Motor effects of calcitonin administered intracerebroventricularly in the rat

European Journal of PharmacologY, 121 (1986) 189--198

189

Elsevier

M O T O R E F F E C T S O F C A L C I T O N I N A D M I N I S T E R E D I N T R A C E R E B R O V E N T R I C U L A R L Y IN THE RAT MICHAEL J. TWERY l, BRIAN KIRKPATRICK 2.3, ELIZABETH C. CRITCHER 3. MARK H. I,EWIS 3 RICHARD B. MAILMAN 1,2,3and CARY W. COOPER 4.. Departments of / PharmacoloKv. " Psychiatry and ~ Biological Sciences Research Center. Unit,er~'ityof North Carolina School of Medicine, Chapel Itill. NC 27514 and 4 Department of Pharmacology and Toxicology. Unit'ersity of Texas Medical Branch. Gah'eston, TX. U.S.A.

Received 2 October 1985, accepted 12 November 1985

M.J. TWERY, B. KIRKPATRICK, E.C. CR1TCHER, M.H. LEWIS. R.B. MAILMAN and C.W. COOPER, Motor effects of calcitonin administered intracerebrot:entricularl.v in the rat. European J. Pharmacol. 121 (1986) 189-198.

In rats treated with salmon calcitonin (CT) administered intracerebroventricularly (i.c.v., 85 or 8.5 pmol), spasmodic body movements, bopping and tail jerks, collectively termed dyskinesia, appeared within 1 h of administration and persisted for at least 24 h. In addition, spontaneous grooming, rearing and locomotion occurred less often in CT-treated rats than in vehicle-injected animals, while the incidence of both sniffing and nose poking remained essentially unchanged. The CT failed to displace either [3 H]dopamine or [3 H]spiperone from striatal membranes, and the behavioral effects were not blocked by haloperidol or SCH 23390, suggesting that the peptide did not directly affect dopamine receptors. The dyskinesia was not blocked by scopolamine, atropine, muscimol, diazepam or ketanserin. These data are consistent with the hypothesis that a compound with recognition characteristics similar to those of salmon CT may function as a neurotransmitter-modulator in the central nervous system. Calcitonin

Behavior

Motor function

Dopamine

I. Introduction

Calcitonin (CT) is a peptide hormone synthesized by C-cells in the thyroid gland of mammals and the ultimobranchial gland of lower vertebrates. Embryologically, C-cells are thought to be derived f r o m neuroectodermal tissue (Pearse et al., 1972). The hormone contributes to calcium homeostasis by controlling the efflux of calcium from skeletal sites, and. in coordination with vitamin D and parathyroid hormone, influences the concentration of serum calcium (Munson, 1976). In rats, salmon CT administered intracerebroventricular (i.c.v.) leads to decreases in gastric acid secretion (Morley et al., 1981), prolactin release (Pecile et al., 1981; Olgiati et al., 1982), food * To whom all correspondence should be addressed: Department of Pharmacology and Toxicology. University of Texas Medical Branch, Galveston. TX 77550. U.S.A. 0014-2999/86/$03.50 ~: 1986 Elsevier .Science Publishers B.V.

Binding

Dyskinesia

consumption (Freed et al., 1979; Perlow et al., 1980), water intake (Twery et al., 1982), response to painful stimuli (Pecile et al., 1975} and the secretion of several pituitary hormones (Pecile et al., 1981: Leicht et al., 1974). In addition, calcitonin is also reported to affect haloperidol-induced catalepsy (Nicoletti et al., 1982) and active avoidance behavior (Clementi et al., 1984). It has been suggested that the effects of centrally administered salmon CT mimic the actions of endogenous CTlike substances located within the CNS, and salmon CT-like immunoreactivity (Deftos et al., 1978a,b; Fischer et al., 1981b; Cooper et al., 1980; Fischer et al., 1983), a calcitonin gene-related peptide ( C G R P ) structurally similar to C T (Amara et al., 1982; Rosenfeld et al., 1983), and high affinity binding sites for salmon CT and C G R P (Fischer et al.. 1981a,b; Henke et al., 1983: G o l t z m a n and Mitchell, 1985) have been reported. Our previous report that calcitonin caused a

