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Behavioural effects of tachykinins and related peptides
l. l~c,,ic!
BRE 11943
Behavioural Effects of Tachykinins and Related Peptides P.J. ELLIOTI" and S.D. IVERSEN
Department of Experimental Psychology, Cambridge University, Cambridge ( U.K. Key words: substance P - - locomotor activity - - grooming - - wet dog shakes - - tachykinin -lateral ventricle - - ventral tegmental area
Substance P (SP) and related tachykinins administered either intracerebroventricularly or directly into the ventral tegmental area of the mesencephalon of rat brain caused increased locomotor activity, grooming behaviour and wet dog shakes. Kassinin, eledoisin, neurokinin A and DiMe-C7, agonists with some selectivity for the SP-E-receptor elicited the greatest locomotor activity and wet dog shake responses, whereas SP and physalaemin which are more selective for the SP-P-receptor were most effective in eliciting the grooming response. INTRODUCTION The tachykinins are a group of related peptides 6, which share a c o m m o n C-terminal sequence of amino acids. Substance P (SP) was the first of the family to be demonstrated in mammalian CNS which is now known to contain two further tachykinins, neurokinin A and neurokinin B. In non-mammalians and invertebrates further related tachykinins have been identified: kassinin and physalaemin in amphibia and eledoisin in molluscs. A substantial amount of biochemical and pharmacological work has been done with this range of endogenous tachykinins and it is clear that in the mammalian brain and in peripheral organs at least two and probably 3 different SP binding sites can be characterized s'27. The definition of the receptor types has depended on the rank ordering of potencies of various tachykinins on peripheral organ bioassays. Two patterns of agonist response have been most clearly defined: the SP-P-type in which physalaemin and SP act at nanomolar concentrations with eledoisin and kassinin somewhat less potent, and the SP-E-type response being characterized by nanomolar potencies for eledoisin and kassinin but much lower potencies for physalaemin and SP. More recently an SP-K-type response has been differ-
entiated with neurokinin A as the most potent agonist 2. Many questions regarding the definition of these receptor types and their localization in different target organs remain to be clarified. Although it appears that all 3 receptor types exist in the brain and the periphery, it is not clear if a given receptor type is the same at the central and peripheral sites. Despite these problems the proposed classification scheme provides a heuristic approach to the definition of the receptor types involved in different physiological and behavioural responses to the tachykinins. SP and neurokinin A (substance K), are unevenly distributed in the mammalian CNS 12"17. Both peptides are found in high concentrations in areas of brain rich in dopamine (DA)-containing neurones and closely associated with motor behaviouPS: the substantia nigra and the ventral tegmental area (VTA) of the mesencephalon. Bilateral infusions of SP into these areas of the ventral mesencephalon or into the ventricles adjacent to these areas induce a range of m o t o r behaviours, which on the basis of pharmacological and lesion experiments are known to involve ascending D A projections. During the daytime, when the spontaneous activity in the rat is relatively low, bilateral infusion of SP
Correspondence: P.J. Elliott, Neuroscience Research Centre, Merck Sharp & Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
69 into the VTA produces an increase in locomotor activity 24, a response dependent on the integrity of the A10 mesolimbic D A neurones with cell bodies lying in the VTA and axons innervating the nucleus accumbens (NAS) 15. Similar behavioural activation is observed after intracerebroventricular (i.c.v.) administration of SP 21'26 but inhibition of activity is observed if the peptide is infused during the active dark phase of the rat's diurnal cycle 26. Bilateral infusion of SP into the substantia nigra elicits locomotor activity but with repeated infusions grooming behaviour becomes predominant 13'14 whereas unilateral infusions in this nucleus produce contralateral rotation 9`m'19"2s, a motor behaviour mediated by the A9 nigrostriatal DA system. The initial behavioural experiments with SP were complicated by the finding that the effects of this tachykinin are of short duration because of its rapid degradation by brain peptidase enzymes 3,16. An analogue of SP, (Glp 5, MePhe 8, Sar9)-substance P5 11 (DiMe-C7), that is protected from enzymatic degradation is now available 3'22 and has been shown to induce a long-lasting stimulation of locomotor activity when infused into the VTA 4. DiMe-C7 also stimulates locomotor activity when infused i.c.v. 5. This synthetic peptide therefore provides a valuable tool for pursuing the behavioural analysis of the effects of tachykinins. The present experiments were designed first to compare the effects of VTA and i.c.v, infusions of SP and related tachykinins on locomotor activity and on structured behaviour in the open field. A second purpose of the present experiments was to obtain further dose-response data on the behavioural response to the stable analogue of SP, DiMeC7, after VTA and i.c.v, routes of administration and to study the ability of various neurotransmitter antagonists to block the response to DiMe-C7 after i.c.v, infusion. The actions of DiMe-C7 are of particular relevance in this respect, as this synthetic analogue of SP has recently been reported to be highly selective for eledoisin binding sites in brain which resemble SP-E-receptors I .
Sprague-Dawley rats (Olac, Bicester) weighing 300-350 g. Animals were housed in pairs in rooms with light and temperature regulation and food and water available ad libitum.
Measures of motor behaviour Photocell cages. Locomotor activity was measured in a bank of wire cages (40 x 25 x 25 cm), each equipped with two photocells on the long axis, located i cm above the floor and 10 cm from either end of the cage. Interruptions of the light beams were registered on line with a digital microprocessor (Rockwell AIM 65) and collated every 5 min throughout the test period. Testing occurred between 10.00 and 15.00 h to minimize the effect of day-to-day variations of environment on behaviour. This period also represents the quietest period of the rat circadian rhythm of activity. The testing room was fully illuminated and masking noise was present during the experiment. Open field. A white formica-covered wooden enclosure (100 x 100 x 40 cm) with black-lined squares (20 x 20 cm) was used to quantify behaviour. Testing occurred under identical conditions to those described above. Behaviour was recorded using a closed circuit video-tape recorder; film analysis was performed blind at a later date. The following behaviours were noted: locomotor behaviour (squares crossed) in the centre vs periphery of the field, full body grooming (duration) and wet dog shakes (frequency),
Surgery Subjects were anaesthetized with i.p. Equithesin and stereotaxically implanted with bilateral cannulae (23-gauge stainless steel) 1 mm above the desired injection site in the ventricles (i.c.v.) or VTA. With the head above the intra-aural line the coordinates used were: i.c.v. (0.4 mm posterior to bregma, 2.0 mm lateral to midline, 3.1 mm below skull); VTA (3.4 mm posterior to bregma, _+ 0.7 mm lateral to midline, 7.4 mm below skull). Once in position cannulae were affixed to the skull using stainless-steel screws and acrylic cement.
