Pharmacological characterization of grooming induced by a selective NK-1 tachykinin receptor agonist

BRAIN RESEARCH ELSEVIER

Brain Research 700 (1995) 115-120

Research report

Pharmacological characterization of grooming induced by a selective NK-1 tachykinin receptor agonist A. J o n S t o e s s l a, * M u r i e l B r a c k s t o n e a, N a g a l i n g h a m R a j a k u m a r a, C a n d a c e J. G i b s o n b a

Clinical Neurological Sciences, University of Western Ontario and Robarts Research Institute, London, Ont., Canada b Department of Pathology, University of Western Ontario, London, Ont., Canada Accepted 11 July 1995

Abstract

Bilateral intranigral administration of the selective NK-1 tachykinin receptor agonist [AcArg6,Sar9,Met(O2)ll]SP6 11 (0-1 nmol total bilateral dose) selectively induced grooming in rats. This response was blocked by concurrent intranigral administration of the NK-1 tachykinin receptor antagonist RP 67580 (2 nmol), but not by NK-2 (L-659,877) or NK-3 ([TrpT,/3-AlaS]NKA4_10) antagonists. Pretreatment with systemic opioid (naloxone 1.5 mg/kg) and D 1 dopamine (SCH 23390 100/zg/kg) receptor antagonists also attenuated tachykinin-induced grooming, which was unaffected by D2 dopamine (sulpiride 30 mg/kg) or 5-HT2A+C (ritanserin 2 mg/kg) antagonists. Grooming induced by intranigrai [AcArg6,Sar9,Met(O2)aa]SP6_11was also attenuated by bilateral 6-hydroxydopamine lesions of the substantia nigra. These findings indicate that grooming induced by intranigral tachykinins reflects activation of NK-1 receptors and is dependent upon endogenous dopamine and consequent selective stimulation of D 1 dopamine receptors. Keywords: Dopamine; D 1 receptor; Grooming; Substantia nigra; Tachykinin

1. Introduction

There is a dense substance P (SP)-containing projection from the striatum to the substantia nigra [14] and electrophysiological [6,24], neurochemical [26,36] and behavioural [8,16] studies all suggest a functionally important interaction between SP and nigral dopamine neurons. This has been somewhat difficult to explain, as the substantia nigra is virtually devoid of receptors for SP [19,25]. However, there is pharmacological and molecular evidence for at least 3 types of tachykinin receptor [18,22] and whereas SP binds preferentially to the NK1 tachykinin receptor, we [31] and others [5] have demonstrated that the substantia nigra contains NK3 receptors which are reduced following 6-hydroxydopamine lesions [33]. In keeping with this, the NK3 agonist senktide stimulates firing of midbrain dopamine neurons [15] and also stimulates locomotion and rearing following local infusion into this region [32]. Although there appears to be a strong argument for preferential regulation of midbrain dopamine neurons by

* Corresponding author. Clinical Neurological Sciences, University Hospital, 339 Windermere Rd., London, Ont., Canada N6A 5A5. Fax: (1) (519) 663-3162; E-maih [email protected] 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 9 4 0 - X

the NK3 tachykinin receptor, it has additionally been noted that SP induces grooming following infusion into the substantia nigra, which may also be dopamine-dependent [16]. However, unlike locomotion and rearing, grooming is preferentially induced by NK1 agonists [9,32]. As autoradiographic binding studies have indicated that the substantia nigra is devoid of NK1 receptors [19,25], we sought to confirm this finding using [AcArg6,Sar9,Met(O2) 11]SP6_11, a novel, potent and selective NK1 tachykinin receptor agonist [27]. We additionally sought to determine which other transmitter systems might be involved in tachykinininduced grooming and specifically to determine whether this response is dependent upon an interaction with midbrain dopamine neurons.

2. Materials and methods

2.1. Animals

Male Sprague-Dawley rats (Charles River, Montreal) weighing approximately 250 g were used for all experiments. Animals were housed 2 per cage on a 12 h lightdark cycle (lights on at 07.00 h) and allowed tap water and rat chow ad libitum.

