Atypical neuroleptic-like behavioral effects of neurotensin

BrainResearchBulktin, Vol. 32,pp. 487-491.1993 Printed in the USA. All rights reserved.

Copyright

0361-9230/93$6.00+ .OO 0 1993 PergamonPress Ltd.

Atypical Neuroleptic-like Behavioral Effects of

F. B. JOLICOEUR,’

M. A. GAGN6,

R. RIVEST, A. DRUMHELLER

AND S. ST-PIERRE

~~~art~ent of ~s~~hiatr~, Fatuity o~~edi~ine, Un~~~~rsit~ o~~herb~oake, ~herbrooke, 12th Ave. Nerd, Fleurimont, Quebec, Canada JlH-5N4 JOLICOEUR, F. B., M. A. GAGNE, R. RIVEST, A. DRUMHELLER AND S. ST-PIERRE. Alypica/ neuroleptic-like behaviorul efects ~?~~e~~~te~~~~. BRAIN RES BULL 32(S) 487-49 I, 1993.-To better characterize the neuroleptic-like properties of neurotensin, the dose-related effects of the peptide on the followin ~havioral phenomena were examined: a) the yawning-~niie erection syndrome induced by small doses of the dopamine agonists apomorphine and N-propylnom~mo~hine (NPA): b) yawning produced by the anticholinesterase physostigmine, and c) stereotyped climbing and sniffing produced by a larger dose of apomorphine. Several doses of the peptide were injected intraventricularly 30 min prior to drug administration. Results indicate that neurotensin markedly decreased yawning and penile erections produced by both apomorphine and NPA. These effects were seen with relatively small doses (0.9-3.75 gg). Neurotensin also potently decreased physostigmine-induced yawning with the initial inhibitory effect seen with 50 ng of the peptide. Apomorphine-induced climbing was significantly attenuated with 30.0 and 60.0 pg neurotensin, whereas stereotyped sniffing was unaffected, even by doses as large as 120.0 pg. These findings suggest that neurotensin might antagonize dopamine autoreceptors and indicate that the peptide possesses central anticholinergic activity. Furthermore, these results lend support to the hypothesis that neurotensin’s profile of central actions resemble that of atypical neuroleptics. Neurotensin Stereotypy

Apomorphine

Physostigmine

N-propylnorapomorphine

Yawning

Penile erection

mine postsynaptic receptors (2). Whereas typical neuroleptics antagonize apomorphine-induced yawning, penile erections, and stereotypies at similar doses, atypical neuroleptics arc markedly more potent to inhibit yawning and penile erections than to attenuate stereotyped behaviors (23). Also, while typical neuroleptics decrease both stereotyped climbing and oro-facial stereotypies at similar doses, atypical neuroleptics are markedly more potent in reducing the former than the latter stereotyped behavior (7.18). Furthermore. as stereotypies produced by large doses of the dopamine agonist gradually dissipate, yawning responses begin to be emitted by the animals. Such emergence of yawning following disappearance of stereotypy is maintained with typical but is inhibited by atypical neuroleptics (23). The purpose of the present study was to better characterize the atypical neuroleptic-like nature of neurotensin. We examined the effects of several doses of the peptide on yawning and penile erections induced by 100 pg/kg of apomorphine as well as on stereotyped climbing and sniffing produced by 600 @kg of the dopamine agonist. The possible appearance of yawning after administration of the larger dose of a~mo~hine was also closely monitored. The effects of neurotensin on yawning and erections produced by a small dose of N-propylnorapomorphine (NPA) were also assessed. The inclusion ofthis second dopamine agonist was prompted by the report that the peptide specifically and markedly reduces the binding of NPA to neuronal membranes (1). To determine the specificity of the effects of neurotensin on

MANY neurobehavioral effects of the tridecapeptide neurotensin resemble those of neuroleptics. These effects include decreases in body temperature, motor activity, muscular tone, and rates of self-stimulation ( 10,17,2 1). Also, similarly to neuroleptics, neurotensin attenuates the behavioral hyperactivity induced by a variety of substances known to stimulate dopamin~rgic transmission f&11,14). On the other hand, contrary to typical neuroleptics, the peptide does not induce catalepsy in rats and fails to decrease stereotyped sniffing elicited by dopamine stimulating drugs ( 11). Because of this, it has been suggested that the profile of neurotensin’s neurobehavioral effects is more akin to that of so-called atypical neuroIeptics such as sulpiride and clozapine (12). These drugs, while capable of producing all of the aforementioned behavioral effects of typical neuroleptics, also display weak cataleptogenic properties and are relatively impotent in reducing stereotyped sniffing induced by dopaminergic stimulation (30). The examination of drug effects on behaviors induced by small and large doses of apomorphine has been shown to be a simple and reliable procedure to detect potential neuroleptics and to differentiate between typical and atypical neuroleptics (5,23). Among other effects in rats, relatively small doses of apomorphine (25-200 pg kg) induce yawning and penile erections, two responses often attributed to the activation of dopamine autoreceptors (21,3 I). Larger doses produce stereotyped behaviors that are thought to be mediated by the activation of dopa-

’ To whom requests for reprints should be addressed.