MATERIALS AND METHODS
Infusion procedure
Subjects All experiments were performed on male albino
One week after surgery, intracerebral infusions and behavioural testing began. Rats were infused bi-
70 laterally using two 10-¢d syringes mounted on a microdrive infusion pump. Conscious animals were hand held whilst the infusion needles (1 mm longer than cannulae) were inserted. Rats were allowed to move freely during the actual infusion procedure in an open white hemispherical bowl. The subjects were placed in the test apparatus 60 s after the drug administration, i.c.v, drug infusions: 5 ¢d/side at a rate of 2 kd/min; VTA microinjections: 1/d/side at a rate of 0.5/~l/min.
Testing procedures In our previous experience and bearing in mind the limited number of intracerebral infusions that can be made in an experimental animal without inducing non-specific damage, we have not found it reliable to treat one experimental animal with a range of peptides at different doses plus vehicle infusions. Accordingly, a separate group of rats was used for each peptide studied in both behavioural situations and within an experiment, an individual rat received peptide (at a given dose) or vehicle on 3 separate occasions. Animals were not used as their own controls. Independent groups of rats were used for the photocell and open field study. In the former study a given rat received the same dose of peptide or vehicle on 3 occasions at least 48 h apart. Rats were given a 1-h habituation period in the photocell cages before the drug or vehicle infusion. After the intracerebral infusion the rats were returned to the photocell cages for a 2-h test period. For open field behaviour the rats were placed in the apparatus immediately after drug infusion and observed for a period of 15 rain. Rats were exposed to the open field apparatus on only one occasion, and received a single peptide or vehicle treatment.
anaesthetized with pentobarbital and perfused intracardially with saline and finally lOC,;~ formalin, The brains were removed and stored in a 30G sucrose/lO% formalin solution until sucrose saturation had occurred. Using a microtome, sections were made throughout the brain, mounted and stained with Cresyl violet, to verify the cannulae placements.
Drugs SP, eledoisin and physalaemin were obtained from Cambridge Research Biochemicals, kassinin, neurokinin and DiMe-C7 from Peninsula Labs. The peptides were dissolved in artificial cerebrospinal fluid (CSF) which contained the following constituents (mEq per litre): NaCI 126, NaHCO~ 27.5, KCI 2.4, KH2PO4 0.5, CaCI 2 1.1, MgCI2 0.83, Na2SO ~ 0.5, glucose 5.9 (at pH 7.4). Peptide content (nmoi) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I. The antagonists haloperidol (Searle), fluphenazine (Squibb) cimetidine and mepyramine (Sigma), naloxone-HC! (Endo Labs.) and bicuculline (Cambridge Research Biochemicals) were prepared in physiological saline for i.p. injection. The doses of antagonists used and the pretreatment times were selected on the basis of published studies where the antagonists had been shown on i.p. administration to block neurotransmitter-mediated behavioural responses.
Statistics Results were analysed using a one-way analysis of variance ( A N O V A ) and a subsequent post-hoc Newman-Keuls test for group comparisons 29. Two-tailed significance tables were used to obtain significance values.
Histology On completion of the experiments, subjects were TABLE I
Peptide
Molecular weight
Substance P Eledoisin Neurokinin A DiMeC7 Kassinin Physalaemin
1348 1188 1132 880 1333 1266
RESULTS
Dose-response effects of i.c.v, administered SP, eledoisin and DiMe-C7 on locomotor activity SP at doses ranging from 0.1 to 20/~g induced a significant stimulation of locomotor activity of increasing magnitude. (Fig. 1, top). Eledoisin was active at doses of 0.7 and 7.0/~g and the size of the locomotor response was greater than that obtained with equivalent doses of SP. (Fig. 1, centre).
71 WEANPHOTOCELL COUNTS
MF./~I PHOTOCELL COUNTS
9OO
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m
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1.0 10.0 DOSEOF SUBSTANCEP (pg) (n--B)
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0 VEHICLE
0.25
1,0 DOSE OF DiMeC7 (Hg) (n=6)
40
8.0
WEAN PHOTOCELL COUNTS 2500
-Z-
2000
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Fig. 2. Dose-related mean (+_ S.E.M.) increase in locomotor activity measured in photocell cages over a 2-h period after bilateral infusion of 0.25-8.0 ug DiMe-C7 or vehicle CSF into the VTA. Dosage values are given as ug/rat (administered as two bilaterally symmetrical infusions). *P < 0.01 compared with CSF vehicle.
I
1000
1 )
DiMe-C7 at 1 - 8 Fg induced a significant stimulation of l o c o m o t o r activity and elicited a response as great as that observed with the highest dose of eledoisin studied (Fig. 1, bottom).
I
VT,HICLE
7.0
0.07 0.7 DOSEOFE ~ S i H {ff~)
Dose-response effects of V T A infusion of DiMe-C7 on locomotor activity
MCAN PHOTOCELL COUNTS 2500 m
2000
DiMe-C7 induced a significant stimulation of locom o t o r activity at 1 - 8 # g . The size of the response was double that seen with the same dose administered i.c.v. (Fig. 2).
1500
Effect of neurotransmitter antagonists on the locomotor response to DiMe-C7 i. c. v.
n
1000
500
VEHICLE
0.25
I .o
4.0
B0
DOSE OF OiMnC7 ~ag) (.=6)
Fig. 1. Dose-related mean (_+ S.E.M.) increase m locomotor activity measured in photocell cages of a 2-h period after i.c.v. infusion of top: SP, 0.1 #g (0.07 nmol), 1.0,ug (0.74 nmol)', 10.0 ug (7.4 nmo[), 20 ug (14.8 nmol); middle: eledoisin, 0.07 #g (0.059 nmol), /).7 #g (0.59 nmol), 7.0 Mg (5.9 nmol); and bottom: DiMe-C7, 0.25 #g (0.28 nmol), 1.01~g (1.14 nmol), 4.0 #g (4.5 nmoi), 8,0,ug (9.1 nmol), n = 6 per dose and vehicle. Dosage values are given as ug/rat (administered as two bilaterally symmetrical infusions). *P < 0.01 compared with CSF vehicle; 0, P < 0.05 compared with CSF vehicle.