decrcase in the behavioral cffects of amphetamine in rats, as measured by an automated photocell apparatus (Twery et al., 1983a,b; Cooper ct al., 1984) raised the possibility that calcitonin had other behavioral effects. The present report confirms that possibility and describes several behavioral effects, the most pronounced being a pattern of marked and persistent dyskinetic movemcnts.

2. Materials and methods 2.1. Materials and animals

Synthetic salmon CT was purchased from Bachcm Corp., Torrence, CA (4500 MRC U/mg), and also was kindly provided by Dr. Robert Schleuter, Armour Pharmaceutical Co., Kankakee, IL (4700 MRC U/rag). Dopamine hydrochloride was purchased from Sigma Chemical Co. (St. 1,ouis, MO). Research Organics, Inc. (Cleveland, OH) supplied HEPES. The drugs used in these studies, and their suppliers, were: haloperidol (McNeil Laboratories, Fort Washington, PA), SCH 23390 hemimaleate (Schering Corp., Bloomfield. N J), diazepam (Hoffmann-La Roche Inc., Nutley. N J), naloxone (Endo Laboratories, Wilmington, I)L), ketanserin (Janssen Pharmaceutica, New Brunswick, N J) and muscimol (Research Organics Inc., Cleveland, OH). Other reagents were purchased from Fisher Scientific Co. (Pittsburgh, PA). Cannulas for permanent implacement were purchased from Plastic Products. The rats used in these studies were obtained commercially from Charles River Breeding Laboratories (Wilmington. MA, U.S.A.). As noted below, both the Sprague-Dawley and Long-Evans strains were used in specific experiments. Rats were generally received at a weight of 125-150 g, and housed at 25 _+ 2°C with lights on from 7 : 00 to 19:00 h. Food (Wayne Lab Blox) and water were available ad libitum, except as otherwise noted. 2.2. Drug administration

Salmon CT was administered intracerebrovcntricularly (i.e.v.), using the method of Popick

(1975) as well as permanently indwelling lateral ventricular cannulas. When administered bv the method of Popick, salmon CT was dissolved in 1 mM HCI-0.15 M NaCI and delivered in a 10 /~1 w)lume. In previous studies from this laboratory, the Popick technique was thoroughly characterized and shown to produce results essentially identical to those obtained using indwelling cannulas (I,ewis et al.. 1983: Kilts et al.. 1984). Cannulas for permanent placement were surgically implanted under pentobarbital anesthesia, with placement into the lateral ventricle. The coordinates used for stereotaxic surgery were those of Patinos and Watson (1982). Placement of the cannula into the lateral ventricle and its patency were confirmed by the injection of 10 #1 of methylene blue before the animals were killed. All drugs other than CT were injected intraperitoneally (i.p.) except for muscimol. The latter was injected by the i.c.v, method (Popick, 1975) to minimize peripheral and spinal effects. Naloxone, ketanserin and muscimol were dissolved in isotonic saline. Haloperidol and SCH 23390 were dissolved in 0.5 or 0.1% tartaric acid, respectively. The diazepam used was the Valium Tel-E-Jet ~' preparation. For i.p. injections, drug concentrations were adjusted so final injection volumes were approximately 2 m l / k g body weight, except for the diazepam, in which half this volume was used. All challenge drugs were administered 30 rain prior to administration of salmon CT. and with this pretreatment time, and with the doses used, reasonably complete receptor occupancy is known to occur with these drugs (e.g. Van Nueten et al., 1981; Mailman et al., 1984: Breese et al., 1979: Lewis et al., 1983). 2.3. Behat,ioral ohsert'ations

