Anxiolytic-like effect of neuropeptide Y (NPY), but not other peptides in an operant conflict test

Regulatory Peptides, 41 (1992) 61-69 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00

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REGPEP 01215

Anxiolytic-like effect of neuropeptide Y (NPY), but not other peptides in an operant conflict test Markus Heilig a, Sarah M c L e o d b, George K. Koob a and Karen T. Britton b ~Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA (USA) and b Department of Psychiatry, San Diego Veterans' Administration Medical Center, and University of California at San Diego, La Jolla, CA (USA) (Received 18 March 1992; Revised version received and accepted 15 June 1992)

Key words: Neuropeptide Y; Anxiety; Conflict test; Rat

Summary The peptide messengers neuropeptide Y (NPY), growth hormone-releasing hormone (GHRH), atrial natriuretic peptide (ANP) and [3-endorphin (BEND) were tested in an animal model of anxiety, the Geller-Seifter conflict test. Rats were subjected to a multiple schedule consisting of three components: in the first component, lever-pressing produced food-reward ('unpunished responding'). The second component was a timeout period, during which lever-pressing had no consequences. During the third component, lever-pressing produced food-reward, but was also punished by an incremental foot-shock ('punished responding'). After establishing a stable baseline of both unpunished and punished responding, animals were injected with various doses of NPY, GHRH, ANP, BEND, or with saline into the lateral cerebral ventricle, and testing was repeated. While changes in unpunished responding can reflect alterations in performance factors or motivational strength, increases in punished responding have previously been shown to be highly specific for anxiety-reducing drugs, such as the bensodiazepines. NPY markedly and dose-dependently increased punished responding. A smaller increase of unpunished responding was also seen. These results add further support to the hypothesis that NPY may be an endogenous anxiolytic. GHRH, ANP and END did not affect punished responding.

Correspondence to: M. Heilig, Dept. of Neuropharmacology, CVN-7, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA.

62 Introduction

Numerous peptide messengers, acting both as hormones and neurotransmitters, have been isolated and characterized in the last years. While many of these have been shown to profoundly affect various physiologic functions, considerably less is known about their psychotropic actions. The parallel occurrence of psychological, autonomic and endocrine events in various emotional states prompts the question whether peptides affecting hormonal and autonomic nervous system homeostasis are also involved in modulating the concomitant psychological states. Such a dual role has, e.g., recently been proposed for the neuropeptide Corticotropin-Releasing Factor (CRF) (see, e.g., Ref. 1). In particular, anxiety is strongly correlated with autonomic and endocrine changes (for a review, see Ref. 3). Little is known about the role of several hormonally and autonomically active peptides in this emotional state. Human studies of anxietyproducing (anxiogenic) or anxiety-decreasing (anxiolytic) actions of peptide messengers are made difficult by the chemical nature of this class of neurotransmitters, since peptides will normally not pass the blood-brain barrier to enter the central nervous system upon peripheral administration. Pharmacologically validated animal models of anxiety are available as a means to assess anxiolytic or anxiogenic actions of neurotransmitters and transmitter candidates. In the present study, we have used such a model, the Geller-Seifter conflict test, to examine possible effects of several neuropeptides on anxiety. [3-Endorphin (BEND), a processing product of the pro-opiomelanocortin (POMC) molecule is synthesized and released in parallel with adrenocorticotropin (ACTH) (see, e.g., Ref. 4). Furthermore, endogenous opioids have been implicated in anxiety mechanisms [ 10]. Growth hormone-releasing hormone (GHRH), named for its action as a hypothalamic release factor for growth hormone [17] has in addition been shown to stimulate food intake, and this is thought to represent a central action of the peptide [ 18]. Little is known about other possible behavioural effects o f G H R H . Atrial natriuretic polypeptide (ANP) is present in the central nervous system as well as in the heart [15]. It has been suggested that ANP participates in the regulation of the hypothalamo-pituitary-adrenal (HPA) axis [13]. Finally, neuropeptide Y (NPY) is abundantly present in both hypothalamic and limbic brain areas, and produces a host of endocrine and autonomic effects upon central administration. In addition, significant evidence suggests NPY to be involved in mechanisms of anxiety and depression (for a review, see Ref. 8). We have therefore examined BEND, G H R H , ANP and NPY for anxiety-modifying effects in the Geller-Seifter paradigm. Materials and Methods

Subjects Male albino Wistar rats, weighing 200-275 g at the start of the experiment were used. Animals were housed three per cage, in a light- and temperature-controlled environment. For operant training, rats were food deprived to 85 To of their free-feeding weight, and then maintained on 15 g food per day in addition to that earned during testing.

