood pressure, locomotor activation and release of ACTH and corticosterone (11,32,37,39,43). The use of telemetry in the present study enabled us to preserve the full expression of the intruder cardiovascular and locomotor responses. Our results indicate that these stress-induced effects are more prominent when animals are tested in their inactive, i.e. lights on, period. In this period, the rats show a marked vasopressor and locomotor response that persists about an hour following confrontation. When animals were tested during their normally active period, i.e., lights out, these changes were less marked and returned to baseline levels in less than 30 min. These light vs. dark differences were also apparent in the response to the injection procedure itself, and may be due in part to the fact that baseline levels of blood pressure, pulse and locomotor activation are higher during the dark period, thus obscurring moderate stress-induced changes via ‘‘ceiling effects’’. The pressor and tachycardiac responses observed in intruder rats following social confrontation with a resident male were significantly reduced by pretreatment with either NPY itself or the Y1 agonist [Leu31,Pro34]NPY. This result is consistent with prior studies showing potent stress-reducing effects of these compounds in a variety of animal models of anxiety. The anxiolytic-like properties of NPY appear to be mediated by the Y1 receptor (17,20), consistent with the current findings. Y1 agonists produce a marked release of punished responding in the conflict test (5), elevated plus maze, and startle paradigms (8). Stress-induced mesenteric vasoconstriction also appears to be mediated via the NPY Y1 receptor subtype (52). Overall, these findings support the hypoth-
esis that Y1 receptors in the brain were involved in central cardiovascular control to aversive stimuli. In contrast to the ‘‘anti-stress’’ action of the Y1 agonist, the Y2 agonist NPY(13–36) not only failed to blunt the cardiovascular effects of stress, but appeared to potentiate them. The pressor effects of stress produced a higher peak systolic, diastolic and pulse pressure than that of controls and a slower return to baseline levels following stress, remaining elevated for nearly 3 h. Vasopressor actions of NPY(13–36) have been previously reported (3,35). Furthermore, NPY(13–36) can counteract the vasodepressor action of NPY itself (51). We did not observe a vasopressor response with this Y2 agonist in non-stressed animals. However, it is possible that a higher dose might have produced this intrinsic action. Previous studies have implicated corticotropin releasing factor (CRF) as a modulator of autonomic, endocrine and behavioral responses to stressful stimuli (23,28). The CRF antagonist alpha-helical7– 41 CRF reduces the anxiogenic and endocrine effects of exposure to an aggressive resident rat. Furthermore, functional studies have provided some evidence that NPY acts in the brain to modulate the central action of endogenous CRF. For example, exogenous NPY inhibits CRF-induced inhibition of gastric acid secretion (16) and the anticonflict (‘‘anxiogenic-like’’) action of CRF in the conflict test (K. Britton, unpublished results). These findings suggest that NPY and CRF may exert a reciprocal modulation of autonomic and behavioral responses to aversive stimuli. In conclusion, the results of the present study confirm and extend the previously reported finding that centrally administered NPY produces a vasodepressor and bradycardic action in resting animals. These cardiovascular effects appear to be due to activation of the Y1 receptor subtype. Social stress, using the resident intruder stress paradigm, produced a significant and lingering increase in blood pressure and heart rate relative to control animals which was inhibited by NPY and the selective Y1 receptor agonist [Leu31,Pro34]-NPY. In contrast, the selective Y2 agonist NPY13–36 produced a marked and prolonged poststress vasopressor action relative to stressed control rats. Thus, central Y1 and Y2 NPY receptor subtypes may exert opposing effects on central cardiovascular control, which are modulated by stressful stimuli. ACKNOWLEDGEMENTS The authors wish to thank Dr. Jean Rivier of the Salk Institute for supplying NPY and NPY receptor agonists, and Dr. Stephen Heinrichs of Neurocrine Biosciences for advice concerning the resident-intruder procedure. This work was supported by a VA Merit Review to KTB.
REFERENCES 1. Adrian, T. E.; Allen, J. M.; Bloom, S. R.; Ghatei, M. A.; Rossnor, M. N.; Roberts, G. W.; Crow, T. J.; Tatemoto, K.;
Polak, J. M. Neuropeptide Y distribution in human brain. Nature (Lond.). 306:584 –586; 1983.
STRESS, NPY, AND BLOOD PRESSURE 2. Aguirre, J. A.; Fuxe, K.; Agnati, L. F. Increased vasopressor potency of the centrally injected neuropeptide Y fragment 13–36 in the adult spontaneously hypertensive awake male rat of the Wistar–Kyoto strain. Acta Physiol. Scand. 139:609 – 610; 1990. 3. Aguirre, J. A.; Fuxe, K.; Agnati, L. F.; von Euler, G. Centrally injected neuropeptide Y (13–36) produces vasopressor effects and antagonizes the vasodepressor action of neuropeptide Y (1–36) in the awake male rat. Neurosci. Lett. 118:5– 8; 1990. 4. Aguirre, J. A.; Fuxe, K.; Andbjer, B.; Eneroth, P.; Agnati, L. F. Changes in pituitary-adrenal activity regulate the central vasodepressor responses induced by neuropeptide Y and adrenaline in the awake unrestrained male rat. Acta Physiol. Scand. 141:35– 44; 1991. 5. Aguirre, J. A.; Hedlund, P. B.; Narvaez, J. A.; Bunnemann, B.; Ganten, D.; Fuxe, K. Increased vasopressor actions of intraventricular neuropeptide Y-(13–36) in spontaneously hypertensive versus normotensive Wistar–Kyoto rats. Possible relationship to increases in Y2 receptor binding in the nucleus tractus solitarius. Brain Res. 684:159 –164; 1995. 6. Boulenger, J. P.; Jerabek, I.; Jolicoeur, F. B.; Lavallee, Y. J.; Leduc, R.; Cadieux, A. Elevated plasma levels of neuropeptide Y in patients with panic disorder. Am. J. Psychiatry 153:114 –116; 1996. 7. Britton, K. T.; McLeod, S.; Koob, G. F.; Hauger, R. Pregnane steroid alphaxolone attenuates anxiogenic behavioral effects of corticotropin releasing factor and stress. Pharmacol. Biochem. Behav. 41:399 – 403; 1992. 8. Broqua, P.; Wettstein, J. G.; Rocher, M. N.; Gauthier–Martin, B. Behavioral effect of neuropeptide Y receptor agonists in the elevated plus maze and fear-potentiated startle procedures. Behav. Pharmacol. 6:215–222; 1995. 9. Cheng, J. T.; Chen, C. F.; Shum, A. Y.; Wang, J. Y.; Chen, H. I. Increase of plasma neuropeptide Y-like immunoreactivity following chronic hypoxia in the rat. Neurosci. Lett. 140: 211–214; 1992. 10. Data Sciences, PA Device Surgical Manual. St. Paul: Data Sciences International; 1994. 11. Fokkema, D. S.; Koolhaas, J. M. Acute and conditioned blood pressure changes in relation to social and psychosocial stimuli in rats. Physiol. Behav. 34:33–38; 1985. 12. Fuhlendorff, J.; Gether, U.; Aakerlund, L.; Langeland-Johansen, N.; Thogersen, H.; Melberg, S. G.; Olsen, U. B.; Thastrup, O.; Schwartz, T. W. [Leu31,Pro34]neuropeptide Y: a specific Y1 receptor agonist. Proc. Natl. Acad. Sci. USA 87:182–186; 1990. 13. Fuxe, K.; Agnati, L. F.; Harfstrand, A.; Zini, I.; Tatemoto, K.; Pich, E. M.; Hokfelt, T.; Mutt, V.; Terenius, L. Central administration of neuropeptide Y induces hypotension bradypnea and EEG synchronization in the rat. Acta Physiol. Scand. 118:189 –192; 1983. 14. Gehlert, D. R. Subtypes of receptors for neuropeptide Y: implications for the targeting of therapeutics. Life Sci. 55: 551–562; 1994. 15. Grundemar, L.; Hakanson, R. Multiple neuropeptide Y receptors are involved in cardiovascular regulation. Peripheral and central mechanisms. Gen. Pharmacol. 24:785–796; 1993. 16. Gue, M.; Junien, J. L.; Reeve, J. R. J.; Rivier, J.; Grandt, D.; Tache, Y. Reversal by NPY, PYY, and 3-36 molecular forms of NPY and PYY of intracisternal CRF-induced inhibition of gastric acid secretion in rats. Br. J. Pharmacol. 118:237–242; 1996. 17. Harfstrand, A. Brain neuropeptide Y mechanisms. Basic as-
91
18.
