Neurotransmitter-mediated open-field behavioral action of CGRP

PII s0024-3205(!39)oooo2-8

ELSEVIER

NEUROTRANSMITTER-MEDIATED Annamaria

OPEN-FIELD

Life Sciences, Vol. 64, No. 9, pp. 73%740, 1999 Copyright 0 1999 Ekvicr Science Inc. Printed in the USA. All rights resemd 0024-3205/99/$see front matter

BEHAVIORAL

ACTION OF CGRP

Kovacs, Gyula Telegdy, Gabor T&h, Botond Penke

Departments of Pathophysiology and Medical Chemistry, Albert Szent-Gyorgyi University, H-6701 Szeged, Semmelweis u. 1, P.O.Box 53 1, Hungary

(Received

in final form November

Medical

13, 1998)

Summary The effects of central administration of calcitonin gene-related peptide (CGRP) on open-field activity were examined in male rats. Three doses (250 ng, 500 ng and 1 pg) of CGRP given intracerebroventricularly (i.c.v.) were tested on the ambulatory, rearing and grooming activities of the animals. One pg of peptide significantly decreased the ambulatory activity and increased the rearing and grooming activities 30 min after the treatment. The animals were pretreated with different receptor antagonists in doses which by itsels did not affect the behavioural paradigm. The decrease in ambulation induced by CGRP was antagonized by acetylcholine-, opioid-, SHT-receptor and B-adrenoceptor antagonists. CGRP induced increase in rearing activity was blocked by naloxone, phenoxybenzamine and propranolol. The CGRP-induced increase in grooming behavior was prevented by atropine, haloperidol, naloxone, methysergide and propranolol. The results suggest that different neurotransmitter systems are involved in the action of CGRP on open-field behavior in rats. Key Words: calcitonin neurotransmitters

gene-related

peptide,

intracerebroventricular

administration,

open-field

behavior,

CGRP is synthesized in the central nervous system (1,38) and is expressed in a variety of central (21,40) and peripheral (17,34) neuronal structures. CGRP has multiple target tissues (13). including the heart and arterial blood vessels (39), where CGRP is a potent vasodilator (2,ll). CGRP receptors have been recognized in the central and peripheral nervous tissues (49). Specific binding receptors have been identified in the brain (18,37), where they are present in highest density in the cerebellum (13,48), the substantia gelatinosa of the spinal cord (15,41), the heart muscle, the vascular smooth muscle cells, the endothelial cells (16,32), and the human neuroblastoma cell line (33). Various immunohistochemical and electrophysiologic evidence suggests that CGRP may play an important role in neurotransmission as either a neurotransmitter or a neuromodulator in the central nervous system. CGRP influences neurobehavior in rodents. It has analgesic effect. induces hyperthermia, causes catalepsy, decreases food intake (19). Corresponding author: Gyula Telegdy, Department of Pathophysiology, Albert Szent-Gyorgyi Medical University, H-6701 Szeged, Semmelweis u. 1, P.O.Box 53 1, Hungary

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CGRP improves the fear-motivated learning-associated memory formation in passive avoidance paradigms. The action of the peptide is mediated by P-adrenergic, serotonergic and opiate mechanisms (22). Electroconvulsive shock-induced partial amnesia can be prevented by CGRP. The antiamnesic action of CGRP is mediated by cholinergic, opiate, and a- and P-adrenergic systems (23). CGRP antiserum administered i.c.v. attenuates the passive avoidance responses (24), and facilitates the extinction in active avoidance behavior of rats (25). The data obtained by administration of CGRP antiserum support the role of endogenous CGRP in the behavioral processes. The possible involvement of dopaminergic transmission in the action of CGRP was demonstrated in an in vivo microdialysis study (26). Since changes in open field activity can also modify the fear-motivated learning-associated memory processes of animals (22) in the present study the action of i.c.v. administration of CGRP was investigated on open field behavior in rats. The open field test has been designed to measure the reaction of rodents to a novel environment. The new environment evokes not only exploratory activity, to obtain information about the environment, but also fear (44). The possible roles of neuroneurotransmitters in mediating the open field behavioral action of CGRP were tested in animals pretreated with different receptor antagonists. Methods Animals and surgery

The animals were kept and handled during the experiments in accordance with the instructions of the Albert Szent-Gyorgyi Medical University Ethical Committee for the Protection of Animals in Research. Adult male Wistar rats (LATI, Gddiillo, Hungary) weighing 150-200 g were housed five per cage in a light- and temperature-controlled room (lights on between 6:00 a.m. and 6:00 p.m.; 23 “C) and had free access to food and water. Under pentobarbital-Na anesthesia (Nembutal 35 mg/kg i.p.), a stainless steel cannula, with an external diameter of 0.7 mm, was stereotaxically implanted into the right lateral brain ventricle at a point 1.O mm posterior, 1.5 mm lateral and 3.5 mm ventricular according to the atlas of Pellegrino et al. (36). The cannula was fixed with dental cement and acrylate resin. The animals were used in experiments after a recovery period of 5 days. The correct positioning of the cannula was checked by dissection of the brain. Experiments were performed in the morning. Open-field test