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Surgical procedure and injections Under halothane anesthesia, animals were stereotactically implanted with 23 gauge guide cannulas aimed 1 m m dorsal to a planned injection site in the lateral cerebral ventricle ( - 0 . 6 m m posterior and 2.0 m m lateral to bregma, 3.2 m m ventral to skull surface; tooth bar at + 5.0 mm). Cannulas were secured to the skull using stainless steel screws and acrylic cement, and were closed with obturators when not used. At least 7 days of recovery were allowed after surgery. Peptides or vehicle were injected over one minute, through a 30 gauge injector connected to a Hamilton syringe, in a volume of 5 ~tl.

Conflict test Training and testing of animals was performed in sound-attenuated operant chambers (Coulbourn Instruments, Lehigh Valley, PA). Chambers were equipped with stainless steel bar floors, through which electric shock could be delivered. Animals were first trained to lever-press for 45 mg of Noyes food pellets on a continuous reinforcement schedule. They were subsequently switched to a random interval 30 s reinforcement schedule, and finally trained on a multiple-schedule conflict test with incremental shock [ 14]. The conflict test consisted of three components: a pure reward (unpunished) component, a time-out component, and a conflict (punished) component. Responses made during the reward component were reinforced on a random interval 30 s schedule in a darkened chamber. The chamber was illuminated with a house light during the time-out component, and responses were not reinforced. The third component (conflict) was signaled by three flashing lights above the lever (I/s) and responses were both rewarded with food and punished with footshocks on a continuous reinforcement schedule. Footshock consisted of a scrambled biphasic square-wave produced by a SGS-003 stimulator (BRS/L VE Division of Tech. Serv., Laurel, MD). During the conflict component, shock was incremented in 0.15 mA steps to a maximum of 3.3 mA with delivery of every reinforcer. A testing session consisted of a 5 rain reward period, a 2 min time out, and a 2 min conflict period presented in succession. Testing sessions were repeated on successive days, at the same time of day. For each animal, baseline responding during both unpunished and punished components of the test was determined over two to three sessions preceding the session during which drug effects were studied. For each subject, responding during the actual testing session (number of lever presses) was expressed as percentage of this individuals baseline.

Tail flick test In the N P Y experiment, a tail-flick test was performed immediately following the session during which drug effects had been studied. In this test, rats were held, and the tail was dipped 3.5 cm into 55°C water. The latency for the tail to flick was measured [9].

Chemicals The source of the peptides, and time interval between injection and testing was: NPY, Bachem California, Torrance, CA; 60 min. G H R H was provided by Dr. Jean

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Rivier (The Salk Institute, La Jolla, CA); 30 rain. ANP was provided by Dr. Roger Guillemin; 30 min. END was provided by Dr. Nicholas Ling (The Whittier Institute, La Jolla, CA); 15 rain. Vehicle for all peptides was normal saline. For each peptide, control injection of vehicle was given at a pretreatment interval equal to that of the peptide tested. Statistics

ANOVA was performed for each peptide with respect to the treatment effect. Unpunished and punished responding were analyzed separately. Individual groups were compared to the vehicle treated controls using Tukey's multiple comparison test. Results NPY

In this experiment, average baseline responding on the unpunished component was 213.4+ 11.2, and on the punished component 19.3 +0.8 lever presses per session (mean + S.E.M.). Effects of NPY (0.2-5.0 nmol) on unpunished and punished responding are shown in Fig. 1. NPY increased punished responding in a dose dependent manner (F(3,26)= 9.0, P < 0.001). At the highest dose, punished responding was doubled (P< 0.001 vs. controls on Tukey's test). An increase in unpunished responding

280

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.

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---0-

UNPUNISHED

-~-

PUNISHED

,~ 200 z

160 z

1,0o 150

0 i0

~ 02.

~ 1.0

5 i0 NPY

0 i0

~ 02.

~ 1.0

6,0

(nmoO

Fig. I. Unpunished (left panel) and punished (right panel) responding after icv injection of vehicle, or NPY (0.2-5 nmol). Responding, i.e., the number of lever-presses during the 5 min unpunished, or the 2 rain punished test component, respectively, is expressed as percentage of a baseline value obtained over two to three sessions preceding the session in which injections were made. Values are means of 6 - 8 animals, and error bars represent S.E.M. For statistical analysis, see Results.