The stimulation of l o c o m o t o r activity induced by 4 #g (2 ~g/side) of DiMe-C7 was not antagonized by naloxone (1 rng/kg), bicuculline (1 mg/kg), cimetidine (50 mg/kg) or m e p y r a m i n e (50 mg/kg) administered i.p. 30 rain before the i.c.v, infusion of D i M e C7. H a l o p e r i d o l (0.05 and 0.2 mg/kg) and fluphenazine (0.23 mg/kg) administered 30 rain before DiMeC7 significantly attenuated the locomotor response to the SP analogue (Fig. 3). A t the lower dose of haloperidol baseline spontaneous activity was not reduced from control levels but DiMe-C7-induced activity, although still significantly above vehicle control levels (P < 0.01), was significantly attenuated from DiMe-C7 control levels (P < 0.01). A t higher
72 doses of haloperidol and 0.23 mg/kg fluphenazine, the neuroleptic-induced attenuation of the DiMe-C7 response was greater but these doses of neuroteptic also significantly reduced baseline levels of activity: therefore only at 0.05 mg/kg haloperidol can a selective antagonism of DiMe-C7 be claimed.
EFFECT OF TACHYKININS (icy) ON LOCOMOTOR ACTWITY
MEAN PHOTOCELL COUNTS 1800
160O 14,00 1200
Comparison of the effect of i. c. v. and VTA infusion of equivalent doses of tachykinins on locomotor activity The locomotor responses to equivalent (0.45 nmol) doses of the 5 peptides infused i.c.v, are presented in Fig. 4 (upper part). Peptide content (nmol) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I. All the tachykinins except SP at this dose increased locomotor activity when compared with CSF vehicle: the rank order of effectiveness was eledoisin = kassinin = physalaemin = neurokinin A (NK-A) < SP. The same peptides were also administered bilaterally directly into the VTA. All the compounds significantly stimulated locomotor activity (Fig. 4, lower part). In agreement with results obtained after i.c.v, infusion, eledoisin and kassinin gave significantly
II
4-
1000
800 600 40O 200 0 VEHICLE
PHY SP NK-A KASS COMPOUND TESTED MEAN OF 6 ANIMALS EXCEPT SP AND VEH. ( n - 12 )
EL
EFFECT OF TACHYKININS (VTA) ON LOCOMOTORACTIVITY MEANpHOTOCELLCOUNTS 1800 1600 1400 1200
NEUROLEPTIC ANTAGONISM OF BEHAVIOURAL EFFECTSOF DiMeC7 (icy) CSF VEHICLE
OiMeC7
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i
600 MEAN PHOTOCELL COUNTS 1600 1400 ~
4`00 t
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0 VEHICLE
1000 800 600 4.00 200 0 CON'n~OL
+ 0,05 klAL. 4- 0.2 HAL. DRUG TREATMENT (m~lk9-1 )
4- 0.23 FLU.
(n-S)
Fig. 3. Antagonism of the locomotor stimulation induced by 4 pg or 4.5 nmol (2 ~g/side) DiMe-C7 infused i.c.v, by haloperidol (HAL; 0.05 and 0.2 mg/kg) and fluphenazine (FLU; 0.23 mg/kg) administered 1 per 30 min before peptide. Peptide content (nmol) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I. Mean locomotor activity (+ S.E.M.) measured in photocell cages over a 2-h period after i.c.v, infusion of DiMe-C7. * significant (P < 0.01) stimulation of locomotor activity compared with CSF vehicle.
PHY
t
l
SP NK-A COMPOUND TESTED
KASS
EL
(n=6) Fig. 4. Upper: mean (+ S.E.M.; n = 6) increase in locomotor activity recorded over 2 h in photocell cages after bilateral i.c.v, infusion of CSF or doses of physaelamin (PHY), substance P (SP), neurokinin A (NK-A), kassinin (KASS) and eledoisin (EL) calculated equimolar to 4 jzg (4.5 nmol) DiMe-C7; Peptide content (nmol) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I; lower: as above after bilateral VTA infusion. *P < 0.01 compared with CSF vehicle.
greater responses than NK-A, physataemin and SP. Comparison of the effect of i. c. v, or VTA infusions of equivalent doses of tachykinins on open field behaviour Locomotor activity. Locomotion in the centre of
73 MEAN SQUARES CROSSED
MEANSQUARESCROSSED
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~00
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2O0
%(30
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PHY
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NK-A KASS COMPOUNDTESTED (n-B)
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/EHICLE
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NK-A KASS COMPOUND TESTED
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(~=6)
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NK-A KASS COMPOUND TESTED
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NK-A KASS COMPOUND TESTED (n=6)
(n=6)
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GROOMING
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MEAN SECONDS 300
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NR -A KASS COMPOUNDTESTED (n-B)
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VEHICLE
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SP
NK - A KASS COMPOUND TESTED [n--6)
EL
Dill,Ill C7
Fig. 5. Left panel: mean (± S.E.M.; n = 6) increase in peripheral field locomotor activity, wet dog shakes and grooming behaviour recorded in the open field apparatus after bilateral i.c.v, infusion of CSF or doses of physaelamin (PHY), substance P (SP), neurokinin A (NK-A), kassinin (KASS) and eledoisin (EL) calculated equimolar to 4#g (4.5 nmol) DiMe-C7. Peptide content (nmol) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I. Right panel: as above after bilateral VTA infusion. *P < 0.01 compared with CSF vehicle. ~kP < 0.05 compared with CSF vehicle.