The effect of salmon CT on spontaneous behavior was studied by systematically recording and analyzing observational data for selected behaviors using the method of Lewis et al. (1985). The observer recorded previously defined behavioral topographies (see table 1) observed during each interval by entering the appropriate codes on an electronic data collecting device (Datalnyte 900. Electro General Corp., Minneapolis, MN). The

191 TABI,E 1 Behavioral categories of the rat monitored in these experiments. Name of behavior

Description

Asleep/ inactive Inactive

In resting position with eyes closed

Rearing

Grcx~ming I,ocomotion

Circling

Sniffing Licking Gnawing

Body gnawing

Nose poking

Dyskinesia

Tongue protrusion

In resting position with eyes open. Occasional sniffing, shifts in body position, and dyskinetic movements may occur while the animal is 'inactive'. Both front paws are raised off the cage floor and are either in the air or against the cage wall. Engaged in flank scratching, face-, paw-, or genital washing. Movement of all four paws at least once resulting in change in the animal's location within the cage (not just a shift in body position. Rotations around a tight axis in either clockwise or counter-clockwise direction: must occur more than once during an interval. Vibrissae movement in the air along the cage floor or wall. Protrusion of tongue resulting in contact with body or other objects. Wire of the cage floor is gripped between the teeth or the cage wall is contacted with the teeth. Tail, paws or other body parts are taken between the teeth, in the absence of normal grooming behaviors. Protrusion of snout and vibrassae through openings in the cage floor or wall. Small and large jerky movements of limbs, body. head or tail. These movements may appear as shaking, hoping or tail waggins,depending on the parts of the body involved and the severity of the episode. Protrusion of tongue against the cage floor or wall.

behavioral categories scored included those that occur with drug-free rats as well as u n u s u a l behaviors induced by drug treatment (e.g. dyskinesia). This system quantifies nearly all behaviors observed d u r i n g the scoring interval. In all experi-

ments, observers were not aware of the order in which treatments were given. After treatment, each rat was placed u n d e r an inverted, clear Plexiglas cage (21 × 37 cm) with a wire mesh floor. I l l u m i n a t i o n was provided by overhead fluorescent lighting, and a white noise b a c k g r o u n d was generated electronically. The end of an interval was signaled by an electronic timer through headphones. Results were tabulated as the p r o p o r t i o n of intervals d u r i n g each 1 min period of observation that the behavior occurred (percentage occurrence). Unless noted otherwise, data were transformed prior to statistical analysis using a two-factor analysis of variance for repeated measures, and the transform p' = 2 . arcsin[sqrt(p)] was used to stabilize the variances (Lewis et al., 1985).

2.4. Experimental design For experiments in rats with p e r m a n e n t c a n n u las. both male Sprague-Dawley or L o n g - E v a n s rats were used when they weighed 350-450 g. Salmon C T or vehicle (0.9% saline) was injected, a n d the a n i m a l s were immediately placed in the Plexiglas cages. A n i m a l s were then videotaped for a 5 rain period 60 m i n after injection. The videotapes were then scored, using 27, 10 s intervals for each animal. Using the method of Popick (1975), male Sprague-Dawley rats (175-200 g) were administered salmon C T or vehicle i.c.v. Thirty m i n u t e s after treatment, each animal was placed in a Plexiglas observation cage, and the behavior of the animal was scored d u r i n g 10 equally spaced, 1 min observation periods over the next 60 min. The scoring was done by direct observation and not from videotapes. Each 1 min observation period consisted of four consecutive 15 s scoring intervals. In all experiments, interobserver reliability was examined to indicate the adequacy of behavioral definitions. Data collected simultaneously by two i n d e p e n d e n t observers were compared within each period of observation. Interobserver scoring agreement was summarized by the kappa statistic. The

lq2

kappa statistic is less sensitive than percentage agreement to the frequency' and the ease with which either occurrence or nonoccurrence can be scored (Hartman, 1977). Values of kappa greater than zero indicate that the number of observed agreements is greater than attributable to chance alone: values less than zero occur when the number of observed agreements is less than the proportion of agreements expected due to chance. Therefore, kappa has the value of + 1.0 when no disagreement are present and 0.0 when observed agreements equal the number due to chance.