487

488

.lOi.iC’oFI.JR I: i Ai

yawning produced by dopamine agonists, and because a dopamine-cholinergic link has been implicated in yawning behavior (3 I). the effects ofthe peptide on physostigmine-induced yawning were also examined. Finally, to verify the possible implication of chohnergic transmission in penile erections produced by dopamine agonists, the effects of atropine on erections induced by 100 pg/kg apomorphine were also studied. Some preliminary results of the present study have been presented previously (13.16).

Results obtained in each experiment were analyzed by means of individual one-way ANOVAs for inde~ndcnt groups. Each dose administered constituted one level of the main factor. Following a significant main effect. individual group comparisons between controls and treated animals were performed by means of Dunnett tests (29). Number ofanimals in a group displaying a particular behavior constituted nonparametric data that were analyzed with Fisher exact printability tests (76).

METHOD

Male hooded rats (2.50-300 g) were obtained from Canadian Breeding Farm (St-Constant, Quebec). They were housed in a temperature-controlled room having a 12L: 12D cycle. Food (Purina Rat Chow) and water were available ad lib. Under pentobarbital anesthesia. animals were impianted with a 23 ga stainless steel indwelling guide cannula using the foliowing stereotaxic coordinates: A.P. -0.08, D.V. 2.0, and L. I .5 (22). so that the tip would rest 2.0 mm above the left lateral cerebroventricle. A protective stainless steel 30 ga metal styiet was kept in the guide cannuia until the time of injection.

After at least 4 days of recovery following surgery. animals were separated into different groups (n = 8). Experiments were performed between 9 and I9 h in a room with an ambient temperature of approximately 22°C. Separate groups first received one dose of neurotensin via the intravent~cuiar (IV) route. Depending on the nature of individual experiments, doses were selected from a wide range: 0.025 to 120.0 pg. Groups ofcontrol animals were given 0.99, NaCl IV. Other groups of rats were first injected subcutaneously (SC) with either 1.O . 5.0, or 10.0 mg/kg of atropine. Control animals for these groups received a SC injection of 0.9% NaCI. Thirty minutes following these pretreatments, separate groups of animals were administered either a~rno~h~ne (100 or 600 @g/kg), NPA ( 1.88 pg/kg), or physostigmine (100 pg/kg). All these drugs were given SC. immediately following these injections, animals were placed in individual observation wire mesh circular cages (diameter: 20 cm; height: 30 cm). Five minutes later, behavioral monitoring was initiated and endured for 1 h. Total number of yawning responses and penile erections observed during the 1-h test period was recorded. Number of animals displaying yawning or penile erections in each group was also noted. The presence of sniffing and cage climbing was verified for approximately IO s every 2 min of the test period, and total sniffing and climbing episodes were recorded. Neurotensin, obtained from Sigma Chemical Company. was dissolved in 0.9% NaCI. At the time of injection, protective styiets were removed and a 30 ga needle was inserted into the guide cannula. The length of the injection needle was adjusted so that the tip protruded 2 mm beyond the guide cannuia into the left lateral ventricle. Volume of injection was 10 ~1 administered over a 30 s period by means of a 50 ~1 Hamilton syringe. Apomorphine hydrochloride and NPA hydrochloride were obtained from Research Bj~hernic~s Incorporated; atropine suiphate and physostigmine salicyiate from Sigma Chemical Corporation. Apomorphine and NPA were dissolved in oxygen-free boiled 0.9% NaCi solutions containing, respectively, 0.1% ascorbic acid and 0.1% metabisulfite to prevent oxidation. Physostigmine and atropine were dissolved in 0.9% NaCl. Ail these drugs were injected in volumes of 1 ml/kg under the loose skin of the back of the animals’ neck.