74 the open field was not consistently increased by either i.c.v, or VTA infusions of the tachykinins. Locomotion in the periphery of the field, however, proved to be a reliable indicator of the stimulatory effect of tachykinins on locomotor behaviour. The 5 natural tachykinins and DiMe-C7 all significantly increased peripheral locomotor activity when administered into the VTA (Fig. 5: right panel). The order of effectiveness was DiMe-C7 > kassinin = eledoisin = NK-A > SP = physalaemin. After i.c.v, infusion DiMe-C7, kassinin, eledoisin and NK-A produced a signficant stimulation of locomotion (Fig. 5: left panel) and the order of effectiveness of the tachykinins in this model was the same as that seen after VTA infusions. Wet dog shakes. Wet dog shakes were observed after peptide administration via the i.c.v, and the intracerebral routes of administration; more consistently by the former route. The order of effectiveness of the tachykinins in inducing wet dog shakes via the i.c.v, route was DiMe-C7 > eledoisin > NK-A > kassinin: the same order observed for the tachykinins on locomotor activity in the open field and in the photocell cages after i.c.v, and VTA infusion. Only DiMe-C7, the stable analogue of SP, induced wet dog shakes when peptides were administered by the VTA infusion route. Grooming behaviour. The tachykinins induced full body grooming behaviour when administered via the i.c,v, and intracerebral route; again more consistently following administration by the i.c.v, route. This behaviour showed a different order of effects than locomotor behaviour and wet dog shakes. DiMe-C7 failed to stimulate grooming, whereas SP and physalaemin, which did not markedly stimulate either locomotion or wet dog shakes when administered i.c.v. produced a marked stimulation of grooming behaviour. When administered into the VTA only SP and physalaemin significantly stimulated grooming. DISCUSSION The experiments confirm earlier studies in which SP or DiMe-C7 infusions into the lateral ventricles 4":~ or the VTA 24 stimulated locomotor activity. D o s e response studies showed that eledoisin and DiMe-C7 were more potent that SP in eliciting locomotion. DiMe-C7 induced a response of longer duration than
either SP or eledoisin when administered i.c.v, or directly into the VTA, confirming earlier findings. '[he response to DiMe-C7 administered i.c,v, was blocked by low doses of neuroleptics which did not themselves significantly reduce spontaneous locomotor activity. This supports the view that specific blockade of DA receptors rather than non-specific motor impairment accounts fl)r the ability of neuroleptics to block the effects of SP and related analogues on forebrain motor circuits inw~lving DA neurones 15 From earlier studies it was known that SP modulates DA neurones of the mesolimbic system to affect locomotion when the peptide is administered to VTA. The pattern of response to the various tachykinins suggests that an SP-E-type receptor s is involved in this SP dopaminergic interaction The conclusion that in the ventral tegmental area SP-E receptors are more important than SP-P receptors for the modulation of those dopamine neurones innervating nucleus accumbens and involved in locomotor activity is borne out by recent findings of Kalivas et al.ll. These authors observed a higher density of NK-A than SP receptors in the VTA and found that NK-A was 10 times more potent than S P m producing an increase in locomotor activity when injected into the VTA. SP was previously reported to stimulate various aspects of open field behaviour 15 when infused into the VTA and in the present stud}' this has also been found after i.c.v, administration of the peptide. The present study extends these obselvations to a range of tachykinins and their order of effectiveness m stimulating locomotor behaviour again suggests that motor arousal in both familiar (photocell cages) and unfamiliar (open field) environments involves the activation of an SP-E-type of receptor~ Tachykinins elicited wet dog shakes with the same order of effectiveness, again suggesting the inw)lvement of SP-E-type receptors, although DiMeC7 was far more effective than other SP-E-selective peptides. The brain substrate mediating this SP-elicited response remains unidentified. However, the site is readily accessible from the ventricles since i.c.v, infusions induced a high frequency of wet dog shakes whereas VTA infusions did not. By the latter route only DiMe-C7, the SP analogue known to be protected from enzymatic degradation, and to spread
75 undegraded from V T A sites of injection to remote
ments with a range of tachykinins. Alternatively the
brain sites, induced wet dog shakes, confirming ear-
rank order of effectiveness of the tachykinins in elic-
lier studies a. It is interesting that SP and eledoisin ad-
iting grooming may imply that an SP-P-type receptor
ministered to mice by the intracisternal or intrathecal
is more important for this behaviour as has been shown for salivation in the rat 25.
route giving ready access to the spinal cord do not induce wet dog shakes but instead cause intense biting and scratching behaviour 7'2°'23. G r o o m i n g behaviour
receptors mediate the stimulation of locomotor ac-
was stimulated by tachykinin infusions i.c.v, and into
tivity induced by V T A infusions of tachykinins. The
the VTA. However, this behaviour showed a differ-
same class of SP receptor is involved in the stimula-
ent range of sensitivity to the different tachykinins.
tion of wet dog shake behaviour, although at basal
SP and physalaemin were the most active whereas they were the least active in stimulating locomotion
of receptor appears to mediate grooming behaviour
and wet dog shakes. It is possible that stimulation of locomotion interferes with long sequences of inte-
In summary the present results suggest that SP-E
ganglia sites distinct from the VTA. A different class although its nature and anatomical location need to be determined.
grated grooming behaviour and those tachykinins inducing most locomotion, fail to stimulate grooming because of response incompatability. In earlier studies SP infusion into the substantia nigra elicited progressively more intense grooming behaviour and less locomotion with successive substantia nigra infusions 13. It would be interesting to repeat these experi-
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ACKNOWLEDGEMENTS P.J.E. received an M.R.C. training award and the research was supported by M.R.C. Grant G978/290/N to S. D. 1.
effects of substance P injected into the substantia nigra of the rat, J, Pharm. Pharmacol., 29 (19771 181-182. l/) James, T.A. and Starr, M.S., Behavioural and biochemical effects of substance P injected into the substantia nigra, Br. J. Pharmacol., 65 (1979) 423-429. 11 Kalivas, P.W., Deutch, A+Y., Maggio, J.E., Mantyh, P.W. and Roth, R.H.+ Substance K and substance P in the ventral tegmental area, Neurosci. Lett_ 57 (1985) 241-246. 12 Kanazawa, I., Ogawa, T., Kimura, S. and Munekata, E+, Regional distribution of substance P, neurokinin ct neurokinin fl in rat central nervous system, Neurosci. Res., 2 (1982) 111-120. 13 Kelley, A.E. and Iversen, S.D., Behavioural responses to bilateral injections of substance P into the substantia nigra of the rat, Brain Research, 158 (1978) 474-478. 14 Kelley, A.E. and Iversen, S.D., Substance P infusion into substantia nigra of the rat: behavioural analysis and involvement of striatal dopamine+ Eur. J. Pharmacol., 60 (1979) 171-179. 15 Kelley, A.E., Stinus, L. and Iversen, S.D., Behavioural activation induced in the rat by substance P infusions into the VTA: implications of dopaminergic All) neurones, Neurosci. Lett., 11 (1979) 335-339. 16 Lee, C.M., Sandberg, B.E.B., Hanley, M.R. and lversen, L.L., Purification and characterization of a membrane bound substance P degrading enzyme from human brain, Eur. J. Biochem., 114 (1981) 315-327. 17 Ljungdahl, A., H6kfelt, T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-944. 18 Ljungdahl, A., HOkfelt, T., Nilsson, G. and Goldstein, M.,
76 Distribution of substance P-like immunoreactivity in the central nervous system of the rat. II. Light microscopic localization in relation to catecholamine-containing neurons, Neuroscience, 3 (1978) 945-976. 19 Olpe, H.R. and Koelta, W.P., Rotatory behaviour in rats by intranigral application of substance P and an eledoisin fragment, Brain Research, 126 (1977) 576-579. 20 Rackham, A., Therriarlt, M. and Wood, P.L., Substancc P: evidence for spinal mediation of some behavioural effects, Neuropharmacoloy, 20 (1981) 753-755. 21 Rondeau, D.B., Jolicoeur, F.B., Belanger, F. and Barbeau, A., Motor activity induced by substance P in rats, Pharrnacol. Biochem. Behav., 9 (1978) 769-775. 22 Sandberg, B.E.B., Lee, C.M., Hanley, M.R. and Iversen, L.L., Synthesis and biological properties of enzyme-resistant analogues of substance P, Eur. J. Biochem., 114 (1981 ) 329-337. 23 Share, N.N. and Rackham, A., Intracerebral substance P in mice: behavioural effects and narcotic agents, Brain Research, 211 (1981)379-386.