116

A.J. Stoessl et al./ Brain Research 700 (1995) 115-120

2.2. Surgery All surgery was performed under ketamine (40 m g / k g ) + xylazine (7 m g / k g ) anaesthesia. Stainless steel (22gauge) guide cannulae were stereotactically implanted bilaterally, 1.5 mm above the substantia nigra (5.3 mm posterior, 2.4 mm lateral, 7.6 mm ventral) using coordinates derived from Paxinos and Watson [23], with Bregma, the midline and skull surface as the respective reference points. Following surgery, patency of the guide cannulae was maintained using stainless steel obturators. Cannula placement was verified with conventional histology following all experiments.

2.3. Behavioural observations Animals were removed from their home cages and allowed to habituate for at least 60 min to perspex observation boxes (50 × 50 × 30 cm, with mirrors fixed to the rear walls) prior to drug administration. Following drug administration, behavioural responses were recorded for 3 min out of each 6 min block for a total of 10 blocks (60 rain), using a microcomputer with custom-designed software (BEBOP; Dr. M.T. Martin-Iverson, Univ. of Alberta) which allows simultaneous frequency and duration measurements of up to 30 categories of response. In addition to grooming, the following responses were recorded: locomotion (defined as forward motion from 1 of 9 imaginary cage sectors to another), rearing, sniffing and vacuous chewing movements (all non-directed mouth movements, including tongue protrusions and jaw tremor, but excluding gnawing and yawning). Grooming was categorized into face washing, body fur licking and scratching, each recorded separately. Penile grooming was not recorded.

2.4. Statistical analysis Univariate, repeated measures analysis of variance was performed, followed where appropriate by Bonferroni-adjusted paired t-tests comparing each treatment with vehicle-vehicle or vehicle-NK1 agonist conditions.

2.5. Drugs [AcArgO,Sarg,Met(O2)11]SP6_la (Bachem; Torrance, CA) was dissolved in DMSO (final concentration 5% v / v ) and brought to volume in 0.9% saline. RP 67580 was a generous gift from Rhone-Poulenc; L-659,877 was purchased from RBI (Natick, MA) and [TrpY,/3-AlaS]NKA4.1o from Peninsula. These drugs were dissolved in 0.1 N HC1, brought to volume in d H 2 0 and the pH brought to 6.2 with sodium bicarbonate. Naloxone and ( + ) - S C H 23390 (RBI, Natick, MA) were dissolved in 0.9% saline. (-)-Sulpiride and ritanserin (RBI, Natick, MA) were dissolved in 0.1 N HC1. 6-Hydroxydopamine-HBr (RBI, Natick, MA) was

dissolved in 0.05% ( w / v ) ascorbate in 0.9% saline and kept on ice. Desipramine (Sigma, St. Louis, MO) was dissolved in water.

2.6. Dose-response grooming

studies

of NK1

agonist-induced

[AcArg6,Sarg,Met(O2)ll]SP6_11 (0, 0.2, 0.5, 1 nmol in 1 /zl total bilateral dose) was administered into the substantia nigra bilaterally over 60 s via infusion cannulae inserted through the indwelling guide cannulae. Infusion cannulae were left in place another 60 s to allow diffusion of drug, then removed and replaced with the obturators. Each animal received all 4 doses of drug, separated by at least 48 h, with dose order randomized according to a Latin square design. The dose range chosen was based on preliminary studies (data not shown) which revealed robust grooming at a bilateral dose of 1 nmol, not significantly different at higher doses up to 5 nmol.

2.7. Effects of tachykinin antagonists on NK1 agonist-induced grooming In order to determine the specificity of the response for the NK1 tachykinin receptor, grooming induced by intranigral [AcArg6,Sarg,Met(O2) n ]SP6_ n (1 nmol total bilateral dose) was assessed as described above. Immediately prior to administration of the NK1 agonist, selective NK1 (RP 67580 [12]), NK2 (L-659,877 [21]) or N K 3 ( [ T r p 7 , j 3 AIa8]NKA4_10 [7]) antagonists or their vehicle were administered into each substantia nigra in a total bilateral dose of 2 nmot per/zl. Each animal received 5 treatments (vehicle + vehicle; vehicle + NK1 agonist; antagonists ([3] + NK1 agonist) separated by at least 48 h, with treatment order randomized using a Latin square design.