3. Results

3.1. I:ffects on spontaneous activity q/ the rat Vehicle-injected control animals (30 min pretreatment) typically maintained a resting posture and. like untreated control rats (data not shown), showed little locomotion, rearing or grooming (see fig. IA-C). Rats pretreated with 85 pmol (ca. 300 ng) salmon CT via the method of Popick. 30 rain prior to observation, exhibited decreases in several behavioral categories: these differences were detec-

2.5. Radioligand binding studies 1"00 1

Because of the involvement of dopamine systems in motor function, radioligand binding studies were used to determine whether salmon CT could compete for binding sites for ligands believed to interact with dopamine receptors. Homogenatcs of rat striatal tissue were prepared m approximately 100 volumes of ice cold 50 mM tIEPES buffer (pH 7.55 at 25°C) using a hand-held glass homogenizer with teflon pestle. The tissue homogenate was centrifuged at 25000 x g for 10 rain, and the pellet was washed twice by suspending it in fresh HEPES buffer and recentrifuging it. The final washed pellet was added to glass tubes in triplicate to give a final concentration of 5 mg original wet weight/ml in a final volume of 1 ml containing approximately 0.25 mg protein. Labelled ligand. 0.2 nM [3H]spiperone (31.3 C i / mmol) or 7 nM [3Hldopaminc (44.4 C i / m m o l ) , and unlabelled test compounds (salmon CT, apomorphine, or haloperidol, dissolved in 0.5~ tartaric acid') were added as appropriate. After samples were incubated for 20 rain at 37°C, they were filtered under vacuum through glass fiber filters (Gelman Sciences, Inc., Type A / E , 25 ram) which had been previously soaked in HEPES buffer. After the filters had dried, 10 ml scintillalion cocktail (Scintiverse I, Fisher Scientific) were added to each vial, and radioactivity quantified by liquid scintillation spectrometry. Efficiency was ca. 45% for all samples.

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Fig. 1. The effects of salmon CT pretreatment on the incidence of spontaneous locomotion (A), spontaneous rearing (By, spontaneous gr¢×)ming (C) and inactivity/asleep (D). Bars represent the mean incidence (')~ occurrence over a 1 min period. Vehicle (unshaded bars) or 85 pmol of salmon CT obtained from Bachem Corp. (shaded bars) was administered i.c.~. 30 min prior t,a observation (18 rats/group). Where vehicle and salmon CT values were similar, half-shaded bars are used. Analysis of variance for repeated measures revealed the follov,ing significant changes: spontaneous locomotion (A): effects due to treatment (F(1.34) = 7.58, P < 0.01) and time (F(9.306) 189.9. I) < 0.001); spontaneous rearing (By: effects due to treatment 0:(1,34) = 46.9. P < 0.001), time (F (9,306)- 14,:1..8, P < 0.001) and treatment by time interaction (F(9,306)= 21.5. P < 0.01 ): spontaneous grooming (C): effects due to treatment (F(1,34) = 22.8. P < 0.001). time (F(9.306) = 5.92, P < 0.01). and treatment by time interaction (F(9,306)= 3.31. P < 0.01): inactivity/asleep (D): effects due to treatment (F(1,34)~ 6.83, P < 0.05), time (F(9.306) = 7.92. P < 0.001) and treatment b? time interaction (F(9.306) = 4.10, P < 0.00l ). One factor analysis of variance for simple main effects indicated a significant effect due to treatment: spontaneous locomotion (A): at 6 rain (P < 0.01): spontaneous rearing (By: at 6 and 12 rain (P < 0.01 )" spontaneous grooming (C): at 6 and 12 rain (P .~ 0.t)5): inacti'.i t y / a s l e e p (D): at 30. 42.48 and 60 rain (P < 0.05).