19. 20.
21. 22.
23. 24.
25.
26.
27.
28.
29. 30.
31. 32. 33.
34.
pects and involvement in cardiovascular and neuroendocrine regulation. Acta. Physiol. Scand. Suppl. 565:1– 83; 1987. Heilig, M. Antisense inhibition of neuropeptide Y (NPY)-Y1 receptor expression blocks the anxiolytic-like action of NPY in amygdala and paradoxically increases feeding. Regul. Pept. 59:201–205; 1995. Heilig, M.; Koob, G. F.; Ekman, R.; Britton, K. T. Corticotropin-releasing factor and neuropeptide Y: role in emotional integration. Trends. Neurosci. 17:80 – 85; 1994. Heilig, M.; McLeod, S.; Brot, M.; Heinrichs, S. C.; Menzaghi, F.; Koob, G. F.; Britton, K. T. Anxiolytic-like action of neuropeptide Y: mediation by Y1 receptors in amygdala, and dissociation from food intake effects. Neuropsychopharmacology. 8:357–363; 1993. Heilig, M.; Murison, R. Intracerebroventricular neuropeptide Y protects against stress-induced gastric erosion in the rat. Eur. J. Pharmacol. 137:127–129; 1987. Heilig, M.; Soderpalm, B.; Engel, J. A.; Widerlov, E. Centrally administered neuropeptide Y (NPY) produces anxiolytic-like effects in animal anxiety models. Psychopharmacology (Berl) 98:524 –529; 1989. Heinrichs, S. C.; Menzaghi, F.; Merlo Pich, E.; Britton, K. T.; Koob, G. F. The role of CRF in behavioral aspects of stress. Ann. NY Acad. Sci. 771:92–104; 1995. Heinrichs, S. C.; Menzaghi, F.; Pich, E. M.; Baldwin, H. A.; Rassnick, S.; Britton, K. T.; Koob, G. F. Anti-stress action of a corticotropin-releasing factor antagonist on behavioral reactivity to stressors of varying type and intensity. Neuropsychopharmacology. 11:179 –186; 1994. Heinrichs, S. C.; Pich, E. M.; Miczek, K. A.; Britton, K. T.; Koob, G. F. Corticotropin-releasing factor antagonist reduces emotionally in socially defeated rats via direct neurotropic action. Brain Res 581:190 –197; 1992. Irwin, M.; Brown, M.; Patterson, T.; Hauger, R.; Mascovich, A.; Grant, I. Neuropeptide Y and natural killer cell activity: findings in depression and Alzheimer caregiver stress. FASEB J. 5:3100 –3107; 1991. Junien, J. L.; Gue, M.; Bueno, L. Neuropeptide Y and sigma ligand (JO 1784) act through a Gi protein to block the psychological stress and corticotropin-releasing factor-induced colonic motor activation in rats. Neuropharmacology 30:1119 –1124; 1991. Koob, G. F.; Heinrichs, S. C.; Pich, E. M.; Menzaghi, F.; Baldwin, H.; Miczek, K. A.; Britton, K. T. The role of corticotropin-releasing factor in behavioral responses to stress. Ciba. Found. Symp. 172:277– 89; discus; 1993. Larhammar, D. Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide. Regul. Peptides 62:1–11; 1996. Macrae, I. M.; Reid, J. L. Cardiovascular effects of neuropeptide Y in the caudal ventrolateral medulla. In: Stumpe, K. O.; Kraft, K.; Faden, A. I., Eds. Opioid peptides in blood pressure control. New York: Springer–Verlag; 1988;112–116. Martin, J. R.; Kneupfer, M. M.; Westfall, T. C. Hemodynamic effects of posterior hypothalamic injection of neuropeptide Y in awake rats. Am. J. Physiol. 261:H814 –H824; 1991. Meehan, W. P.; Tornatzky, W.; Miczek, K. A. Blood pressure via telemetry during social confrontations in rats: effects of clonidine. Physiol. Behav. 58:81– 88; 1995. Michel, M. C. Receptors for neuropeptide Y: multiple subtypes and multiple second messengers [published erratum appears in Trends Pharmacol. Sci. 1991 Dec; 12(12):448]. Trends. Pharmacol. Sci. 12:389 –394; 1991. Morris, M.; Kapoor, V.; Chalmers, J. Plasma neuropeptide Y concentration is increased after hemorrhage in conscious rats:
92
35.
36.
37.
38.
39. 40. 41. 42. 43. 44.