2.8. Effects of dopamine, opioid and 5-HT antagonists G room ing

induced

by

intranigral

[AcArg6,Sar9,Met(O2) 11]8P6_11 (1 nmol bilateral dose) was assessed as described above, 15 min following administration of naloxone (1.5 m g / k g s.c.), (-)-sulpiride (30 m g / k g i.p.), ritanserin (2 m g / k g s.c.) or their vehicle. Each animal received 5 treatments separated by 48 h, in randomized order. In a separate group of animals, the effects of pretreatment (15 min) with SCH 23390 (0-100 /xg/kg s.c.) on grooming induced by intranigral administration of the NK1 agonist were assessed. There were again 5 treatments per animal.

2.9. 6-hydroxydopamine lesions Animals were observed following the bilateral intranigral infusion of [AcArg6,Sar9,Met(O2)n]SP6_la or its vehicle, given in randomized order. After a delay of 48 h,

A J . Stoessl et al. / B r a i n Research 700 (1995) 115-120

animals then received either bilateral intranigral infusions of 6-hydroxydopamine (8 /zg in 2 /xl), following pretreatment with desipramine (20 m g / k g i.p.), or sham lesions performed by infusion of the ascorbate/saline vehicle. Following recovery from surgery and of preoperative weight (approximately 2 weeks) all animals were then observed following a second intranigral infusion of [AcArg6,Sar9,Met(O2 )u]sP6_ll. Following completion of this experiment, animals were perfused with 4% paraformaldehyde and sections through the substantia nigra and striatum were stained for tyrosine hydroxylase, using a rabbit primary antibody, goat secondary antibody and the avidin-biotin peroxidase technique (ABC, Vector).

3. Results In all cases, analysis was restricted to those animals with cannulae correctly positioned in the substantia nigra, as demonstrated by conventional histology. 3.1. Dose-response grooming

117

Table 1 Effects of nigral [AcArg6,Sar9,Met(O2)ll ]SP6_11 on behavioural responses other than grooming Vehicle

0.25 nmol

0.5 nmol

Locomotion

20+5

22+6

32+14

11+3

Rearing Sniffing

17+7

11+3

7+2

2_+1

316_+53 43 _ 13

294_+48 44 + 20

377+51 29 _+8

320_+75 25 _+5

Vacuous chewing

1.0 nmol

Values are the mean + S.E.M. duration of response (seconds), recorded for 30 out of the 60 min immediately following drug administration.

nist L-659,877 or the NK3 antagonist [Trp 7,/3AlaS]NKA4 10 (F4,36 = 19.56, P < 0.001; Fig. 2a; one of 10 animals excluded from analysis following histological examination). The antagonists alone did not affect grooming compared to intranigral vehicle (Fig. 2b), nor did they

A

EFFECTS OF NK1, NK2, NK3 RECEPTOR

ANTAGONISTS

2000

curve for NK1 agonist-induced 1500

No animals were discarded from this analysis. [AcArg6,Sar9,Met(Oz)ll]SP6_ll dose-dependently increased the duration of grooming (F3,27 = 4.38, P = 0.01; Fig. 1). All components of grooming were increased to a similar extent (data not shown). Locomotion, rearing, sniffing and vacuous chewing movements were unaffected (Table 1).