24 Stinus, L., Kelley, A.E. and Iversen, S.D., Increased spontaneous activity following substance P infusion into A 10 dopaminergic area, Nature (London), 276 (1976) 616-618. 25 Takano, Y., Takeda, Y., Doteuchi, M., lnouye, K. and Kamiya, H.O., Effect of a novel tachykinin, substance K, on salivation in rats, Eur. J. Pharmacol., 1ll (1985) 381-383. 26 Treptow, K., Oehme, P., Gabler, [i. and Bienerf, M., Modulation of locomotor activity by substance P in rats, ReguL Peptides, 5 (1983) 343-351. 27 Watson, S.P., Are the proposed substance P receptor subtypes, substance P receptors, Life Sci., 25 (1984) 797-808. 28 Welzl, H., Flack, G.H.G. and Husttm, J.P., Contraversive circling and facilitation of the perioral biting reflex by injection of substance P or D-AIa2 Met 5 enkephalinamide into the substantia nigra, Behav. NeuroL BioL, 34 (1982) 104-108. 29 Winer, B.J., Statistical Principles it1 £[r.pertrnental Design, MacGraw-Hill, New York, 1971.
BRE 11943
Behavioural Effects of Tachykinins and Related Peptides P.J. ELLIOTI" and S.D. IVERSEN
Department of Experimental Psychology, Cambridge University, Cambridge ( U.K. Key words: substance P - - locomotor activity - - grooming - - wet dog shakes - - tachykinin -lateral ventricle - - ventral tegmental area
Substance P (SP) and related tachykinins administered either intracerebroventricularly or directly into the ventral tegmental area of the mesencephalon of rat brain caused increased locomotor activity, grooming behaviour and wet dog shakes. Kassinin, eledoisin, neurokinin A and DiMe-C7, agonists with some selectivity for the SP-E-receptor elicited the greatest locomotor activity and wet dog shake responses, whereas SP and physalaemin which are more selective for the SP-P-receptor were most effective in eliciting the grooming response. INTRODUCTION The tachykinins are a group of related peptides 6, which share a c o m m o n C-terminal sequence of amino acids. Substance P (SP) was the first of the family to be demonstrated in mammalian CNS which is now known to contain two further tachykinins, neurokinin A and neurokinin B. In non-mammalians and invertebrates further related tachykinins have been identified: kassinin and physalaemin in amphibia and eledoisin in molluscs. A substantial amount of biochemical and pharmacological work has been done with this range of endogenous tachykinins and it is clear that in the mammalian brain and in peripheral organs at least two and probably 3 different SP binding sites can be characterized s'27. The definition of the receptor types has depended on the rank ordering of potencies of various tachykinins on peripheral organ bioassays. Two patterns of agonist response have been most clearly defined: the SP-P-type in which physalaemin and SP act at nanomolar concentrations with eledoisin and kassinin somewhat less potent, and the SP-E-type response being characterized by nanomolar potencies for eledoisin and kassinin but much lower potencies for physalaemin and SP. More recently an SP-K-type response has been differ-
entiated with neurokinin A as the most potent agonist 2. Many questions regarding the definition of these receptor types and their localization in different target organs remain to be clarified. Although it appears that all 3 receptor types exist in the brain and the periphery, it is not clear if a given receptor type is the same at the central and peripheral sites. Despite these problems the proposed classification scheme provides a heuristic approach to the definition of the receptor types involved in different physiological and behavioural responses to the tachykinins. SP and neurokinin A (substance K), are unevenly distributed in the mammalian CNS 12"17. Both peptides are found in high concentrations in areas of brain rich in dopamine (DA)-containing neurones and closely associated with motor behaviouPS: the substantia nigra and the ventral tegmental area (VTA) of the mesencephalon. Bilateral infusions of SP into these areas of the ventral mesencephalon or into the ventricles adjacent to these areas induce a range of m o t o r behaviours, which on the basis of pharmacological and lesion experiments are known to involve ascending D A projections. During the daytime, when the spontaneous activity in the rat is relatively low, bilateral infusion of SP
Correspondence: P.J. Elliott, Neuroscience Research Centre, Merck Sharp & Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
69 into the VTA produces an increase in locomotor activity 24, a response dependent on the integrity of the A10 mesolimbic D A neurones with cell bodies lying in the VTA and axons innervating the nucleus accumbens (NAS) 15. Similar behavioural activation is observed after intracerebroventricular (i.c.v.) administration of SP 21'26 but inhibition of activity is observed if the peptide is infused during the active dark phase of the rat's diurnal cycle 26. Bilateral infusion of SP into the substantia nigra elicits locomotor activity but with repeated infusions grooming behaviour becomes predominant 13'14 whereas unilateral infusions in this nucleus produce contralateral rotation 9`m'19"2s, a motor behaviour mediated by the A9 nigrostriatal DA system. The initial behavioural experiments with SP were complicated by the finding that the effects of this tachykinin are of short duration because of its rapid degradation by brain peptidase enzymes 3,16. An analogue of SP, (Glp 5, MePhe 8, Sar9)-substance P5 11 (DiMe-C7), that is protected from enzymatic degradation is now available 3'22 and has been shown to induce a long-lasting stimulation of locomotor activity when infused into the VTA 4. DiMe-C7 also stimulates locomotor activity when infused i.c.v. 5. This synthetic peptide therefore provides a valuable tool for pursuing the behavioural analysis of the effects of tachykinins. The present experiments were designed first to compare the effects of VTA and i.c.v, infusions of SP and related tachykinins on locomotor activity and on structured behaviour in the open field. A second purpose of the present experiments was to obtain further dose-response data on the behavioural response to the stable analogue of SP, DiMeC7, after VTA and i.c.v, routes of administration and to study the ability of various neurotransmitter antagonists to block the response to DiMe-C7 after i.c.v, infusion. The actions of DiMe-C7 are of particular relevance in this respect, as this synthetic analogue of SP has recently been reported to be highly selective for eledoisin binding sites in brain which resemble SP-E-receptors I .
Sprague-Dawley rats (Olac, Bicester) weighing 300-350 g. Animals were housed in pairs in rooms with light and temperature regulation and food and water available ad libitum.