65 TABLE I Unpunished and punished responding after icv administration of various doses of BEND, GHRH or ANP, expressed as percentage of previously obtained baseline values Values are means + S.E.M. n = 4-6 Peptide

Dose

Unpunished responsing

Punished responsing

BEND

0.0 nmol 0.2 nmol 0.4 nmol 0.8 nmol

103.8 + 5.4 56.0 ± 12.8 24.2 ± 8.0 0.3 + 0.3

96.7 ± 8.0 94.3 ± 12.6 82.5 ± 15.3 0.0 ± 0.0

GHRH

0.0 pmol 0.2 pmol 2.0 pmol 20.0 pmol 200.0 pmol

112.8 _+7.5 115.1 ± 17.4 114.5 + 10.3 100.4 + 10.4 91.6 ± 9.9

105.5 + 12.6 115.8± 15.3 112.2 + 15.7 110.5 ± 20.7 112.9 + 3.8

0.0 nmol 1.5 nmol 3.0 nmol 6.0 nmol

103.3 + 6.4 83.6 _+15,7 108.3 ± 4.0 113.7 +__7.8

99.1 ± 7.5 99.3 ± 4.7 94.4 ± 3.8 96.9 + 5.8

ANP

was also seen ( F ( 3 , 2 6 ) - 4 . 2 , P = 0.015). This increase, however, was at m o s t about 30~o, and h a d already reached a plateau at 1.0 nmol. In the tail flick experiment which followed administration of N P Y in order to determine possible effects on pain threshold, no difference in latency to tail flick was seen ( d a t a not shown). BEND

R e s p o n d i n g after administration o f B E N D (0.2-0.8 nmol) is shown in Table I. Unpunished r e s p o n d i n g was d e c r e a s e d in a d o s e - d e p e n d e n t manner. Punished r e s p o n d i n g was not affected except for the highest dose. A t this dose, both unpunished and punished r e s p o n d i n g were eliminated, and visible signs o f m o t o r i m p a i r m e n t were seen. GHRH

R e s p o n d i n g after injection o f G H R H (0.2 p m o l - 0 . 2 nmol) is shown in Table I. Neither unpunished, nor p u n i s h e d r e s p o n d i n g was affected. ANP

R e s p o n d i n g after A N P ( 1 . 5 - 6 . 0 nmol) is shown in Table I. Neither unpunished nor punished r e s p o n d i n g was affected.

Discussion NPY

N P Y m a r k e d l y increased p u n i s h e d r e s p o n d i n g in the current conflict p a r a d i g m . Such an 'anti-conflict' effect is typically seen after administration o f clinically effective anx-

66 iolytics, such as the bensodiazepines (see, e.g., Ref. 10). Thus, the action of NPY resembled that of anxiolytics, and can therefore be termed 'anxiolytic-like'. In control experiments, pain threshold was not affected by NPY, making it unlikely that altered pain perception accounts for the observed action of the peptide. The inability of several peptides of similar molecular size to affect punished responding indicates that the anxiolytic-like effect of NPY is directly related to the molecular structure of this peptide, and probably involves receptor-specific events. Powerful orexigenic effects of NPY have been shown upon central administration (see, e.g., Ref. 11). In addition, it has recently been reported that NPY given i.c.v, in mice increased the number of shocks accepted in order to obtain milk reward [5]. This action of NPY was interpreted as the result of an increased motivation to eat. In the present study, unpunished lever pressing for food reward was also somewhat elevated by NPY, raising the question whether the observed increases in both unpunished and punished responding can be attributed to increased appetitive drive, rather than to an anxiolytic action of the peptide. While the minor increase in unpunished responding could indeed be explained by increased appetite, this is less likely to be the case for the increase in punished responding. First, the increase in punished responding was markedly larger than that in unpunished responding, a difference which can not easily be explained by increased appetite. Secondly, G H R H , which is also a powerful stimulant of food intake, failed to increase punished responding. Clearly, an increased appetitive drive is not sufficient to produce a release of punished behaviour such as that seen after NPY administration. In fact, a pattern similar to that produced by NPY has been seen in the presently used test with low to moderate doses (2-6 mg/kg) of the prototypical bensodiazepine anxiolytic chlordiazepoxide. In addition to markedly and dose-dependently increasing punished responding, chlordiazepoxide also produced a slight (up to 30% ) increase in the unpunished component [2]. Observations of an anxiolytic-like action of NPY have previously been made in two other animal models of anxiety, the elevated plus-maze, and Vogels punished drinking test [7]. These observations were made in Sprague-Dawley rats, while the present results were obtained using the Wistar strain. Thus, an anxiolytic-like effect seems to be produced by NPY regardless of animal strain or experimental paradigm chosen, strengthening the hypothesis that NPY is, in fact, an endogenous anxiolytic. The dose-response curve observed for NPY in the current experiment, however, differed from that obtained in the Vogel-test. In that model, the maximal increase in responding was observed at 1 nmol, and a smaller increase was seen after 5 nmol. In parallel, clear signs of sedation were seen at the higher dose. This prompted the interpretation that an anxiolytic-like effect was produced at low doses of NPY, and that sedation was added to the spectrum of effects at the highest dose, interfering with performance in the test apparatus. In the current experiment, punished responding increased with increasing dose throughout the dose range tested, and no signs of sedation were observed. This may be due to the low response requirement of the present incremental shock paradigm. Alternatively, the ratio of sedative/anxiolytic dose may differ between different rat strains. Doses of chlordiazepoxide only slightly higher than those required for maximum anxiolytic effect have been reported to produce clear signs of sedation in the present test. Thus, at 8 mg/kg chlordiazepoxide became less effective