~

5

**

1000

500

I NKI (lnmol) : ANTAG

I 0 0

1 0

3.2. Effects of selective tachykinin antagonists

1 NK3

EFFECTS OF I N T R A N I G R A L NEUROKININ ANTAGONISTS ON GROOMING 300

Grooming induced by [AcArg6,Sarg,Met(O2)U]sP6_u was selectively blocked by intranigral infusion of the NK1 antagonist RP 67580, but unaffected by the NK2 antago-

250

~" 2 0 0

NK1AGONIST-INDUCED

1 1 NK1 NK2 TREATMENTS

GROOMING

L

C9

z

700

150

o o

600

:z

100

5OO

50

400 300

ANTAGONIST

2OO

I00 olo

015 DOSE (nmol)

1.'o

Fig. 1. Dose-response curve for total grooming following igral infusion of [AcArg6,Sarg,Met(O2)U]SP6_la (total Each point is the mean _+S.E.M. duration of grooming (n each dose) recorded for 30 of the 60 min following * P < 0.05, * * P < 0.01 compared to vehicle.

bilateral intranbilateral dose). = 10 animals at drug infusion.

VEH

NK1

NK2

NK3

Fig. 2. (a) Effects of concurrent nigral administration of NK1 (RP 67580), NK2 (L-659,877) and NK3 ([TrpT, fl-AlaS]NKA4_10) antagonists on grooming induced by intranigral [AcArg6,Sar9,Met(O2)U]SP6_u . Each bar is the mean_S.E.M, duration of total grooming (n = 9 per group) recorded for 30 out of the 60 min immediately following drug administration. * * P < 0.01, NK1 agonist alone vs. vehicle; ÷+ P < 0.01, NK1 agonist + NK1 antagonist vs. NK1 agonist alone. (b) Effects of intranigral NK1, NK2 and NK3 antagonists (2 nmol) alone on grooming. Each bar is the mean+S.E.M, duration of grooming ( n = 10 per group) recorded over 30 out of the 60 min immediately following drug administration.

118

A.J. Stoessl et al. / Brain Research 700 (1995) 115-120 EFFECTS OF SYSTEMIC D2, OPIATE, 5-HT ANTAGONISTS !500-

NK1 AGONIST INDUCED GROOMING EFFECTS OF 6 @HDA LESIONS

!500[ ~ V E H I C L E NK1 AGGNIST PRE LESION NK~AGONIST POST-LESION '~" 1000 I

c-

~2 c~ ~ olisoooo, si ' '

I

SO0 i

NK1 (lnmol) ANTAG

[ i NAL SULP TREATMENTS

1 R!T

1000

z cc~ c3

gO0

NK] (lqmoJ) SCH

0 0

t

affect locomotion, rearing, sniffing or vacuous chewing movements (no animals excluded; data not shown).

3.3. Effects of dopamine, opioid and 5-HT antagonists 1

.

10 ug 50 ug TqEATMENTS

, 100 u c,

Fig. 3. (a) Effects of pretreatment (15 min) with systemic opioid (naloxone 1.5 m g / k g ) , D e dopamine (sulpiride 30 m g / k g ) or 5-HTzA_ c (ritanserin 2 m g / k g ) antagonists on grooming induced by intranigral [AcArg6,Sarg,Met(O2) u ]SP~_ll (1 nmol total bilateral dose). Each bar is the mean _+S.E.M. duration of grooming (n = 10 per group) recorded for 30 out of the 60 min immediately following intranigral drug adminstration. * P <0.05, NK1 agonist alone vs. vehicle; + P <0.05, NK1 agonist +naloxone vs. NKI agonist alone. (b) Effects of systemic pretreatment (15 min) w i t h the D 1 dopamine receptor antagonist SCH 23390 (0-100 / . z g / k g ) on g r o o m i n g i n d u c e d by i n t r a n i g r a l [AcArg6,Sarg,Met(O2) n ]SP~_ I I (1 nmol total bilateral dose). Each bar is the mean -+ S.E.M. duration of grooming (n = 7 per group) recorded for 30 out o f the 60 min immediately following intranigral drug adminstration. * P <0.05, NK1 agonist alone vs. vehicle; + P < 0.01, NK1 agonist + SCH 23390 vs. NK1 agonist alone.