Measures of motor behaviour Photocell cages. Locomotor activity was measured in a bank of wire cages (40 x 25 x 25 cm), each equipped with two photocells on the long axis, located i cm above the floor and 10 cm from either end of the cage. Interruptions of the light beams were registered on line with a digital microprocessor (Rockwell AIM 65) and collated every 5 min throughout the test period. Testing occurred between 10.00 and 15.00 h to minimize the effect of day-to-day variations of environment on behaviour. This period also represents the quietest period of the rat circadian rhythm of activity. The testing room was fully illuminated and masking noise was present during the experiment. Open field. A white formica-covered wooden enclosure (100 x 100 x 40 cm) with black-lined squares (20 x 20 cm) was used to quantify behaviour. Testing occurred under identical conditions to those described above. Behaviour was recorded using a closed circuit video-tape recorder; film analysis was performed blind at a later date. The following behaviours were noted: locomotor behaviour (squares crossed) in the centre vs periphery of the field, full body grooming (duration) and wet dog shakes (frequency),
Surgery Subjects were anaesthetized with i.p. Equithesin and stereotaxically implanted with bilateral cannulae (23-gauge stainless steel) 1 mm above the desired injection site in the ventricles (i.c.v.) or VTA. With the head above the intra-aural line the coordinates used were: i.c.v. (0.4 mm posterior to bregma, 2.0 mm lateral to midline, 3.1 mm below skull); VTA (3.4 mm posterior to bregma, _+ 0.7 mm lateral to midline, 7.4 mm below skull). Once in position cannulae were affixed to the skull using stainless-steel screws and acrylic cement.
MATERIALS AND METHODS
Infusion procedure
Subjects All experiments were performed on male albino
One week after surgery, intracerebral infusions and behavioural testing began. Rats were infused bi-
70 laterally using two 10-¢d syringes mounted on a microdrive infusion pump. Conscious animals were hand held whilst the infusion needles (1 mm longer than cannulae) were inserted. Rats were allowed to move freely during the actual infusion procedure in an open white hemispherical bowl. The subjects were placed in the test apparatus 60 s after the drug administration, i.c.v, drug infusions: 5 ¢d/side at a rate of 2 kd/min; VTA microinjections: 1/d/side at a rate of 0.5/~l/min.
Testing procedures In our previous experience and bearing in mind the limited number of intracerebral infusions that can be made in an experimental animal without inducing non-specific damage, we have not found it reliable to treat one experimental animal with a range of peptides at different doses plus vehicle infusions. Accordingly, a separate group of rats was used for each peptide studied in both behavioural situations and within an experiment, an individual rat received peptide (at a given dose) or vehicle on 3 separate occasions. Animals were not used as their own controls. Independent groups of rats were used for the photocell and open field study. In the former study a given rat received the same dose of peptide or vehicle on 3 occasions at least 48 h apart. Rats were given a 1-h habituation period in the photocell cages before the drug or vehicle infusion. After the intracerebral infusion the rats were returned to the photocell cages for a 2-h test period. For open field behaviour the rats were placed in the apparatus immediately after drug infusion and observed for a period of 15 rain. Rats were exposed to the open field apparatus on only one occasion, and received a single peptide or vehicle treatment.
anaesthetized with pentobarbital and perfused intracardially with saline and finally lOC,;~ formalin, The brains were removed and stored in a 30G sucrose/lO% formalin solution until sucrose saturation had occurred. Using a microtome, sections were made throughout the brain, mounted and stained with Cresyl violet, to verify the cannulae placements.
Drugs SP, eledoisin and physalaemin were obtained from Cambridge Research Biochemicals, kassinin, neurokinin and DiMe-C7 from Peninsula Labs. The peptides were dissolved in artificial cerebrospinal fluid (CSF) which contained the following constituents (mEq per litre): NaCI 126, NaHCO~ 27.5, KCI 2.4, KH2PO4 0.5, CaCI 2 1.1, MgCI2 0.83, Na2SO ~ 0.5, glucose 5.9 (at pH 7.4). Peptide content (nmoi) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I. The antagonists haloperidol (Searle), fluphenazine (Squibb) cimetidine and mepyramine (Sigma), naloxone-HC! (Endo Labs.) and bicuculline (Cambridge Research Biochemicals) were prepared in physiological saline for i.p. injection. The doses of antagonists used and the pretreatment times were selected on the basis of published studies where the antagonists had been shown on i.p. administration to block neurotransmitter-mediated behavioural responses.
Statistics Results were analysed using a one-way analysis of variance ( A N O V A ) and a subsequent post-hoc Newman-Keuls test for group comparisons 29. Two-tailed significance tables were used to obtain significance values.
Histology On completion of the experiments, subjects were TABLE I
Peptide
Molecular weight
Substance P Eledoisin Neurokinin A DiMeC7 Kassinin Physalaemin
1348 1188 1132 880 1333 1266
RESULTS
Dose-response effects of i.c.v, administered SP, eledoisin and DiMe-C7 on locomotor activity SP at doses ranging from 0.1 to 20/~g induced a significant stimulation of locomotor activity of increasing magnitude. (Fig. 1, top). Eledoisin was active at doses of 0.7 and 7.0/~g and the size of the locomotor response was greater than that obtained with equivalent doses of SP. (Fig. 1, centre).
71 WEANPHOTOCELL COUNTS
MF./~I PHOTOCELL COUNTS
9OO
4500
BOO
47)00
700
3500
600
E
m
3000
•
500
2500
4,00
2000
300
1500
200
1000
100 0
I
5000
1000
Z 5-
500 VEHICLE
0.1
1.0 10.0 DOSEOF SUBSTANCEP (pg) (n--B)
20.0
0 VEHICLE
0.25
1,0 DOSE OF DiMeC7 (Hg) (n=6)
40
8.0
WEAN PHOTOCELL COUNTS 2500
-Z-
2000
1500
Fig. 2. Dose-related mean (+_ S.E.M.) increase in locomotor activity measured in photocell cages over a 2-h period after bilateral infusion of 0.25-8.0 ug DiMe-C7 or vehicle CSF into the VTA. Dosage values are given as ug/rat (administered as two bilaterally symmetrical infusions). *P < 0.01 compared with CSF vehicle.
I
1000
1 )
DiMe-C7 at 1 - 8 Fg induced a significant stimulation of l o c o m o t o r activity and elicited a response as great as that observed with the highest dose of eledoisin studied (Fig. 1, bottom).