193 table p r i m a r i l y while the rats were actively exp l o r i n g i m m e d i a t e l y after being placed in the cage. "Salmon C T treated rats exhibited a lower incidence of l o c o m o t i o n (25% decrease, P < 0.01: fig. 1A), rearing (55% decrease, P < 0.001: fig. 1B) and g r o o m i n g (52% decrease, P < 0 . 0 5 : fig. IC), whereas the occurrence o f sniffing a n d nose p o k ing was not different from control levels. However, the behavioral category of inactivity (absence of a n y observed behavior) was m a r k e d l y different in the s a l m o n C T - t r e a t e d animals at later times in the o b s e r v a t i o n p e r i o d (fig. 1D). Rats injected with s a l m o n C T also d i s p l a y e d piloerection, increased muscle tone, and a greater t e n d e n c y for vocalization d u r i n g h a n d l i n g when c o m p a r e d to vehicle-inj e c t e d rats. Essentially identical results were obtained in a n i m a l s of both S p r a g u e - D a w l e y and L o n g - E v a n s strains using p e r m a n e n t l y i m p l a n t e d cannulas.

3.2. CT-induced dyskinetic mot~,ements P r o m i n e n t episodes of unusual m o v e m e n t s which we have t e r m e d dyskinesia were present in a n i m a l s a d m i n i s t e r e d CT. These m o v e m e n t s consist of quick, j e r k y m o v e m e n t s consisting of tailflicking, limb and trunk c h o r e i f o r m movements, or head shaking, but were not associated with loss of consciousness or ataxia. The dyskinesia occurs in m o r e than 95% of animals in which c a n n u l a placem e n t is c o n f i r m e d by the injection of dye p r i o r to killing. N o seizure activity was ever detected in treated animals, and there were no a b n o r m a l i t i e s in posture or changes in righting or reaching reflexes. A single m o v e m e n t usually lasted no more than 1 s a n d occurred d u r i n g locomotion, sleep, inactivity or grooming. These m o v e m e n t s were very rare in vehicle-injected control rats c o m p a r e d to rats injected with 85 p m o l of salmon C T (t-test, P < 0.001, N = 26, for rats with p e r m a n e n t l y indwelling cannulas). The nature and frequency of the dyskinesia was not d e p e n d e n t on the m e t h o d of a d m i n i s t r a t i o n of s a l m o n CT. Using the m e t h o d of Popick, an 8.5 p m o l dose of s a l m o n C T also elicited dyskinesia (P < 0.05), but episodes of dyskinesia occurred in a b o u t 25% of scoring intervals at this dose c o m p a r e d to 40% or m o r e at 85 p m o l (fig. 2). In

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Fig. 2. The effects of salmon CT pretreatment on the incidence of dyskinesia. Bars represent the mean incidence of C'I" dyskinesia over a 1 min period. Two doses of salmon CT were used, 85 pmol (A top) or 8.5 pmol (B bottom). Vehicle (unshaded bars) or salmon CT obtained from Bachem Corp. (shaded bars) was administered i.c.v. 30 min prior to observation (18 rats/group). For 85 pmol dose: Analysis of variance for repeated measures revealed a significant effect due to treatment (F(1,34) = 66.5, P < 0.001), time (F(9,306) = 4.2, P < 0.001) and treatment by time interaction (F(9,306) = 3.86. P < 0.001). One factor analysis of variance for simple main-effects indicated a significant effect due to treatment in each period of observation (P < 0.01). For 8.5 pmol dose: Analysis of variance for repeated measures revealed a significant effect due to treatment (F(1,34) = 6.95. P < 0.05).