67 in increasing punished responding, and produced decreased responding in the unpunished test component [2]. Our present results would therefore seem to indicate that NPY has a markedly higher sedative/anxiolytic dose ratio than presently available anxiolytics. NPY is decreased in the CSF of depressed human subjects [ 19]. In these patients, an inverse correlation between scored anxiety-levels and concentration of NPY in the CSF has been found, suggesting a deficit in NPY to be a possible pathogenetic factor behind the anxiety symptoms observed [8]. In addition, tricyclic antidepressants, which provide effective anti-anxiety therapy in humans and produce anxiolytic-like effects in animal conflict models (see, e.g., Ref. 12) elevate NPY levels in the rat forebrain (Ref. 6). On the basis of these findings, we have hypothesized that NPY may be important in anxiety mechanisms, and that disturbances in central NPY systems may produce anxiety. The present result provides additional support for such a hypothesis. BEND

BEND suppressed unpunished responding in a dose-dependent manner. Punished responding was not affected until the highest dose of BEND was reached. At this dose, both unpunished and punished responding were entirely absent, and motor performance of the animals was visibly impaired. It is therefore likely that the decrease is to be attributed to performance factors, with the punished test component being markedly less sensitive to such non-specific effects. The lack of effect of BEND on punished responding is consistent with extensive results showing that opioid drugs fail to produce an anxiolytic-like effect in operant conflict tests [ 14,16]. It has previously been shown that the opioid receptor antagonist, naloxone, blocks the anxiolytic effects of chlordiazepoxide, a prototypical bensodiazepine anxiolytic, in the Geller-Seifter paradigm. In addition, naloxone blocked the anxiolytic action of alcohol. On the basis of these findings, it was hypothesized that endogenous opioids could be a common final pathway for various drugs with anxiolytic actions [ 10]. The present findings do not support such a hypothesis. It is, however, conceivable that a subpopulation of opioid receptors not activated by BEND (but by some other endogenous ligand) could be involved in an anxiolytic action of endogenous opioids. GHRH

In a wide dose range, G H R H affected neither punished nor unpunished responding. In the same dose range, G H R H has been shown to be a potent inducer of feeding [ 18]. The current finding makes it unlikely that G H R H would be involved in central anxiety mechanisms, and reemphasizes the specificity of the Geller-Seifter paradigm for anxiolytic drug actions. ANP

No effects on punished or unpunished responding were seen after central administration of ANP. Increased plasma levels of corticosterone have been reported after peripheral injection of ANP. Since abundant evidence supports the notion that central CRH is anxiogenic (see, e.g., Ref. 1), hormones acting on the HPA axis could affect

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anxiety. The hypercortisolaemia seen in ANP treated animals was, however, observed in hypophysectomized subjects [13], and may represent a peripheral action of thc peptide. Summary and conclusion Four peptide neurotransmitters involved in endocrine stress responses were tested for anxiety modifying properties in a highly specific, pharmacologically validated animal model of anxiety. Among NPY, BEND, G H R H and ANP, NPY produced a marked anxiolytic-like effect, while the other peptides were ineffective. This finding provides further support for the hypothesis that NPY is a potent endogenous anxiolytic.

Acknowledgements M. Heilig was sponsored by a fellowship from the UCLA Programme for Psychoneuroimmunology. This work was supported by NIDDK grant AM 26741 (G.F.K.), and a VA Merit Award (K.T.B.). We gratefully thank Dr. Nicholas Ling of the Whittier Institute, La Jolla, CA, for providing the 13-endorphin, Dr. Jean Rivier of the Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA for providing the GHRH, and Dr. Roger Guillemin for providing the ANP. This is publication 7221-NP from the Scripps Research Institute.

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