KLEMFUSS ET AL. relative contributions of sympathetic nerves and the adrenal medulla. J. Cardiovasc. Pharmacol. 9:541–545; 1987. Narvaez, J. A.; Aguirre, J. A.; Van der Pleog, I.; Fuxe, K. Intracerebroventricularly administered pertussis toxin blocks the central vasopressor action of neuropeptide Y(13–36) in the awake unrestrained male rat. Neurosci. Lett. 140:273–276; 1992. Penner, S. B.; Smyth, D. D.; Glavin, G. B. Effects of neuropeptide Y and [Leu31,Pro34] neuropeptide Y on experimental gastric lesion formation and gastric secretion in the rat. J. Pharmacol. Exp. Ther. 266:339 –343; 1993. Pich, E. M.; Heinrichs, S. C.; Rivier, C.; Miczek, K. A.; Fisher, D. A.; Koob, G. F. Blockade of pituitary-adrenal axis activation induced by peripheral immunoneutralization of corticotropin-releasing factor does not affect the behavioral response to social defeat stress in rats. Psychoneuroendocrinology. 18:495–507; 1993. Sato, K.; Crofton, J. T.; Wang, Y. X.; Share, L. Effects of gender on the central actions of neuropeptide Y and norepinephrine on vasopressin and blood pressure in the rat. Brain Res. 689:71–78; 1995. Sgoifo, A.; Stilli, D.; Aimi, B.; Parmigiani, S.; Manghi, M.; Musso, E. Behavioral and electrocardiographic responses to social stress in male rats. Physiol. Behav. 55:209 –216; 1994. Sheikh, S. P.; Hakanson, R.; Schwartz, T. W. Y1 and Y2 receptors for neuropeptide Y. FEBS Lett. 245:209 –214; 1989. Tatemoto, K.; Carlquist, M.; Mutt, V. Neuropeptide Y—a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296:659 – 660; 1982. Tseng, C. J.; Mosqueda-Garcia, R.; Appalsamy, M.; Robertson, D. Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ. Res. 64:55– 61; 1989. Viken, R. J.; Johnson, A. K.; Knutson, J. F. Blood pressure, heart rate, and regional resistance in behavioral defense. Physiol. Behav. 50:1097–1101; 1991. Wager-Page, S. A.; Raizada, E.; Veale, W.; Davidson, J. S. Peripheral modulation of duodenal and colonic motility and
45.
46.
47.
48.
49.
50.
51.
52.
arterial pressure by neuropeptide Y, neuropeptide Y fragment 13–36, peptide YY, and pancreatic polypeptide in rats: cholinergic mechanisms. Can. J. Physiol. Pharmacol. 71:768 – 775; 1993. Wahlestedt, C.; Edvinsson, L.; Ekblad, E.; Hakanson, R. Neuropeptide Y potentiates noradrenaline-evoked vasoconstriction: mode of action. J. Pharmacol. Exp. Ther. 234:735–741; 1985. Wahlestedt, C.; Grundemar, L.; Hakanson, R.; Heilig, M.; Shen, G. H.; Zukowska–Grojec, Z.; Reis, D. J. Neuropeptide Y receptor subtypes, Y1 and Y2. Ann. NY Acad. Sci. 611:7– 26; 1990. Wahlestedt, C.; Hakanson, R.; Vaz, C. A.; Zukowska–Grojec, Z. Norepinephrine and neuropeptide Y: vasoconstrictor cooperation in vivo and in vitro. Am. J. Physiol. 258:R736 –R742; 1990. Wahlestedt, C.; Pich, E. M.; Koob, G. F.; Yee, F.; Heilig, M. Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides. Science 259:528 –531; 1993. Walker, P.; Grouzmann, E.; Burnier, M.; Waeber, B. The role of neuropeptide Y in cardiovascular regulation. Trends. Pharmacol. Sci. 12:111–115; 1991. Westfall, T. C.; Martin, J.; Chen, X. L.; Ciarleglio, A.; Carpentier, S.; Henderson, K.; Knuepfer, M.; Beinfeld, M.; Naes, L. Cardiovascular effects and modulation of noradrenergic neurotransmission following central and peripheral administration of neuropeptide Y. Synapse 2:299 –307; 1988. Yang, S. N.; Narvaez, J. A.; Bjelke, B.; Agnati, L. F.; Fuxe, K. Microinjections of subpicomolar amounts of NPY(13–36) into the nucleus tractus solitarius of the rat counteract the vasodepressor responses of NPY(13–36) and of a NPY Y1 receptor agonist. Brain Res. 621:126 –132; 1993. Zukowska–Grojec, Z.; Dayao, E. K.; Karwatowska–Prokopczuk, E.; Hauser, G. J.; Doods, H. N. Stress-induced mesenteric vasoconstriction in rats is mediated by neuropeptide Y-Y1 receptors. Am. J. Physiol. 270:H796 –H800; 1996.
PII S0196-9781(97)00266-0
Cardiovascular Actions of Neuropeptide Y and Social Stress HARRY KLEMFUSS,1 SCOTT SOUTHERLAND AND KAREN T. BRITTON Department of Psychiatry, Veterans Affairs Medical Center and University of California at San Diego, San Diego, CA 92161 Received 10 April 1997; Accepted 28 August 1997 KLEMFUSS, H., S. SOUTHERLAND AND K. T. BRITTON. Cardiovascular actions of Neuropeptide Y and social stress. PEPTIDES 19(1) 85–92, 1998 —The role of central neuropeptide Y (NPY) in the cardiovascular response to social stress was evaluated in freely moving rats using telemetry. In unstressed rats, intracerebroventricular (ICV) administration of NPY and the selective Y1 receptor agonist [Leu31, Pro34]-NPY decreased blood pressure and heart rate, while the selective Y2 agonist NPY13–36 transiently raised blood pressure. NPY and [Leu31,Pro34]-NPY blunted elevations in blood pressure and pulse rate following exposure to the resident-intruder procedure, an established social stress paradigm. In contrast, the Y2 agonist significantly augmented stressinduced pressor effects. These observations indicate that the hypotensive effects of ICV NPY appear to be mediated by the Y1 receptor subtype and the NPY receptor subtypes may mediate opposing cardiovascular actions in response to stressful stimuli. © 1998 Elsevier Science Inc. Stress Resident-intruder Neuropeptide Y Heart rate Locomotor activity
Y1 receptor
NEUROPEPTIDE Y (NPY), a 36-residue member of the pancreatic polypeptide family, is one of the most abundant peptides in the central nervous system (1,41) and among the most highly conserved peptides phylogenetically (29), implying that it may play an important physiological role. At least two subtypes of NPY receptors, designated Y1 and Y2, have been characterized, although other forms have been proposed (14,40). The substituted analogue [Leu31,Pro34]NPY is an agonist displaying high affinity for the Y1 receptor and low affinity for the Y2 receptor (12,46), while the truncated analogue NPY(13–36) is an agonist with low affinity for the Y1 receptor and high affinity for the Y2 receptor type (33). NPY may be a component of the autonomic and endocrine responses to acute stress. Peripherally, NPY directly mediates vasoconstriction and augments the pressor action of vasoconstrictors such as norepinephrine (15,45,49). Receptors of the Y1 subtype mediate vasoconstriction postsynaptically, while those of the Y2 type act presynaptically in the regulation of catecholamine release (46,47). Physical
Y2 receptor
Blood pressure