similar experiments, the lower dose of s a l m o n C T (8.5 pmol) p r o d u c e d no a p p r e c i a b l e effects on rearing, l o c o m o t i o n , sniffing, or nose poking, although the incidence of g r o o m i n g was reduced (P < 0.05, d a t a not shown). T h e dyskinesia p r o d u c e d by C T (i.c.v.) was not seen following subc u t a n e o u s (s.c.) a d m i n i s t r a t i o n of doses as high as 15 n m o l / k g s a l m o n CT. l n t e r r a t e r reliability for the d y s k i n e s i a was assessed using the k a p p a statistic. C a l c u l a t e d from a c o m p a r i s o n of 1440 o b s e r v a t i o n periods, it had a value of 0.88 for animals a d m i n i s t e r e d salmon C T by the m e t h o d of Popick. The k a p p a statistic was + 0 . 8 2 for o b s e r v a t i o n s on animals a d m i n i s t e r e d s a l m o n C T through p e r m a n e n t l y i m p l a n t e d cannulas; this represents interrater agreement on the presence or absence of the dyskinesia in 93% of scoring intervals. Even this percentage is mislead-

1 ~)4

ingl 3 Imv. duc to the use of short (lCI or 25 s) scoring intervals: i f a dvskinetic movement occurs near the beginning or end of a scoring interval, the raters may agree on its presence but place it in different intervals, which would lower the kappa for that period. Several pharmacological challenges were used to determine if these effects of salmon ( ' T were markedly affected by several well characterized receptor systems. Halopcridol (0.5 mg/kg). S('tl 2339(I (0.1 mg/kg), diazepam (5 mg/kg), naloxone (10 mg/kg), kctanserin (1 m g / k g ) and muscimol (50 /~g i.c.v.) all failed clearly either to block or increase the intensity of the dyskinesia.

.?.3. Time cour~e of d.vskinesia The time coursc of CT-induced dyskincsia was determined using synthetic sahnon C'I" from either Bachetn Inc. or Armour Pharmaceutical Co. The salmon ( " I (85 pmol) was administered to rats which had habituated to the observation environment. Prior to treatmcnt, these animals exhibited almost no behavior scored as dyskinesia, whereas after salmon C'I" (Bachem). dyskincsia became evident by 5-20 rain after treatment. With both salmon C'Is, however, dyskinetic movemcnts were still present, but not prominent, 40 h after treatment (table 2), although the potency and duration of action were somcwhat greater for the Armour than the Bachem preparation.

3.4. Radioligand binding studies The ability of salmon CT to attenuate some behavioral responses induced by amphetamine TABI.I" 2 l i m e course of salmon ('T-induced dyskinesias. l'reatment

N

(l'lBach(mpreparation ~ll) Armour preparation

9 4

Percent ~ c u r r e n c e "' () h

2h

16 h

40 h

6#-4

36+13 50_+ 7

10+19 56+ 7

11k6 15±3

" The proportion of 15 s intervals per minute in which dyskinesia was observed (times 100). Each animal was scored three times during a 15 min scoring period. Data are expressed as means ± S.E.M. t, Salmc, n CT (85 pmol i.c.v.).

('lwer 3 ct al.. 1983a,h), and the documented involvement of dopaminc systems in motor and posrural functions (Fink and Smith. 1980: And6n st al., 1978), suggested that the peptide might interact with dopaminergic systems. These experiments tested the possibility that salmon CT might directly affect dopaminc receptors, and examined whether sahnon CT would compete for the binding of a radiolabelled dopamine agonist or antagonist to striatal homogenates. Salmon ( ' T (Armour Pharmaceutical Co.), in concentrations from 0.3 to 300 nM, failed to alter the binding of [3HIspiperone (0.2 taM), whereas in the same experiment, haloperidol decreased binding as much as 809,: over the same range of ligand concentralions (data not shodn). In another experiment, ths binding of [~Hldopamine (7 taM) was similarly unaffected by salmon ( ' T (0.05-500 nM). whereas apomorphinc was a potent competitor (data not shown). These results suggest that binding sites for d o p a m m e agonists or dopamine antagonists are not blocked by' salmon ("IF in vitro.