Table 2 Effects of SCH 23390 on behavioural responses other than grooming S C H 23390

Vehicle

Vehicle

10

50

100

Vehicle 36_+20 8+4 416_+139 27-+10

1 nmol

1 nmol

1 nmol

1 nmol

80_+20 19_+6 454+92 22-+10

31_+10 25_+11 318_+58 59-+29

5+3 *

8+0

1_+1 *

1_+1 *

(/zg/kg) NK1 agonist Locomotion Rearing Sniffing Vacuous chewing

Values are the mean + S.E.M. the 60 min immediately [AcArg6,Sarg,Met(O2) 11]SP6 * Significantly different from

60HDA

Fig. 4. Effects of bilateral nigral 6-hydroxydopamine lesions on grooming induced by intranigral [AcArg6,Sar°,Met(Oz)ll]SP~, 12 (1 nmol total bilateral dose). Each bar is the mean±S.E.M, duration of grooming (n = 8 per group) recorded for 30 out of the 60 min immediately following intranigral drug administration. * / * * P < 0.05/0.01, respectively, NK1 agonist vs. vehicle; + P = 0.054, post-lesion vs. pre-lesion.

EFFECTS OF SCH 23390 ON NK1 AGONIST-INDUCED GROOMING

B

SHA~I LESION

205_+66 64-+31" 47_+17 56_+21

duration of response, recorded over 30 out following administration of intranigral 11 or its vehicle. SCH 23390 = 0.

Grooming induced by intranigral [AcArg6,Sar9,Met(O2)ll]SP,_ll was attenuated by systemic pretreatment with naloxone (F5,45 = 5.83, P < 0.001; Fig. 3a; no animals exluded) or SCH 23390 (F4,zs = 3.60, P = 0.02; Fig. 3b; 3 of 10 animals excluded following histology), but unaffected by the D 2 antagonist ( - ) sulpiride or the 5-HT 2 antagonist ritanserin (Fig. 3a). In addition to its effects on grooming, SCH 23390 also suppressed sniffing (F4,2s = 3.57, P = 0.02), locomotion (F4,28 = 7.96, P < 0.001) and rearing (F4.28 = 3.90, P = 0.01) (Table 2).

3.4. 6-hydroxydopamine lesions Analysis of the lesioned group was restricted to those animals shown by immunocytochemistry to have sustained total or near total depletion of nigral and/or striatal tyrosine hydroxylase activity (n = 8 out of 12). Grooming induced by intranigral [AcArg6,Sarg,Met(O2) u ]SP6 11 was attenuated nearly to control levels following bilateral 6-hydroxydopamine lesions (F2,24 = 8.33, P = 0.004; P = 0.054, pre- vs. post-lesion), but the response was unaffected by sham lesions (F2,14 = 6.29, P = 0.011; P = n.s., pre- vs. post-lesion)(Fig. 4).

4. Discussion These studies suggest that stimulation of NK1 receptors within the substantia nigra is responsible for tachykinin-induced grooming and furthermore that this effect is in turn