stressors such as immobilization, cold, hypoxia, hemorrhage, and handling increase plasma NPY concentrations in animals (9,34,52). Increased plasma levels of immunoreactive NPY are also reported in humans experiencing emotional stress (26) and panic disorder (6). Central administration of NPY causes behavioral changes similar to benzodiazepines in several animal models of anxiety including the conflict test (20,22), elevated plus-maze (8,20), and fear-potentiated startle test (8). Y1 receptors in the amygdala are thought to be a critical site for the anxiolytic action of NPY (8,19,20). Intracerebroventricular (ICV) NPY injection protects against some stress-induced pathology, such as gastric lesions (21,27,36) and fear-induced stimulation of colonic motility (16,44). On the other hand, behavioral signs of anxiety are elicited by an antisense oligodeoxynucleotide to NPY messenger ribonucleic acid injected ICV (48) or directly into the amygdala (18). These findings are generally consistent with a role of NPY in stress reduction. In contrast to systemic treatment, ICV administration of NPY lowers blood pressure and heart rate in anesthetized or
1
Requests for reprints should be addressed to Harry Klemfuss, Ph.D., Research Service -151, San Diego VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161; E-mail: [email protected]
85
86
awake, catheterized rats (4,13,30,38). Hypotension and bradycardia are also reported following injection into nucleus of the solitary tract, area postrema, and caudal ventrolateral medulla (15,30,42). However, increases in blood pressure have been reported following NPY injection into area postrema (42), nucleus of the solitary tract, or posterior hypothalamus (31,50). It has been reported that intraventricular administration of the Y1 receptor agonist [Leu31,Pro34]-NPY produces hypotension, while the Y2 agonist NPY(13–36) elicits a vasopressor response in awake, catheterized rats (3). The idea that Y1 and Y2 receptors mediate opposing cardiovascular actions is further supported by the observation that low doses of NPY(13–36) antagonize the hypotension produced by NPY itself in catheterized rats (2,3,35,51). The purpose of the present study was to characterize the effects of NPY on the cardiovascular and locomotor responses to a social stressor, using the ‘‘resident-intruder’’ procedure, a social stressor in which a socially naive male ‘‘intruder’’ rat is placed into the home territory of a resident counterpart, resulting in a variety of agonistic interactions (11,25). The intruder rat typically exhibits autonomic changes consistent with a ‘‘stress response’’, i.e. increased heart rate, blood pressure, and renal and mesenteric resistance to blood flow, during the procedure (11,32,39,43). Heart rate may remain elevated for as long as one h after the procedure, but blood pressure tends to return to normal within 10 – 40 min after the intruder is returned to his home cage (32,43). After the preliminary dose-response study, we tested whether central administration of NPY would attenuate the stress-induced increases in heart rate and blood pressure displayed by the intruder rats during and after the resident-intruder procedure. We then tested the Y1 receptor agonist [Leu31,Pro34]-NPY and the Y2 receptor agonist NPY(13–36) to determine possible receptor heterogeneity in the acute cardiovascular and locomotor effects of NPY. METHOD Subjects and Surgical Procedures Male Wistar rats, weighing 200 –250 g at the time of surgery, were implanted with a sterile Data Sciences (St. Paul, MN) PA-C40 device under halothane anesthesia. This device consists of a transmitter capsule and a fluid-filled catheter coated with antithrombogenic film. The tip of the catheter, which is filled with a biocompatible gel, was inserted into the abdominal aorta just cranial to the bifurcation of the aorta and distal to the renal arteries, and secured with tissue adhesive and a fiber patch according to manufacturer’s recommendations (10). The transmitter capsule was sutured to the inside surface of the peritoneum. The device could be turned on and off without disturbing the rat by passing a magnet beneath the cage. Radiofrequency signals from this device were collected by a receiver located under the shoebox cage for 10 s every min. Mean values for systolic pressure, diastolic pressure, heart rate, and locomo-
KLEMFUSS ET AL.
tor activity (gross changes in position, as detected by the receiver) were calculated, using algorithms provided by Data Sciences, for each 10 s epoch and were recorded using the Dataquest IVt data collection and analysis system. About three weeks after transmitter implant surgery, each rat was again anesthetized with halothane and stereotaxically implanted with an indwelling stainless steel cannula directed at the lateral ventricle, as previously described (7). Each implanted rat was housed singly in a standard shoebox cage (45 3 22 3 22 cm), in a sound- and light-attenuating isolation enclosure, on a 12:12 light-dark cycle, for at least one week between procedures. Food and water were provided ad lib at all times, and litter was changed twice per week. Study 1: NPY Dose–Response Procedure Six implanted rats were maintained on a reverse light schedule with lights on from 1800 to 0600 h. By 0700 h on test days telemetry devices were magnetically activated, and recording of blood pressure, heart rate, and activity began in the test rats’ home cages. At about 0930 h (2.5 h after lights off) each test rat was removed from the home cage and injected icv with porcine NPY (0.5, 1, 8, or 32 ug/rat 5 0.12, 0.24, 1.9, or 6.5 nmol/rat) or an equal volume (2 ul) of saline vehicle, injected over 60 s. These doses cover the range of concentrations at which ICV NPY has been reported to have anxiolytic actions in the rat. All injections were performed by an experienced technician careful to minimize the potential stress of the injection procedure. After injection, each test rat was returned to the home cage. Rats were left undisturbed for at least 6 h after injection while blood pressure and activity were continuously recorded. During injections and transfers between cages, 1 or 2 data time points were lost. Each rat was injected 5– 6 times at approximately weekly intervals. This separation of treatment times was chosen since even mild stress such as sham injection or social interaction may produce changes in behavior and blood pressure lasting as long as several days (Seifritz et al., submitted; Klemfuss et al., in preparation). There was no indication that hemodynamic parameters changed over the 6 week period. Study 2: NPY versus Social Stress The same animals were subjects in the second study which followed generally the same procedures. Each rat received either NPY (8 ug/rat) or vehicle control. Following each drug treatment, rats were either left undisturbed for 6 h or else subjected to the resident-intruder procedure starting 30 min after injection. Each rat was tested once with vehicle and once with 8 ug NPY under both unstressed and social stress conditions, at approximately weekly intervals.