4. Discussion

The most significant aspect of the present study is the finding that salmon ('T-treated rats exhibit spasmodic movements of the head, body and tail, an effect which cannot be readily quantified by automated measuring devices. This effect, previously noted in abstract form (Twery et al.. 1983a; Cooper et al.. 1984), is quite robust, and excellent interrater reliability of these movements was established. This dyskinesia is detectable at very low doses of CT, and does not depend on manufacturer, batch, age or strain of rat, or details of administration or scoring. Moreover, our data indicate that the salmon CT induced-dyskinetic movements are long-lasting, but not permanent. Although other investigators have studied certain behavioral aspects of CT administered centrally to rats (e.g. suppression of spontaneous or stress-induced feeding), most have not indicated that CTtreated rats exhibited unusual behaviors (Freed et al., 1979; Levine and Morley, 1981; Nicoletti et al., 19821. The movements we describe are probably the same as the tail-flicking noted anecdotally

195

in rats given salmon CT (Morimoto et al.. 1985). They may also resemble the spontaneous running movements noted in rabbits given salmon CT i.c.v. (Pecile et al., 1975). These movements are interesting not only in themselves, but also because they may have relevance for the study of salmon CTs antinociceptive properties. The tail-flick test is a commonly used animal model for the study of central pain mechanisms, and these dyskinetic movements may affect the data on CT obtained with this or related tests. In any event, our observations confirm the importance of not relying solely on automated apparatus. The trained human observer remains an essential part of behavioral pharmacology. The salmon CT-induced dyskinesia syndrome is distinguishable from the pharmacological effects of other putative neuropeptides, such as thyrotropin-releasing hormone (wet dog shakes) and ,8-endorphin (generalized rigidity). Salmon CT-induced dyskinesia does not resemble the rapid rotations along the longitudinal body axis (barrel rotations) which are reportedly produced by somatostatin (Cohn and Cohn, 1975) and sulfated C-terminal fragments of cholecystokinin (Mann et al., 1980). Moreover, while the spasmodic-like activity induced by salmon CT (85 pmol) is frequently discernible for as long as 36 h after treatment, the barrel rotations induced by somatostatin have a relatively short duration. Microinjection of 29 nmol of ACTH-(1-24) (an active fragment of adrenocorticotrophic hormone) into the locus coeruleus produces a movement disorder which is long-lasting and, like CT, disrupts locomotor activity (Jacquet and Abrams, 1982). However, in contrast to CT, treatment with ACTH-(1-24) results in a contorted posture and an uncoordinated or wobbly gait during locomotion. Superficially, the effect of salmon CT on behavior appears to resemble that of neurotensin, an endogenous brain peptide with neurotransmitterlike qualities, Both neurotensin (0.18 nmol, intranucleus accumbens injection) and salmon CT (i.c.v.) reduce the incidence of rearing and locomotion induced by amphetamine (Ervin et al., 1981; Twery et al., 1983b). However, neurotensin does not produce any of the dyskinesia reported here. Moreover, centrally administered neurotensin in-