I
VT,HICLE
7.0
0.07 0.7 DOSEOFE ~ S i H {ff~)
Dose-response effects of V T A infusion of DiMe-C7 on locomotor activity
MCAN PHOTOCELL COUNTS 2500 m
2000
DiMe-C7 induced a significant stimulation of locom o t o r activity at 1 - 8 # g . The size of the response was double that seen with the same dose administered i.c.v. (Fig. 2).
1500
Effect of neurotransmitter antagonists on the locomotor response to DiMe-C7 i. c. v.
n
1000
500
VEHICLE
0.25
I .o
4.0
B0
DOSE OF OiMnC7 ~ag) (.=6)
Fig. 1. Dose-related mean (_+ S.E.M.) increase m locomotor activity measured in photocell cages of a 2-h period after i.c.v. infusion of top: SP, 0.1 #g (0.07 nmol), 1.0,ug (0.74 nmol)', 10.0 ug (7.4 nmo[), 20 ug (14.8 nmol); middle: eledoisin, 0.07 #g (0.059 nmol), /).7 #g (0.59 nmol), 7.0 Mg (5.9 nmol); and bottom: DiMe-C7, 0.25 #g (0.28 nmol), 1.01~g (1.14 nmol), 4.0 #g (4.5 nmoi), 8,0,ug (9.1 nmol), n = 6 per dose and vehicle. Dosage values are given as ug/rat (administered as two bilaterally symmetrical infusions). *P < 0.01 compared with CSF vehicle; 0, P < 0.05 compared with CSF vehicle.
The stimulation of l o c o m o t o r activity induced by 4 #g (2 ~g/side) of DiMe-C7 was not antagonized by naloxone (1 rng/kg), bicuculline (1 mg/kg), cimetidine (50 mg/kg) or m e p y r a m i n e (50 mg/kg) administered i.p. 30 rain before the i.c.v, infusion of D i M e C7. H a l o p e r i d o l (0.05 and 0.2 mg/kg) and fluphenazine (0.23 mg/kg) administered 30 rain before DiMeC7 significantly attenuated the locomotor response to the SP analogue (Fig. 3). A t the lower dose of haloperidol baseline spontaneous activity was not reduced from control levels but DiMe-C7-induced activity, although still significantly above vehicle control levels (P < 0.01), was significantly attenuated from DiMe-C7 control levels (P < 0.01). A t higher
72 doses of haloperidol and 0.23 mg/kg fluphenazine, the neuroleptic-induced attenuation of the DiMe-C7 response was greater but these doses of neuroteptic also significantly reduced baseline levels of activity: therefore only at 0.05 mg/kg haloperidol can a selective antagonism of DiMe-C7 be claimed.
EFFECT OF TACHYKININS (icy) ON LOCOMOTOR ACTWITY
MEAN PHOTOCELL COUNTS 1800
160O 14,00 1200
Comparison of the effect of i. c. v. and VTA infusion of equivalent doses of tachykinins on locomotor activity The locomotor responses to equivalent (0.45 nmol) doses of the 5 peptides infused i.c.v, are presented in Fig. 4 (upper part). Peptide content (nmol) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I. All the tachykinins except SP at this dose increased locomotor activity when compared with CSF vehicle: the rank order of effectiveness was eledoisin = kassinin = physalaemin = neurokinin A (NK-A) < SP. The same peptides were also administered bilaterally directly into the VTA. All the compounds significantly stimulated locomotor activity (Fig. 4, lower part). In agreement with results obtained after i.c.v, infusion, eledoisin and kassinin gave significantly
II
4-
1000
800 600 40O 200 0 VEHICLE
PHY SP NK-A KASS COMPOUND TESTED MEAN OF 6 ANIMALS EXCEPT SP AND VEH. ( n - 12 )
EL
EFFECT OF TACHYKININS (VTA) ON LOCOMOTORACTIVITY MEANpHOTOCELLCOUNTS 1800 1600 1400 1200
NEUROLEPTIC ANTAGONISM OF BEHAVIOURAL EFFECTSOF DiMeC7 (icy) CSF VEHICLE
OiMeC7
1000 800
i
600 MEAN PHOTOCELL COUNTS 1600 1400 ~
4`00 t
200
1200
0 VEHICLE
1000 800 600 4.00 200 0 CON'n~OL
+ 0,05 klAL. 4- 0.2 HAL. DRUG TREATMENT (m~lk9-1 )
4- 0.23 FLU.
(n-S)
Fig. 3. Antagonism of the locomotor stimulation induced by 4 pg or 4.5 nmol (2 ~g/side) DiMe-C7 infused i.c.v, by haloperidol (HAL; 0.05 and 0.2 mg/kg) and fluphenazine (FLU; 0.23 mg/kg) administered 1 per 30 min before peptide. Peptide content (nmol) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I. Mean locomotor activity (+ S.E.M.) measured in photocell cages over a 2-h period after i.c.v, infusion of DiMe-C7. * significant (P < 0.01) stimulation of locomotor activity compared with CSF vehicle.
PHY
t
l
SP NK-A COMPOUND TESTED
KASS
EL
(n=6) Fig. 4. Upper: mean (+ S.E.M.; n = 6) increase in locomotor activity recorded over 2 h in photocell cages after bilateral i.c.v, infusion of CSF or doses of physaelamin (PHY), substance P (SP), neurokinin A (NK-A), kassinin (KASS) and eledoisin (EL) calculated equimolar to 4 jzg (4.5 nmol) DiMe-C7; Peptide content (nmol) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I; lower: as above after bilateral VTA infusion. *P < 0.01 compared with CSF vehicle.
greater responses than NK-A, physataemin and SP. Comparison of the effect of i. c. v, or VTA infusions of equivalent doses of tachykinins on open field behaviour Locomotor activity. Locomotion in the centre of
73 MEAN SQUARES CROSSED
MEANSQUARESCROSSED
500
600
500
~00
3O0
2O0
%(30
0 VEH
PHY
SP
NK-A KASS COMPOUNDTESTED (n-B)
EL
/EHICLE
DiMeC7
PHY
SP
NK-A KASS COMPOUND TESTED
EL
OiMaC7
(~=6)
WET DOG SHAKES
WET DOG SHAKES WEANNUMBER
MEANNUMBER
40
30
I
i
25
20
15
10
I
S
[
0 VEH
PHY
SP
NK-A KASS COMPOUND TESTED
EL
,
"
,
VEHICLE
OiMeC7
i
i
PHY
I
I
SP
NK-A KASS COMPOUND TESTED (n=6)
(n=6)
I
L
l
l "I- , EL
DiMaC7
GROOMING
GROOMING ,IEAN SECONDS
MEAN SECONDS 300
300
250
250
200
200
±
150
150
I
! lOO
I00
S0
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0
i
9
VEH
PHY
SP
NR -A KASS COMPOUNDTESTED (n-B)
OiMeC7
VEHICLE
PHY
SP
NK - A KASS COMPOUND TESTED [n--6)
EL
Dill,Ill C7
Fig. 5. Left panel: mean (± S.E.M.; n = 6) increase in peripheral field locomotor activity, wet dog shakes and grooming behaviour recorded in the open field apparatus after bilateral i.c.v, infusion of CSF or doses of physaelamin (PHY), substance P (SP), neurokinin A (NK-A), kassinin (KASS) and eledoisin (EL) calculated equimolar to 4#g (4.5 nmol) DiMe-C7. Peptide content (nmol) was calculated on a weight basis using the molecular weights of the peptides as shown in Table I. Right panel: as above after bilateral VTA infusion. *P < 0.01 compared with CSF vehicle. ~kP < 0.05 compared with CSF vehicle.