STRESS, NPY, AND BLOOD PRESSURE
Resident–Intruder Procedure The social stress consisted of a confrontation between the telemetry-implanted ‘‘intruder’’ rat and an unoperated ‘‘resident’’ rat, following procedures previously described (24,37). Residents were adult male Long–Evans rats, housed in a large shoebox cage (45 3 38 3 42 cm) with a female Wistar rat. Residents were used only when pups were between the ages of 5–21 days. Bedding was changed approximately once per week, after a resident-intruder confrontation. One half hour before a confrontation, females and pups were removed from the residence cage and housed separately. Thirty min after injection, each intruder rat was gently removed from the home cage and introduced into the residence cage. At the same time the home cage telemetry receiver was placed under the residence cage. For the next 15 min of confrontation, behavior of the two rats was continuously monitored for attacks and evasive behaviors. In the event of a clear attack, or if no attack had occurred by 15 min after confrontation, the intruder rat was placed in a protective wire mesh cage (30 3 18 3 13 cm) within the residence cage. The resident could sense the intruder, and could threaten him, but could not physically attack. Thirty min after the beginning of the confrontation the intruder was returned to his home cage, and blood pressure, heart rate, and locomotor activity were recorded for at least another 6 h. Study 3: Y1 and Y2 Receptor Agonists This study was identical to Study 2, with the following exceptions: 1) Drug treatments consisted of either 8 ug/rat of the Y1 agonist [Leu31,Pro34]-NPY or 6 ug/rat of the Y2 agonist NPY(13–36), or saline vehicle. The molar concentration of each dose was approximately equal to the 8 ug dose of NPY; 2) Lights were on from 0400 to 1600 h, so rats were injected 5.5 h after lights on, and 3) the protective cage for intruders was a perforated plastic container (diameter 16 cm 3 10 cm high). Data Analysis Systolic and diastolic pressure, heart rate, and motor activity means were collapsed into 30 min bins using Dataquestt software and analyzed further using Quattro Pro and SPSS. Pulse pressure (systolic– diastolic) was calculated and analyzed as well. Locomotor activity means were log transformed to normalize variance. Unless otherwise stated, data are expressed as a percent of each rat’s baseline during the 120 min prior to confrontation, to reduce within-group variance. Drugs Porcine NPY (MW 5 4252), the Y1 agonist [Leu31,Pro34]NPY (MW 5 4221), and the Y2 agonist NPY(13–36) (MW 5 2862) were generously supplied by Dr. Jean Rivier of the
87
Salk Institute. All drugs were dissolved in saline immediately before injection. Statistics Analysis of variance (ANOVA) was used to test for effects of NPY dose on pressure, heart rate, and locomotor activity. Repeated measures ANOVA was used to evaluate effects of NPY or NPY receptor agonists in relation to exposure to social stress and time post-injection as within-subjects variables. Bonferroni-corrected ANOVA, with planned comparisons (Tukey HSD) when appropriate, were used to localize effects. Significance is defined as p , 0.05. All results in the text are expressed as mean 6 SD. RESULTS Control Rats Freely moving rats had a baseline systolic pressure of 131 6 9 mmHg and diastolic pressure of 95 6 8 mmHg (n 5 60 observations in 6 rats). Baseline heart rate was 325 6 32 beats/min. Immediately following ICV injection of either drug or saline all rats showed a transient increase in blood pressure and heart rate, which returned toward baseline levels within 15 min. Study 1: NPY Dose Response in Unstressed Rats All four doses of NPY (0.5 to 32 ug/rat) significantly lowered systolic, diastolic and pulse pressure, and heart rate, averaged across 3 h beginning 30 min after injection to avoid injection artifacts. Averaged locomotor activity was not significantly affected by NPY treatment. Figure 1 shows that pulse pressure was significantly decreased at all timepoints, after all doses, compared to control (F(4, 24) . 2.8; p , 0.05). Systolic and diastolic pressure were significantly decreased at the first and third timepoint at all doses, and at the last two time points by the 8 and 32 ug doses. Heart rate was decreased at all time points by the 8 and 32 ug doses. Locomotor activity was statistically decreased at the last three time points only by the lowest (0.5 ug) dose of NPY (p , 0.05, planned comparison to vehicle). Social Stress Following Saline Injection (Study 2) During exposure to threat in the resident-intruder confrontation, blood pressure was not significantly increased relative to baseline in saline-pretreated intruders (Fig. 2; F(1, 20) , 2.5). Both heart rate and locomotor activity rose significantly during the confrontation (F(1, 20) . 8.0). Although most residents attacked the intruder within a few minutes of confrontation, clear defeats (defined by submissive posture of the intruder following attack) occurred in fewer than 10% of confrontations. Defeat did not make any apparent difference in acute or chronic pressure or heart rate response, so data from defeated and undefeated intruders are combined.
88
KLEMFUSS ET AL.
2 (left side), and also decreased gross activity 90 –150 min after injection. Graphs on the right side of Figure 2 show comparable data
FIG. 1. Time course of blood pressure, heart rate, and locomotor activity following graded doses of NPY. All data are presented as percent of each animal’s baseline 6 SEM. Nine rats were injected at time 0 with saline vehicle (E), and 5 rats were given each dose of NPY: 0.5 ug/rat (E), 1.0 ug/rat (h), 8 ug/rat (Œ), and 32 ug/rat (■).
Effect of NPY Pretreatment on Responses to Social Stress The effects of the 8 ug/rat dose were generally similar in unstressed rats in Study 2 as in Study 1: NPY produced significant reductions in mean systolic pressure, diastolic pressure, pulse pressure, and heart rate, as shown in Figure
FIG. 2. Effects of NPY and social stress on blood pressure, heart rate, and activity. Rats were injected at time 0 with saline E or 8 ug NPY Œ. Graphs on the left show data from unstressed animals in Study 2. Graphs on the right show comparable data from rats exposed to social stress 30 – 60 min after injection (shaded bar). *Indicates a significant difference from vehicle control (p , 0.05, planned comparisons).