creases the concentration of HVA and DOPAC in certain regions of rat brain (e.g. striatum; Nemeroff et al., 1983), whereas Nicoletti et al. (1982) have failed to detect similar changes following the central administration of salmon CT. Following our observation that salmon CT inhibited amphetamine-induced locomotion as measured by a photocell apparatus (Twery et al., 1983b), the present study was designed to provide a detailed characterization of other behavioral eff e c t s o i calcitonin, and to explore some of the possible mechanisms involved. The present results show that salmon CT treatment decreased the incidence of rearing and locomotion, a finding consistent with our previous studies. It appears unlikely, however, that sedation, gross motor impairment, or the dyskinesia itself, completely underlie these effects, as the incidence of sniffing and nose poking during habituation was not materially reduced by CT treatment. These findings suggest that centrally administered salmon CT affects only certain categories of behavior. Since the locomotor response to low doses of amphetamine is predominantly dopamine-mediated (Hollister et al., 1974; Creese and lversen, 1975), and since nigrostriatal dopamine neurons are involved in a variety of motor and postural functions, our earlier report that salmon CT inhibited the locomotor response to amphetamine (Twery et al.. 1983b) raised the possibility of a direct interaction of CT with dopamine receptors. However, in radioligand binding studies with rat striatal membrames, CT failed to compete for the binding of either a dopamine receptor agonist ([ 3H]dopamine) or a dopamine rcceptor antagonist ([3H]spiperone). These findings complement those of Nakamuta et al. (1981) who reported that dopamine and other monoamines do not antagonize the binding of [~25l]salmon CT to brain tissue, and indicate that CT does not act directly via dopamine receptors. This conclusion is also consistent with the differing anatomical distributions of salmon CT and dopamine binding sites (Koida et al., 1980; Fischer et ai., 1981a,b: Leysen et al., 1978; Henke et al., 1983). Superficially, the present data are at odds with the report of Nicoletti et al. (1983) that 100 I.U. of salmon CT ameliorated both spontaneous and tardive dyskinesia in hu-

19,'~

marts. However, the differences in dose of ('T. the route of ('T administration, and animal species studied, as well as the fact that the Nicoletti sltldv apparently did not use observers unaware of treats e n t . make comparisons difficuh. Further studies are needed to determine whether ('T affects dopaminergic activity indirectly through pathways afferent or efferent to dopamine neurons. For example. Nicoletti et al. (1982) suggested that GABAergic pathways might mediate the locomotor effects of ("I'. Since G A B A pathways are both affercnts and efferent to some dopamincrgic neurons (Domesick. 1981 ), and since G A B A attenuates the locomotor activity produced in response to microinjection of dopamine into the nucleus accumbcns (Jones ct al., 1981 ), it is possible that ('T may act on GABAergic neurons. Consistent with this hypothesis, Nicoletti ct al. (1982) found that salmon c r produced a significant decrease in the glutamate decarboxvlase activit,, of the substantia nigra but not the corpus striatum. Ho,,vever. after treatment with muscimol and diazepam, two compounds which directly or indirectly potentiate GABA function, wc did not detect an effect on the dyskinesia. In summary, these findings raise the possibility that an endogenous, CT-like material may' bc involved in the regulation of motor function. ('andidatcs include a calcitonin genc-related prodt, ct (CGRP) produced in brain (Amara et al.. 1982: Roscnfeld et al., 1983) and a salmon or human CT-like immunorcactive material(Fisher ct al., 1983). Although blood-borne CT of thyroid origin may be the source of some brain CI-like immt, noreactivity, persistence of the immunoreactivity after thyroidectomy suggests that the CNS produces ('T-like material (Jacobs et al., 1982, ('.W. ('oopcr. unpublished observations). Whether any of these compounds explain our findings is an intrigt, ing, but unanswered, question. Nonetheless. the pronounced behavioral effects and high potency of salmon CT given i.c.v, is additional evidence for a ('T-like molecule's being a chemical messenger (e.g. neuromodulator) in the CNS. -i'hcse data suggest that ('T-like peptides have functional roles beyond simply being involved in feeding and gastric acid secretion.

AcknmvledRements Pt+rtion:, of thi,, v, ork '.,,ere presented at the ]3th Annual gociet,, for St~caet,. Netm-,cience Meeting. November. It, S3. and '.'+'eredone in partial fulfillnlcnt of the requtremcut>, for tire degree of I)tvctor of Philosophy in the Department of Pharmacoh+g?, art the t.'ni,.er,.itv of North Carolina. l'his research v.as supported in part b'. L'SPtIS Grants AM-17743. AM3206(I. 1tl)-03110 and MH-14277.

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