Yawning. induced by 100 &kg apomorphine, was signilicantiy decreased with 1.87 PLgand totally abolished with 3.75 pg neurotensin. Starting with 1.87pg ofthe peptide, the number of yawns produced by I .8 pg/kg NPA was also significantly decreased in a dose-related fashion. These effects are illustrated in Fig. I where yawning responses recorded during the I-h test period for both apomorphine (upper section) and NPA (lower section) are presented as a function of doses of neurotensin administered. Number of apomorphine-induced penile erections was significantly decreased tirst with 0.94 pg. Significant reductions in erections caused by NPA were first obtained with 1.87 pg. These results are presented in Table I where total number of penile erections observed in each group are included. Yawning responses produced by the administration of 100 &kg physostigmine were potently decreased by neurotensin. Starting with the relatively small dose of 0.05 pg, yawning was already significantly reduced. and continued to decrease with larger doses ofthe peptide. These effects are illustrated in Fig. 2, where number of yawns recorded during the l-h test period is presented as a function of doses of neurotensin administered. The effects of

0.0

1.87

0.94 NT

3.75

W

FIG. I. Total number of yawning responses recorded during the l-h test period for both apomorphine (upper section) and NPA (lower section) presented as a function of doses of neurotensin administered. Significant differences from saline-treated animals as revealed by Dunnett tests are indicated by asterisks (*p c 0.05: **p < 0.0 1f.

NEUROLEPTIC

PROPERTIES

489

OF NEUROT~~SIN

TABLE 1

TABLE 2

DOSE-RELATEDEFFECTSOF NEUROTENSINON PENILE

DOSE-RELATEDEFFECTSOF ATROPINE ON

ERECTIONS INDUCED BY A~MORPHINE N-FROPYLNOUA~~ORP~~NE~

Treatment

N-propyIno~pomo~hine

Apomorphine (100 /&kg)

Saline

AND

Treatment

Yawns

Erections

t f .8r&s)

2.5 rtr 1.4

2.0 4 0.7

1.9 ir 1.1

1.5 It 2.0 r 1.0 t 0.4 lr

Saline Atropine (mgikg) 1.0 5.0 10.0

NT (rcg) 0.47 0.94 1.87 3.75

YAWNING AND PENILE ERECTIONS INDUCED BY APOMORPHINE (100 ctpikg)*

0.9 i 0.6’ 0.6 +_0.6* 0.6 * ox)*

1.8 2.1 0.71 o.s*

* Values represent means + SD obtained with each group (n = 8) during the i-h test period. t Significant differences from saline-treated animals, as revealed by Dunnett tests, are indicated by symbols; (*II ==z O.OI; tP < 0.05).

various doses of atropine on number of yawns and penile erections induced by 100 &kg apomo~hine are presented in Table 2. As can be seen, 5 mg/kg of atropine significantly decreased num~r of yawns while the larger dose of 10 mg/kg almost totally inhibited this behavior. On the other hand, none of the doses of atropine sign~~cantl~ a&ted apomo~hine-induced penile erections. None of the doses of neurotensin significantly affected the incidence of sniffmg induced by 600 pg/kg apomorphine. Even a dose as large as 120 &g of the peptide failed to attenuate this stereotyped behavior. However, climbing behavior decreased linearly as a function of dose and statistically significant reductions were obtained with 30.0 and 60.0 pg of neurotensin. These results are illustrated in Fig. 3 where climbing frequency is presented as a function of dose of neurotensin administered. Five of the eight control animals exhibited at least one yawning response and in each case, yawning was detected in the last 15 min of the I-h test period when stereotypies had dissipated. On the other hand, no yawning was seen in animals injected with

13.4 rt 4.8

2.7 i 1.3

12.0 + 6.6 6.2 t 4.7*t 0.9 r 0.4*1

3.2 2 1.7 2.0 +- 1.1 4.5 + 5.0

* Values represent means + SD obtained in each group during the l-h test period. f Significant differences from saline-treated animals as revealed by Dunnett tests (p < 0.0 I ).