STRESS, NPY, AND BLOOD PRESSURE
89
from rats exposed to social stress 30–60 min after injection (shaded bar). During the 30 min confrontation, intruders showed transient increases in systolic pressure (p , 0.05), heart rate (p , 0.01) and activity (p , 0.001) compared to unstressed animals, without significant interaction between social stress and drug treatment during the confrontation. During the 2 h interval following social stress, the hypotensive effects of NPY were attenuated, but still significantly lower than controls (p , 0.05, Tukey’s HSD). NPY effects on other measures were also decreased, and no longer reached statistical significance. The dose vs. social stress interaction term was significant for systolic pressure (F(1, 5) 5 6.72) and pulse pressure (F(1, 5) 5 7.1 by repeated measures ANOVA (p , 0.05). Diastolic pressure (F(1, 5) 5 6.2), heart rate (F(1, 5) 5 0.1), and gross activity (F(1, 5) 5 4.53) fail to show significant interactions between NPY treatment and social stress. Y1 and Y2 Receptor Agonists in Unstressed Rats As shown in Fig. 3 (left side), the Y1 receptor agonist [Leu31,Pro34]-NPY significantly lowered systolic, diastolic, and pulse pressures by 5–10% compared to saline control immediately following injection and for at least 3 h after injection (p , 0.05 at all time points). These actions were comparable in magnitude and duration to the effects of an equimolar dose of NPY. Unlike NPY, the Y1 agonist had no significant effect on heart rate (p . 0.3). In contrast to both NPY and [Leu31,Pro34]-NPY, the Y2 agonist NPY(13–36) did not alter lower systolic pressure or heart rate in unstressed animals, but transiently raised systolic pressure (p , 0.05) compared to vehicle. Neither agonist produced a significant change in gross motor activity. Y1 and Y2 Receptor Agonists in Socially Stressed Rats During the confrontation, intruders showed increases in blood pressure parameters and heart rate (p , 0.001), but not in locomotor activity (p 5 0.17). There were no significant interactions between social stress and drug treatment during the confrontation (Fig. 3 right side). The Y1 receptor agonist [Leu31,Pro34]-NPY blunted the pressor response and tachycardia observed following social stress. In contrast, the systolic pressure of NPY(13– 36)-treated rats remained significantly elevated for at least 90 min after the end of the confrontation, while the systolic pressure of saline controls had returned to baseline levels by 30 min after confrontation. In stressed rats, mean systolic pressure was significantly lower in Y1 agonist-treated animals compared to Y2-agonist-treated rats, during the 2 h after return to the home cage (p , 0.05, Newman–Keuls). The interaction between agonist treatment and social stress on systolic pressure was statistically significant by repeated measures ANOVA (F(2, 10) 5 5.56; p , 0.05). The Y1 receptor agonist [Leu31,Pro34]-NPY also attenu-
FIG. 3. Effects of Y1 and Y2 agonists on responses to social stress. Rats were injected at time 0 with saline E, 8 ug(leu,pro)NPY Œ or 6 ug NPY(13–36)■. Graphs on the left show data from unstressed animals in Study 3. *Indicates a significant difference from vehicle control (p , 0.05, planned comparisons). Graphs on the right side show comparable data from rats exposed to social stress 30 – 60 min after injection (shaded bar).
ated stress-induced tachycardia and blood pressure changes during the first 60 min following stress. No other significant main effects of stress or drug treatment, or interactions
90
KLEMFUSS ET AL.
between agonist treatment and social stress, were found in stressed rats (F(2, 10) , 2.3; p . 0.15). DISCUSSION The results of the present study confirm that central administration of NPY to freely-moving rats produces a marked and prolonged vasodepressor and bradycardic action (4,13,17) at doses that have potent anxiolytic actions in other behavioral paradigms (8,22). These actions appear to be mediated by the Y1 receptor subtype since equimolar doses of NPY and the selective Y1 agonist [Leu31,Pro34]NPY lowered blood pressure to a similar degree and with a similar time course, while the selective Y2 agonist NPY(13– 36) was inactive. Thus, the predominant cardiovascular action of centrally administered NPY is to lower blood pressure via Y1 receptor activation. The resident-intruder stress paradigm is an ethologically relevant procedure that produces behavioral and physiological changes consistent with increased stress, including tachycardia, increased blood pressure, locomotor activation and release of ACTH and corticosterone (11,32,37,39,43). The use of telemetry in the present study enabled us to preserve the full expression of the intruder cardiovascular and locomotor responses. Our results indicate that these stress-induced effects are more prominent when animals are tested in their inactive, i.e. lights on, period. In this period, the rats show a marked vasopressor and locomotor response that persists about an hour following confrontation. When animals were tested during their normally active period, i.e., lights out, these changes were less marked and returned to baseline levels in less than 30 min. These light vs. dark differences were also apparent in the response to the injection procedure itself, and may be due in part to the fact that baseline levels of blood pressure, pulse and locomotor activation are higher during the dark period, thus obscurring moderate stress-induced changes via ‘‘ceiling effects’’. The pressor and tachycardiac responses observed in intruder rats following social confrontation with a resident male were significantly reduced by pretreatment with either NPY itself or the Y1 agonist [Leu31,Pro34]NPY. This result is consistent with prior studies showing potent stress-reducing effects of these compounds in a variety of animal models of anxiety. The anxiolytic-like properties of NPY appear to be mediated by the Y1 receptor (17,20), consistent with the current findings. Y1 agonists produce a marked release of punished responding in the conflict test (5), elevated plus maze, and startle paradigms (8). Stress-induced mesenteric vasoconstriction also appears to be mediated via the NPY Y1 receptor subtype (52). Overall, these findings support the hypoth-
esis that Y1 receptors in the brain were involved in central cardiovascular control to aversive stimuli. In contrast to the ‘‘anti-stress’’ action of the Y1 agonist, the Y2 agonist NPY(13–36) not only failed to blunt the cardiovascular effects of stress, but appeared to potentiate them. The pressor effects of stress produced a higher peak systolic, diastolic and pulse pressure than that of controls and a slower return to baseline levels following stress, remaining elevated for nearly 3 h. Vasopressor actions of NPY(13–36) have been previously reported (3,35). Furthermore, NPY(13–36) can counteract the vasodepressor action of NPY itself (51). We did not observe a vasopressor response with this Y2 agonist in non-stressed animals. However, it is possible that a higher dose might have produced this intrinsic action. Previous studies have implicated corticotropin releasing factor (CRF) as a modulator of autonomic, endocrine and behavioral responses to stressful stimuli (23,28). The CRF antagonist alpha-helical7– 41 CRF reduces the anxiogenic and endocrine effects of exposure to an aggressive resident rat. Furthermore, functional studies have provided some evidence that NPY acts in the brain to modulate the central action of endogenous CRF. For example, exogenous NPY inhibits CRF-induced inhibition of gastric acid secretion (16) and the anticonflict (‘‘anxiogenic-like’’) action of CRF in the conflict test (K. Britton, unpublished results). These findings suggest that NPY and CRF may exert a reciprocal modulation of autonomic and behavioral responses to aversive stimuli. In conclusion, the results of the present study confirm and extend the previously reported finding that centrally administered NPY produces a vasodepressor and bradycardic action in resting animals. These cardiovascular effects appear to be due to activation of the Y1 receptor subtype. Social stress, using the resident intruder stress paradigm, produced a significant and lingering increase in blood pressure and heart rate relative to control animals which was inhibited by NPY and the selective Y1 receptor agonist [Leu31,Pro34]-NPY. In contrast, the selective Y2 agonist NPY13–36 produced a marked and prolonged poststress vasopressor action relative to stressed control rats. Thus, central Y1 and Y2 NPY receptor subtypes may exert opposing effects on central cardiovascular control, which are modulated by stressful stimuli. ACKNOWLEDGEMENTS The authors wish to thank Dr. Jean Rivier of the Salk Institute for supplying NPY and NPY receptor agonists, and Dr. Stephen Heinrichs of Neurocrine Biosciences for advice concerning the resident-intruder procedure. This work was supported by a VA Merit Review to KTB.
REFERENCES 1. Adrian, T. E.; Allen, J. M.; Bloom, S. R.; Ghatei, M. A.; Rossnor, M. N.; Roberts, G. W.; Crow, T. J.; Tatemoto, K.;
Polak, J. M. Neuropeptide Y distribution in human brain. Nature (Lond.). 306:584 –586; 1983.