A.J. Stoessl et al. / Brain Research 700 (1995) 115-120

mediated by selective stimulation of D 1 dopamine receptors. [AcArg6,Sar9,Met(O2)ll]sP6_ll is a selective agonist for the NK1 tachykinin receptor [27] and its ability to induce grooming following nigral infusion is in keeping with the responses seen using the closely related compound [Sar9,Met(O2 )11]SP 32 and other, less selective NK1 agonists [9,16]. Grooming is not induced by the NK2 agonist [Nle1°]NKA4_10 or the NK3 agonist senktide [32]. Finally, groom ing induced by [AcArg6,Sar9,Met(Oz)ll]sP6_11 was selectively antagonized by nigral infusion of the NK1 antagonist RP 67580, but unaffected by selective NK2 or NK3 antagonists. T h e g r o o m i n g i n d u c e d by [ A c A r g 6 , S a r 9, Met(O 2)11]SP6_11 was behaviourally specific, in that other behavioural responses such as locomotion, rearing and sniffing were unaffected. Of particular note, we did not observe the induction of vacuous chewing movements following intranigral administration of this compound, in contrast to Liminga [17], who reported mouth movements following nigral administration of [Pro9]Sp, another preferential NK1 agonist. There was a certain degree of variability in the magnitude of the grooming response between studies (e.g. Figs. 1 and 3b vs. Fig. 2a, Fig. 3a and Fig. 4), but this is not unexpected, given the use of different batches of animals and the fact that these studies were performed over several months, with possible changes in ambient laboratory conditions. The majority of the observations were performed by the same individual and the within-subject repeated measures analysis employed reduces the likelihood that treatment-related differences in response are artefactual. Studies with systemically adminstered antagonists to other transmitters suggest that the grooming induced by [AcArg6,Sar9,Met(Oz)11]SP6_ll is dependent upon stimulation of D 1 dopamine receptors. Thus, the D 2 antagonist sulpiride was without effect, whereas the D 1 dopamine antagonist SCH 23390 dose-dependently blocked tachykinin-induced grooming. This is in keeping with observations of grooming induced by SP and a variety of other neuropeptides [35]. We also found that grooming induced by intranigral administration of the NK1 tachykinin agonist was potently suppressed by systemic administration of the opioid antagonist naloxone. Naloxone blocks grooming induced by a number of different peptides [30] and furthermore attenuates grooming induced by the D a dopamine agonist SKF 38393 [34], suggesting that these effects are mediated downstream to the nigrostriatal pathway, possibly in the periaqueductal grey [29]. SCH 23390 also has 5HT 2 antagonist properties [4], but grooming induced by [AcArg 6'Sar9,Met(O:)11 ]SP6_ 11 was unaffected by the 5HT 2 antagonist ritanserin. This, as well as the attenuation of grooming following 6-hydroxydopamine lesions, suggests that tachykinin-induced grooming is indeed dependent upon stimulation of central D x dopamine receptors, consequent upon release of endogenous dopamine.

119

Receptor autoradiographic [33], electrophysiological [15] and in situ hybridization [37] studies all suggest that nigral dopamine neurons express NK-3 tachykinin receptors. Thus, the locomotion and rearing which result from nigral administration of NK3 tachykinin receptor agonists [32] almost certainly reflect direct stimulation of dopaminergic neurons of the pars compacta, with consequent striatal release of dopamine. However, while NK1 sites have not been seen in this region with receptor binding [19,25] or in situ hybridization [37] studies, more sensitive solution hybridization assays have indeed demonstrated the presence of NK1 receptor mRNA [28,37]. Our behavioural data suggest that this mRNA is indeed translated into functional protein. Nigral NK1 mRNA expression is unaffected by 6-hydroxydopamine lesions [28], thus grooming induced by an NK1 agonist is unlikely to reflect d i r e c t stimulation of nigral dopamine neurons, but may result from stimulation of an i n d i r e c t nigrostriatal pathway, possibly via disinhibition of a nigrothalamocorticostriatal loop. This would be compatible with the observations of Baruch et al. [2], who found that striatal dopamine release elicited by intranigral SP was attenuated by striatal administration of a glutamate antagonist, in contrast to dopamine release following the preferential N K 2 / N K 3 agonist neurokinin A, which was unaffected by such treatment. Why stimulation of this pathway should selectively elicit dopamine-dependent grooming and not locomotion or rearing is unclear, but this may reflect preferential release from terminals in striatal patch regions, where the majority of striatal D 1 receptors are expressed [3]. In summary, these findings confirm the importance of the prominent SP striatonigral projection. This pathway is affected by Parkinson's disease [20] and by Huntington's disease [10], as well as neuroleptic treatment [1]. Furthermore, the pattern of dopaminergic replacement therapy in experimental parkinsonism affects the expression of SP in this projection [11,13]. The evidence that stimulation of different tachykinin receptors likely affects striatal dopamine release via different mechanisms suggests that selective agents targetting the different receptor subtypes may be helpful in modifying these disease states or the response to therapy.

Acknowledgements Supported by the Canadian MRC and a Career Scientist Award from the Ontario Ministry of Health to A.J.S. We are grateful to Dr. C. Garret (Rhone-Poulenc Rorer) for the generous gift of RP 67580.

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