neurotensin. These results are included in Table 3 where number of sniffing episodes recorded following injection of saline and of repre~ntative doses of neurotensin are given. Number of animals displaying yawning in each group is also included in the table. 5lSCUSSfON

Together, the results of the present study extend previous findings that suggested that neurotensin has a profile of neurobehavioral effects similar to that of atypical neuroleptics (12). The peptide s~8nificantly decreased both yawning and erections induced by a relatively small dose of apomorphine. Compared to yawning, penile erections were significantly decreased with a smaller dose of neurotensin {Fig. 1; Table 1). Neurotensin also decreased both yawning and penile erections induced by NPA (Fig. 1: Table 1). The peptide was equipotent in inhibiting these behaviors induced by both NPA and apomo~hine. It has been reported that neurotensin decreases the a~nity of NPA for DA receptors in vitro (1). If this action constitutes the mechanism responsible for our in vivo results with NPA, then it might be generalized to other DA agonists, such as apomo~hine. In any case. a direct action of neurote~sin on the Dz receptors thought

I T

0.0

**

0.025 NT

0.05

h

0.94

W

FIG. 2. Total number of yawning responses recorded during the l-h test period following physostigmine administration presented as a function of doses of neurot~nsin administered. Significant differences from salinetreated animals as revealed by Dunnett tests are indicated by asterisks t**p < 0.01).

0.0

0.9

3.75

30.0

60.0

NT @sf FIG. 3. Dose-related effects of ~eurotensin on climbing induced by 0.6 mg/kg a~mo~hine. Statistically significant differences from controls as revealed by Dunnett tests are indicated by: *p < 0.05: **p < 0.0 1.

490

f0i.K

TABLE 3

snitfing*

i I‘ ,\I

hut

EFFECTS OF VARIOUS DOSES 01; ~~UROTE~SIN ON SNlFt’INf; ANlf YAWNING FOLLOWING ADMfNISTRATlON OF APOMORPWINE (600 &kg)

Treatment

Oi:l!R

\‘awmn@

* Expressed as mean ? SD of number of sniffing observations during the I-hr test period. t Values represent number of animals in each group (n = 8) displaying yawning during the l-h test period. 4 Significant differences from salinetreated animals as found with Fisher exact

probability tests (17< 0.05).

to mediate yawning (32) can be ruled out, because NT has only a weak affinity for the this subtype of receptor (20). As mentioned earfier, the inhibition of the yawning-penile erection syndrome is a common effect of both typical and atypical neuroleptics (3,.5,23). I-Iowever, contrary to typical neuroIeptics, neurotensin failed to reduce stereotyped sniffing induced by the larger dose of apomorphine (Table 31, thus confirming previous findings (11). Sniffing remained unaffected even with 120 pugof the peptide, the largest subtoxic dose we could utilize. The differential influence of the peptide on the penile-erection syndrome and on stereotypy thus seems to be more pronounced than that of atypical neuroleptics which can attenuate apomorphine-induced sniffing, albeit at much larger doses than those required to inhibit yawning and erections. It is noteworthy that yawning was not seen in animals injected with the peptide (Table 3). Again. the inhibition of yawning, a behavior which typically appears as stereotypy dissipates, is a characteristic effect of atypical neuroleptics, The results also demonstrate that neurotensin can antagonize apomorphine-induced climbing. This effect cannot be attributed to a nonspecific motor impairment effect of the peptide because we have shown previously that 60.0 fig of neurotensin, the most effective dose in reducing climbing, does not affect motor activity or muscle tone of animals (10). Also, careful observation of the animaIs during the course of this study did not reveal any behavioral abnormalities. Finally, the maintenance of oral stcreotypies in the experimental animals is further indication that inhibition of climbing is not due to a generalized sedative effect of the peptide.