STRESS, NPY, AND BLOOD PRESSURE 2. Aguirre, J. A.; Fuxe, K.; Agnati, L. F. Increased vasopressor potency of the centrally injected neuropeptide Y fragment 13–36 in the adult spontaneously hypertensive awake male rat of the Wistar–Kyoto strain. Acta Physiol. Scand. 139:609 – 610; 1990. 3. Aguirre, J. A.; Fuxe, K.; Agnati, L. F.; von Euler, G. Centrally injected neuropeptide Y (13–36) produces vasopressor effects and antagonizes the vasodepressor action of neuropeptide Y (1–36) in the awake male rat. Neurosci. Lett. 118:5– 8; 1990. 4. Aguirre, J. A.; Fuxe, K.; Andbjer, B.; Eneroth, P.; Agnati, L. F. Changes in pituitary-adrenal activity regulate the central vasodepressor responses induced by neuropeptide Y and adrenaline in the awake unrestrained male rat. Acta Physiol. Scand. 141:35– 44; 1991. 5. Aguirre, J. A.; Hedlund, P. B.; Narvaez, J. A.; Bunnemann, B.; Ganten, D.; Fuxe, K. Increased vasopressor actions of intraventricular neuropeptide Y-(13–36) in spontaneously hypertensive versus normotensive Wistar–Kyoto rats. Possible relationship to increases in Y2 receptor binding in the nucleus tractus solitarius. Brain Res. 684:159 –164; 1995. 6. Boulenger, J. P.; Jerabek, I.; Jolicoeur, F. B.; Lavallee, Y. J.; Leduc, R.; Cadieux, A. Elevated plasma levels of neuropeptide Y in patients with panic disorder. Am. J. Psychiatry 153:114 –116; 1996. 7. Britton, K. T.; McLeod, S.; Koob, G. F.; Hauger, R. Pregnane steroid alphaxolone attenuates anxiogenic behavioral effects of corticotropin releasing factor and stress. Pharmacol. Biochem. Behav. 41:399 – 403; 1992. 8. Broqua, P.; Wettstein, J. G.; Rocher, M. N.; Gauthier–Martin, B. Behavioral effect of neuropeptide Y receptor agonists in the elevated plus maze and fear-potentiated startle procedures. Behav. Pharmacol. 6:215–222; 1995. 9. Cheng, J. T.; Chen, C. F.; Shum, A. Y.; Wang, J. Y.; Chen, H. I. Increase of plasma neuropeptide Y-like immunoreactivity following chronic hypoxia in the rat. Neurosci. Lett. 140: 211–214; 1992. 10. Data Sciences, PA Device Surgical Manual. St. Paul: Data Sciences International; 1994. 11. Fokkema, D. S.; Koolhaas, J. M. Acute and conditioned blood pressure changes in relation to social and psychosocial stimuli in rats. Physiol. Behav. 34:33–38; 1985. 12. Fuhlendorff, J.; Gether, U.; Aakerlund, L.; Langeland-Johansen, N.; Thogersen, H.; Melberg, S. G.; Olsen, U. B.; Thastrup, O.; Schwartz, T. W. [Leu31,Pro34]neuropeptide Y: a specific Y1 receptor agonist. Proc. Natl. Acad. Sci. USA 87:182–186; 1990. 13. Fuxe, K.; Agnati, L. F.; Harfstrand, A.; Zini, I.; Tatemoto, K.; Pich, E. M.; Hokfelt, T.; Mutt, V.; Terenius, L. Central administration of neuropeptide Y induces hypotension bradypnea and EEG synchronization in the rat. Acta Physiol. Scand. 118:189 –192; 1983. 14. Gehlert, D. R. Subtypes of receptors for neuropeptide Y: implications for the targeting of therapeutics. Life Sci. 55: 551–562; 1994. 15. Grundemar, L.; Hakanson, R. Multiple neuropeptide Y receptors are involved in cardiovascular regulation. Peripheral and central mechanisms. Gen. Pharmacol. 24:785–796; 1993. 16. Gue, M.; Junien, J. L.; Reeve, J. R. J.; Rivier, J.; Grandt, D.; Tache, Y. Reversal by NPY, PYY, and 3-36 molecular forms of NPY and PYY of intracisternal CRF-induced inhibition of gastric acid secretion in rats. Br. J. Pharmacol. 118:237–242; 1996. 17. Harfstrand, A. Brain neuropeptide Y mechanisms. Basic as-
91
18.
19. 20.
21. 22.
23. 24.
25.
26.
27.
28.
29. 30.
31. 32. 33.
34.
pects and involvement in cardiovascular and neuroendocrine regulation. Acta. Physiol. Scand. Suppl. 565:1– 83; 1987. Heilig, M. Antisense inhibition of neuropeptide Y (NPY)-Y1 receptor expression blocks the anxiolytic-like action of NPY in amygdala and paradoxically increases feeding. Regul. Pept. 59:201–205; 1995. Heilig, M.; Koob, G. F.; Ekman, R.; Britton, K. T. Corticotropin-releasing factor and neuropeptide Y: role in emotional integration. Trends. Neurosci. 17:80 – 85; 1994. Heilig, M.; McLeod, S.; Brot, M.; Heinrichs, S. C.; Menzaghi, F.; Koob, G. F.; Britton, K. T. Anxiolytic-like action of neuropeptide Y: mediation by Y1 receptors in amygdala, and dissociation from food intake effects. Neuropsychopharmacology. 8:357–363; 1993. Heilig, M.; Murison, R. Intracerebroventricular neuropeptide Y protects against stress-induced gastric erosion in the rat. Eur. J. Pharmacol. 137:127–129; 1987. Heilig, M.; Soderpalm, B.; Engel, J. A.; Widerlov, E. Centrally administered neuropeptide Y (NPY) produces anxiolytic-like effects in animal anxiety models. Psychopharmacology (Berl) 98:524 –529; 1989. Heinrichs, S. C.; Menzaghi, F.; Merlo Pich, E.; Britton, K. T.; Koob, G. F. The role of CRF in behavioral aspects of stress. Ann. NY Acad. Sci. 771:92–104; 1995. Heinrichs, S. C.; Menzaghi, F.; Pich, E. M.; Baldwin, H. A.; Rassnick, S.; Britton, K. T.; Koob, G. F. Anti-stress action of a corticotropin-releasing factor antagonist on behavioral reactivity to stressors of varying type and intensity. Neuropsychopharmacology. 11:179 –186; 1994. Heinrichs, S. C.; Pich, E. M.; Miczek, K. A.; Britton, K. T.; Koob, G. F. Corticotropin-releasing factor antagonist reduces emotionally in socially defeated rats via direct neurotropic action. Brain Res 581:190 –197; 1992. Irwin, M.; Brown, M.; Patterson, T.; Hauger, R.; Mascovich, A.; Grant, I. Neuropeptide Y and natural killer cell activity: findings in depression and Alzheimer caregiver stress. FASEB J. 5:3100 –3107; 1991. Junien, J. L.; Gue, M.; Bueno, L. Neuropeptide Y and sigma ligand (JO 1784) act through a Gi protein to block the psychological stress and corticotropin-releasing factor-induced colonic motor activation in rats. Neuropharmacology 30:1119 –1124; 1991. Koob, G. F.; Heinrichs, S. C.; Pich, E. M.; Menzaghi, F.; Baldwin, H.; Miczek, K. A.; Britton, K. T. The role of corticotropin-releasing factor in behavioral responses to stress. Ciba. Found. Symp. 172:277– 89; discus; 1993. Larhammar, D. Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide. Regul. Peptides 62:1–11; 1996. Macrae, I. M.; Reid, J. L. Cardiovascular effects of neuropeptide Y in the caudal ventrolateral medulla. In: Stumpe, K. O.; Kraft, K.; Faden, A. I., Eds. Opioid peptides in blood pressure control. New York: Springer–Verlag; 1988;112–116. Martin, J. R.; Kneupfer, M. M.; Westfall, T. C. Hemodynamic effects of posterior hypothalamic injection of neuropeptide Y in awake rats. Am. J. Physiol. 261:H814 –H824; 1991. Meehan, W. P.; Tornatzky, W.; Miczek, K. A. Blood pressure via telemetry during social confrontations in rats: effects of clonidine. Physiol. Behav. 58:81– 88; 1995. Michel, M. C. Receptors for neuropeptide Y: multiple subtypes and multiple second messengers [published erratum appears in Trends Pharmacol. Sci. 1991 Dec; 12(12):448]. Trends. Pharmacol. Sci. 12:389 –394; 1991. Morris, M.; Kapoor, V.; Chalmers, J. Plasma neuropeptide Y concentration is increased after hemorrhage in conscious rats:
92
35.