The exploratory activity of the animals was measured by open-field methods. The animals were placed individually in the center of a square, wooden, white-colored open-field box with 36 squares measuring 10 x 10 cm each. The standard source of illumination was a 60-W bulb from 80 cm. The activity was assessed during 3 min. The ambulatory activity was characterized by the total number of floor units entered. The number of occasions on which the animals stood on their hind legs was the total number of rearings. The grooming frequency was characterized by the numbers of occurrences of face washing, forepaw licking and head stroking. Pretreatment with receptor antagonists

The doses of receptor antagonists were chosen in accordance with our previous experience that the receptor antagonist itself did not influence the behavioral paradigm, but blocked the action induced by a number of neuropeptides (46). The following receptor antagonists were given 30 min before the peptide administration: atropine sulfate (EGYT, Budapest) (2 mg/kg i.p.); haloperidol (G. Richter, Budapest) (5 mg/kg i.p.); methysergide hydrogenmaleate (Sandoz, Germany) (5 mgikg i.p.); naloxone hydrochloride (Endo Lab Inc, USA) (0.3 mg/kg i.p.);

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phenoxybenzamine hydrochloride (Smith, Kline and French, Great Britain) (2 mgkg, i.p.); and propranolol hydrochloride (ICI, Great Britain) (10 mg/kg i.p.). The receptor antagonists have been dissolved in saline in a volume of 2 ml/ kg b. w.

Peptide administration CGRP was purchased from Bachem (California)

and also was synthetized by one of the authors (G.T&h), a solid-phase technique utilizing ‘Boc chemistry (31). The amino acid incorporation was monitored by the ninhydrin test (20). The purification was carried out with reverse-phase HPLC on a Lichrosorb RP-18 10 u column (16x250 mm). The identity of the peptide was confirmed by co-elution with a reference standard and by mass spectrometry (VG ZAB-2SEQ). The purity of the peptide was more than 97%. The biological action of the synthesised peptide was compared with that of the Bachem CGRP. Since there was no difference between the CGRP obtained from two different sources, the results are expressed as one group. The CGRP was dissolved in 0.9% saline and injected in doses of 250 ng, 500 ng or 1 pg in a volume of 2 ~1 into the lateral brain ventricle. One pg CGRP was used in combined treatment. The control animals received physiological saline. The pH of the saline and CGRP was 7.4. The animals were tested 30 min later, in accordance with our earlier experiments with other peptides (44). Each rat was tested only once.

Statistical analysis The computations were performed by the GLM procedure of the SAS system. Repeated measures analysis of variance was used for multiple comparisons; a priori contrast was carried out by comparing experimental groups with the control group, using Tukey’s Studentized Range (HDS) Test. The critical value was generally set at p < 0.05. Results CGRP decreased the ambulatory activity in a dose-dependent manner (F(3,60)=5.55). The significance for the 1 pg dose of CGRP was p < 0.01 (Fig. l/a), establishing the effective dose of CGRP in receptor antagonist studies, 1 pg of CGRP was used.

Fig. 1 Effects of i.c.v. injection of CGRP in different doses on a) ambulatory, b) rearing and c) grooming activity in rats. Values are means *S.E. The numbers of animals are given in bars.

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(F(3,32)=9.65, p < 0.05), naloxone (F(3,28)=6.72, p < 0.05) methysergide (F(3,24)=5.84, p < 0.05) and propranolol (F(3,44)=6.67, p < 0.05) prevented the decrease in ambulation induced by the 1 pg dose of CGRF’ (Fig. 2). Haloperidol and phenoxybenzamine had no effect (not shown).

Atropine

140

Total number of squares 1

* p < 0.05 vs contr + p < 0.05 vs CGRP

+

+

100 +

80

f

60

/

;~ ~

Atrop. (9)

? C?ontrola

Nalox. (8)

CGRP lpg

Methys. (7)

m Block. B

Propr. (12)

Block.+ CGRP lpg

Fig. 2 Effects of different receptor antagonists on CGRP-induced exploratory activity in rats. Atropine, naloxone. methysergide and propranolol prevented the action of an i.c.v. administered 1 pg dose of CGRP. Values are means +S.E. The numbers of animals are given in brackets. The rearing activity was increased by the I pg dose of CGRP (F(3,60)=4.68, p < 0.05) (Fig.l/b). This action was blocked by naloxone (F(3.28)=9.48, p < 0.05) phenoxybenzamine (F(3,28)=7.11, p -: 0.05) and propranolol (F(3,44)=7.43. p < 0.05) (Fig. 3). Atropine, haloperidol and methysergide were ineffective (not shown). Total number of rearings 30

* p < 0.05

*

Control@

contr

*

Naloxon (8) 0

vs

Phenox. (8)

CGRP lpg IBlock.