74 the open field was not consistently increased by either i.c.v, or VTA infusions of the tachykinins. Locomotion in the periphery of the field, however, proved to be a reliable indicator of the stimulatory effect of tachykinins on locomotor behaviour. The 5 natural tachykinins and DiMe-C7 all significantly increased peripheral locomotor activity when administered into the VTA (Fig. 5: right panel). The order of effectiveness was DiMe-C7 > kassinin = eledoisin = NK-A > SP = physalaemin. After i.c.v, infusion DiMe-C7, kassinin, eledoisin and NK-A produced a signficant stimulation of locomotion (Fig. 5: left panel) and the order of effectiveness of the tachykinins in this model was the same as that seen after VTA infusions. Wet dog shakes. Wet dog shakes were observed after peptide administration via the i.c.v, and the intracerebral routes of administration; more consistently by the former route. The order of effectiveness of the tachykinins in inducing wet dog shakes via the i.c.v, route was DiMe-C7 > eledoisin > NK-A > kassinin: the same order observed for the tachykinins on locomotor activity in the open field and in the photocell cages after i.c.v, and VTA infusion. Only DiMe-C7, the stable analogue of SP, induced wet dog shakes when peptides were administered by the VTA infusion route. Grooming behaviour. The tachykinins induced full body grooming behaviour when administered via the i.c,v, and intracerebral route; again more consistently following administration by the i.c.v, route. This behaviour showed a different order of effects than locomotor behaviour and wet dog shakes. DiMe-C7 failed to stimulate grooming, whereas SP and physalaemin, which did not markedly stimulate either locomotion or wet dog shakes when administered i.c.v. produced a marked stimulation of grooming behaviour. When administered into the VTA only SP and physalaemin significantly stimulated grooming. DISCUSSION The experiments confirm earlier studies in which SP or DiMe-C7 infusions into the lateral ventricles 4":~ or the VTA 24 stimulated locomotor activity. D o s e response studies showed that eledoisin and DiMe-C7 were more potent that SP in eliciting locomotion. DiMe-C7 induced a response of longer duration than
either SP or eledoisin when administered i.c.v, or directly into the VTA, confirming earlier findings. '[he response to DiMe-C7 administered i.c,v, was blocked by low doses of neuroleptics which did not themselves significantly reduce spontaneous locomotor activity. This supports the view that specific blockade of DA receptors rather than non-specific motor impairment accounts fl)r the ability of neuroleptics to block the effects of SP and related analogues on forebrain motor circuits inw~lving DA neurones 15 From earlier studies it was known that SP modulates DA neurones of the mesolimbic system to affect locomotion when the peptide is administered to VTA. The pattern of response to the various tachykinins suggests that an SP-E-type receptor s is involved in this SP dopaminergic interaction The conclusion that in the ventral tegmental area SP-E receptors are more important than SP-P receptors for the modulation of those dopamine neurones innervating nucleus accumbens and involved in locomotor activity is borne out by recent findings of Kalivas et al.ll. These authors observed a higher density of NK-A than SP receptors in the VTA and found that NK-A was 10 times more potent than S P m producing an increase in locomotor activity when injected into the VTA. SP was previously reported to stimulate various aspects of open field behaviour 15 when infused into the VTA and in the present stud}' this has also been found after i.c.v, administration of the peptide. The present study extends these obselvations to a range of tachykinins and their order of effectiveness m stimulating locomotor behaviour again suggests that motor arousal in both familiar (photocell cages) and unfamiliar (open field) environments involves the activation of an SP-E-type of receptor~ Tachykinins elicited wet dog shakes with the same order of effectiveness, again suggesting the inw)lvement of SP-E-type receptors, although DiMeC7 was far more effective than other SP-E-selective peptides. The brain substrate mediating this SP-elicited response remains unidentified. However, the site is readily accessible from the ventricles since i.c.v, infusions induced a high frequency of wet dog shakes whereas VTA infusions did not. By the latter route only DiMe-C7, the SP analogue known to be protected from enzymatic degradation, and to spread
75 undegraded from V T A sites of injection to remote
ments with a range of tachykinins. Alternatively the
brain sites, induced wet dog shakes, confirming ear-
rank order of effectiveness of the tachykinins in elic-
lier studies a. It is interesting that SP and eledoisin ad-
iting grooming may imply that an SP-P-type receptor
ministered to mice by the intracisternal or intrathecal
is more important for this behaviour as has been shown for salivation in the rat 25.
route giving ready access to the spinal cord do not induce wet dog shakes but instead cause intense biting and scratching behaviour 7'2°'23. G r o o m i n g behaviour
receptors mediate the stimulation of locomotor ac-
was stimulated by tachykinin infusions i.c.v, and into
tivity induced by V T A infusions of tachykinins. The
the VTA. However, this behaviour showed a differ-
same class of SP receptor is involved in the stimula-
ent range of sensitivity to the different tachykinins.
tion of wet dog shake behaviour, although at basal
SP and physalaemin were the most active whereas they were the least active in stimulating locomotion
of receptor appears to mediate grooming behaviour
and wet dog shakes. It is possible that stimulation of locomotion interferes with long sequences of inte-
In summary the present results suggest that SP-E
ganglia sites distinct from the VTA. A different class although its nature and anatomical location need to be determined.
grated grooming behaviour and those tachykinins inducing most locomotion, fail to stimulate grooming because of response incompatability. In earlier studies SP infusion into the substantia nigra elicited progressively more intense grooming behaviour and less locomotion with successive substantia nigra infusions 13. It would be interesting to repeat these experi-
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ACKNOWLEDGEMENTS P.J.E. received an M.R.C. training award and the research was supported by M.R.C. Grant G978/290/N to S. D. 1.
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