36.
37.
38.
39. 40. 41. 42. 43. 44.
KLEMFUSS ET AL. relative contributions of sympathetic nerves and the adrenal medulla. J. Cardiovasc. Pharmacol. 9:541–545; 1987. Narvaez, J. A.; Aguirre, J. A.; Van der Pleog, I.; Fuxe, K. Intracerebroventricularly administered pertussis toxin blocks the central vasopressor action of neuropeptide Y(13–36) in the awake unrestrained male rat. Neurosci. Lett. 140:273–276; 1992. Penner, S. B.; Smyth, D. D.; Glavin, G. B. Effects of neuropeptide Y and [Leu31,Pro34] neuropeptide Y on experimental gastric lesion formation and gastric secretion in the rat. J. Pharmacol. Exp. Ther. 266:339 –343; 1993. Pich, E. M.; Heinrichs, S. C.; Rivier, C.; Miczek, K. A.; Fisher, D. A.; Koob, G. F. Blockade of pituitary-adrenal axis activation induced by peripheral immunoneutralization of corticotropin-releasing factor does not affect the behavioral response to social defeat stress in rats. Psychoneuroendocrinology. 18:495–507; 1993. Sato, K.; Crofton, J. T.; Wang, Y. X.; Share, L. Effects of gender on the central actions of neuropeptide Y and norepinephrine on vasopressin and blood pressure in the rat. Brain Res. 689:71–78; 1995. Sgoifo, A.; Stilli, D.; Aimi, B.; Parmigiani, S.; Manghi, M.; Musso, E. Behavioral and electrocardiographic responses to social stress in male rats. Physiol. Behav. 55:209 –216; 1994. Sheikh, S. P.; Hakanson, R.; Schwartz, T. W. Y1 and Y2 receptors for neuropeptide Y. FEBS Lett. 245:209 –214; 1989. Tatemoto, K.; Carlquist, M.; Mutt, V. Neuropeptide Y—a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296:659 – 660; 1982. Tseng, C. J.; Mosqueda-Garcia, R.; Appalsamy, M.; Robertson, D. Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ. Res. 64:55– 61; 1989. Viken, R. J.; Johnson, A. K.; Knutson, J. F. Blood pressure, heart rate, and regional resistance in behavioral defense. Physiol. Behav. 50:1097–1101; 1991. Wager-Page, S. A.; Raizada, E.; Veale, W.; Davidson, J. S. Peripheral modulation of duodenal and colonic motility and
45.
46.
47.
48.
49.
50.
51.
52.
arterial pressure by neuropeptide Y, neuropeptide Y fragment 13–36, peptide YY, and pancreatic polypeptide in rats: cholinergic mechanisms. Can. J. Physiol. Pharmacol. 71:768 – 775; 1993. Wahlestedt, C.; Edvinsson, L.; Ekblad, E.; Hakanson, R. Neuropeptide Y potentiates noradrenaline-evoked vasoconstriction: mode of action. J. Pharmacol. Exp. Ther. 234:735–741; 1985. Wahlestedt, C.; Grundemar, L.; Hakanson, R.; Heilig, M.; Shen, G. H.; Zukowska–Grojec, Z.; Reis, D. J. Neuropeptide Y receptor subtypes, Y1 and Y2. Ann. NY Acad. Sci. 611:7– 26; 1990. Wahlestedt, C.; Hakanson, R.; Vaz, C. A.; Zukowska–Grojec, Z. Norepinephrine and neuropeptide Y: vasoconstrictor cooperation in vivo and in vitro. Am. J. Physiol. 258:R736 –R742; 1990. Wahlestedt, C.; Pich, E. M.; Koob, G. F.; Yee, F.; Heilig, M. Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides. Science 259:528 –531; 1993. Walker, P.; Grouzmann, E.; Burnier, M.; Waeber, B. The role of neuropeptide Y in cardiovascular regulation. Trends. Pharmacol. Sci. 12:111–115; 1991. Westfall, T. C.; Martin, J.; Chen, X. L.; Ciarleglio, A.; Carpentier, S.; Henderson, K.; Knuepfer, M.; Beinfeld, M.; Naes, L. Cardiovascular effects and modulation of noradrenergic neurotransmission following central and peripheral administration of neuropeptide Y. Synapse 2:299 –307; 1988. Yang, S. N.; Narvaez, J. A.; Bjelke, B.; Agnati, L. F.; Fuxe, K. Microinjections of subpicomolar amounts of NPY(13–36) into the nucleus tractus solitarius of the rat counteract the vasodepressor responses of NPY(13–36) and of a NPY Y1 receptor agonist. Brain Res. 621:126 –132; 1993. Zukowska–Grojec, Z.; Dayao, E. K.; Karwatowska–Prokopczuk, E.; Hauser, G. J.; Doods, H. N. Stress-induced mesenteric vasoconstriction in rats is mediated by neuropeptide Y-Y1 receptors. Am. J. Physiol. 270:H796 –H800; 1996.