+p
vs CGRP

*

Propran. (12) g Block.+CGRP

lpg

Fig. 3 Effects of different receptor antagonists on CGRP-induced rearing activity in rats. Naloxone. phenoxybenzamine and propranolol prevented the action of an i.c.v. administered 1 pg dose of CGRP. Values are means 5S.E. The numbers of animals are given in brackets. The increase in grooming activity induced by CGRP was also dose-dependent

(F(3,60)=15.93,

p

Neurotransmitters

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< 0.001) (Fig. l/c). In the prevention of increased grooming, cholinergic (F(3,32)=17.22, p < O.OS), dopaminergic (F(3,28)=9.08, p < O.OS), opiate (F(3,28)=19.20, p < 0.05), serotonergic (F(3,24)=6.81, p < 0.0.5), and P-adrenergic (F(3,44)=16.69, p < 0.05) mediation played a role (Fig. 4). Phenoxybenzamine had no effect (not shown). Total number of groomings 16 14

* p < 0.05 vs contr

+ p < 0.05 vs CGRP

*

Atrop. (9) Halop.@) Control

Nalox.(ll)

CGRP lpg

??Block.

Methys. (7) Propr. (12) @ Block. + CGRP 1 pg

Fig. 4 Effects of different receptor antaginists on CGRP-induced grooming activity in rats. Atropine, haloperidol, naloxone, methysergide and propranolol prevented the action of an i.c.v. administered 1 pg dose of CGRF’. Values are means kS.E. The numbers of animals are given in brackets.

Discussion The results show that the i.c.v. administration of CGRP caused dose-dependent changes in the open-field activity of rats. The ambulatory activity decreased, while the rearing and grooming frequencies increased. The reduced spontaneous motor activity induced by CGRP is in agreement with the reported decreased locomotion (19). Single central administration of the peptide induced catalepsy and hypomotility in an open field. CGRP potentiated haloperidol-induced catalepsy (3). To reveal the possible roles of different neurotransmitter systems in the CGRP-induced motor activity changes, we pretreated the animals with different selected receptor antagonists, in doses which by did not modify the behavioral paradigms, but were active in blocking the action of other peptides (42). The decreased locomotion was blocked by muscarinic (atropine)-, 5HT (methysergide)-, opioid and P-adrenoceptor (propranolol) antagonists. Haloperidol and (naloxone)receptor, phenoxybenzamine had no effect on the CGRP-induced decrease in ambulatory activity, despite the fact that haloperidol in the same dose prevented the ambulatory activity of beta(Tyr)‘melanotropin-(9-18) (46). Besides the previously mentioned receptor antagonists, we have shown that nitric oxide is involved in mediation of the CGRP action on locomotion. NW-nitro-L-arginine (L-NA), a nitric oxide synthase (NOS) inhibitor, prevented the action of CGRP on locomotion (28).

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CGRP increased the grooming activity. which could be blocked by all receptor antagonists used except phenoxybenzamine. The grooming activity is considered to be a mild stress response (4,7). It seems that an increased grooming activity can be induced by a number of peptides, such as CRF (corticotropin-releasing factor) (8). ACTH (adrenocorticotropic hormone) (IO). bombesin (45). ceruletide (29). vasopressin (30). osytocin (35). prolactin (6). P-endorphin (4.5) etc. The grooming activities induced by different peptides can be blocked by a dopaminergic receptor antagonist (5). The dopaminergic antagonist haloperidol attenuates ACTH-induced grooming (14). A norepinephrine antagonist had limited effects on ACTH-induced grooming. The padrenergic inhibitor propranolol (in a high dose) and the cholinergic antagonist atropine inhibited the action of ACTH on grooming (7). ACTH-induced grooming can also be reduced by the opioid antagonist naloxone in rats (12.45). Since CGRP-induced action on grooming and ACTHinduced grooming can be blocked by essentially the same receptor antagonists (acetylcholine-. dopamine-, opioid-receptor and fi-adrenoceptor). it is tempting to speculate that CGRP given i.c.v. may activate the pituitary-adrenal system. and that the grooming reaction observed is secondary to the effect of CGRP, and is elicited by CRF or ACTH. This hypothesisis is supported by our previous findings that i.c.v. administration of CGRP elevated the plasma corticosterone level, and that the CGRP-increased grooming activity could be blocked by CRF antiserum (27). I,-NA pretreatment also prevented the effect of C‘GRP on grooming (28), and as NOS is colocalized with CRF in the hypothalamus (43). we presume that endogenous NO released in the action of CGRP on grooming is related to C‘RF

To our knowledge, no data have been published on C’GRP action on rearing activity. The increase in rearing activity caused by the i.c.v. administration of CGRP could be blocked by naloxone, propranolol and phenoxybenzamine. which indicates that not only are the CI and P-adrenergic systems involved in the organization of rearing activity, but endogenous opiates are also important. We conclude that the decreased locomotor and increased grooming activity caused by CGRP in an open field is mediated by the opiate. P-adrenergic, cholinergic and serotonergic neurotransmitter system. In CGRP-induced grooming, dopaminergic mediation too has been demonstrated. Besides to classical neuroneurotransmitter mechanisms. activation of the CRFACTH axis and NO release seem to be involved in the organization of this behavioral action of CGRP. Acknowledgements

The work was supported by grants from the Hungarian Ministry of Social Affairs and Health (ETT T-02-670196). OTKA (T-022230. T 6084), FKFP (009111997) and MTA-AKP (96-330 3,2) References

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