The present results demonstrate for the first time that neurotensin can affect stereotyped behavior induced by a dopamine agonist. Until now examination of the possible influence of the peptide on stereotypy induced by do~rn~ner~c stimulation has been limited to oro-facial stereotypies, and the inability of neurotensin to decrease these behaviors has been reported frequently (6.11). This was confirmed in the present study. The selective inhibitory effect of the peptide on stereotyped climbing is, therefore, interesting, although the exact mechanism underlying this action is difficult to explain at the present time. The neuronal substrates of a~mo~hine-indu~ climbing in rats are unknown

they do not seem to be located in either the striatum or rhc nucleus a~~umhens. as lesions of these regions Jo not ahe&climbing (23). 7‘he dop~nlinc rcccptors implicated in this hchavior also remain to bc clearly: identified (7. f 8). However. the Fact that neurotensin aKcctcd chmbing but not cxW’xinl mowmcnts indicates that distinct receptors and/or nculophysit)logicaI processes underlit these difrerent apomorphinc-induced stereotyped behaviors. Yawning and penile erections induced by small doses ot’dopamine agonists are generally thought to hc mediated hy activation of dowamine aut[~r~c~~tors (4.27.3 1.37). ~~ltl~~~ugt~ data inconsistent with this hypothesis have been presented ( 19.28). ifautoreceptors do underlie yawning and climbing induced by small doses of dopdminc agonists. then our results constitute behavioral evidence that n~ur~~t~Ilsin might act as an antagonist ofdopamine aut~~re~ept(~rs. ~eurotensin also markedly antagonized pllysostigmine-induced yawning. The effect was first seen with 50 ng ofthe peptide. indicating that neurotensin is approximately 37 times more potent in reducing yawning produced by cholinergic stimulation than that elicited b>; ;I dopamine agonist. To our knowledge. this prominent tindmg constitutes the first evidence that neurotensin might possess central anticholiner~~ properties. In agreement with this hypothesis. we have ohserved recently that lleurotensin reduces both tremors and muscular rigidity in an animal model of Parkinson’s disease. two etfects that could possibib be attributed to an anticholincrgic action of the peptide ( 15). As discussed above. a dopamine-cholinergi~ link has been demonstrated for fawning responses induced by dopamine agonists (31). Such a link for penile erections produced by these drugs has been suggested but, as yet. has not been clearly cstablished (9). Our results with atropine confirm that an anticholinergic agent can inhibit efl‘ectively yawning induced by small doses of apomorphine (Table 2). However, as can be seen In this table, atropine did not affect the ii-equency oferections induced by the dopamine agonist. Therefore. the inhibition b> neurotensin of apomorphine- or NPA-elicited erections cannot solely ho attributed to an anti~h(~lin~rgi~ property of the peptide. The behavioral findings of this study are in agreement with our recent neurochetnical data that demonstrate that. contrary to the typical neuroleptics haloperidol and perphenazine whose increasing effects on striatal DOPAC tcvels are amplified by the dopam~nergi~ stimulant amfonelic acid. the elevation ofstr~atal levels of the d~~parni~i~t~~etaboljte produced h> cloLapinc. thioridazinc. and neurotcnsin ~+trrc~ actually decreased by amfonelic acid (25). In conclusion, neurotensin shares many central properties with atypical ncuroleptics. More specifically. our data suggest that the actions of neurotensin resemble more closely those of clozapine because, compared to other atypical neuroleptics such as sulpiride and thioridazine. this compound is the only antipsychotic capable ofantagoni~ing with similar potency ya~niiig induced by both dopamine agonists and by physostigminc (51. This finding is important and warrants further research because it has been reported that clozapine, a drug rapidly being recognized as the treatment of choice in the pharmacological management ofs~hizophrenia, does not produce extrapyramidal side effects in man (8). However, this drug is known to produce agranulocytosis in some patients, limiting it’s clinical usefulness. Therefore. the development of pharmokinetically viable neurotensin derivatives could hold promise for treating this debilitating disease. ,A(“KNOWLEIXXMENT

This work was supported by Cirant MT 9101 ofthe Medical Research Council of Canada.

NEUROLEPTIC

PROPERTIES

491

OF NEUROTENSIN REFERENCES

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