Modulation of the rat mesolimbic dopamine pathway by neurokinins

Behavioural Bra#l Research, 51 (1992) 77-82 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0166-4328/92/$05.00

77

BBR 01356

Modulation of the rat mesolimbic dopamine pathway by neurokinins P e t e r J. Elliott, G l e n n S. M a s o n , E l i z a b e t h A. G r a h a m , M i c h a e l P. T u r p i n a n d Russell M. Hagan Neuropharmacology Department, Glaxo Group Research Ltd., Ware(UK) (Received 18 May 1992) (Revised version received 30 June 1992) (Accepted 21 July 1992)

Key words: Nucleus accumbens; Ventral tegmental area; Dopamine; Locomotor activity; Neurokinin agonist; Neurokinin antagonist; Neurokinin I receptor

The locomotor activity (LMA) response induced after infusion of selective neurokinin (NK) agonists into the cell body (A10) and a terminal region of the mesolimbic pathway of the rat was investigated. Infusion of the NK I receptor-selective agonist, GR73632, into the ventral tegmental area (VTA: AI0) or the nucleus accumbens (NAS) significantly and dose-dependently increased basal LMA. Agonists setective for the NK 2 and NK 3 receptors, GR64349 and senktide respectively, had no effect on LMA after intra-NAS infusion. The LMA induced by GR73632 is mediated via dopamine (DA) since the response was abolished by haloperidol. From these studies it would appear that the elevated LMA reported previously after VTA or NAS administration of substance P probably occurs via NK I receptors. Such data supports the notion that endogenous NKs are likely to be important in modulating the mesolimbic DA pathway and, as a consequence, compounds which antagonise their effects could be useful for the treatment of disorders associated with this system. However, simultaneous infusion of the NK~ agonists, + CP-96,345 and its analogue CPQ, into the VTA did not attenuate the LMA induced after intra-VTA infusion of GR73632. Co-infusion of the NK l antagonist CPQ, but not + CP-96,345, attenuated the LMA response induced by GR73632 in the NAS. The apparent poor susceptibility of these responses to blockade by the recently developed non-peptide NK I antagonists was unexpected but may reflect their poor affinity for the rat variant of the NK I receptor. Further advances in the chemistry of NK 1 antagonists are now required to confirm the apparent importance of NK l receptor activation in the control of the mesolimbic DA pathway.

INTRODUCTION

The A10 dopamine (DA) cell body group, within the ventral tegmental area (VTA), project forward to innervate many cortical and subcortical areas 27. Activation of.the cortico mesolimbic DA pathway is known to result in a number of behaviours including reward, addictive behaviour, stress and in also increased locomotor activity (LMA) ~2. The ascending DA pathway from the VTA which innervates a number of structures including the nucleus accumbens (NAS) is known as the mesolimbic system. This pathway is of clinical importance, as it is believed to be overactive in schizophrenics (for review see ref. 16). However, the cause for this overactivity is as yet unknown. In rodents, many neurotr~msmitters are able to increase LMA after administration into the VTA reflecting the multiple inter-

Correspondence:P.J. EUiott, Neuropharmacology Department, Glaxo Group Research Ltd., Park Road, Ware, tterts., S G I 2 0DP, UK.

actions between DA and other systems in this region (for review see ref. 13). In particular, the neurokinins (NKA and NKB) and substance P (SP) are found in high concentrations in the V T A 15'26 and microinfusion of these endogenous peptides and selective N K agonists, have been shown to elevate LMA 5'25. Recently, we have reported that administration of the NKt agonist, GR73632, in the vicinity of the cell body end (VTA) of the mesolimbic DA pathway increases LMA s. In the rat N A S , NK-containing terminals are found in close proximity to tyrosine hydroxylase-containing terminals, suggesting a potential interaction between NKs and the terminals of the mesolimbic DA system22. This concept is supported by behavioural and biochemical studies which have shown that infusion of SP into the NAS increases the local release of DA and increases LMA ~4. Conversely, intra-NAS administration of N K antibodies reduces the release of DA and LMA observed after injection of amphetamine7. Furthermore, infusion of NKA into the NAS enhances amphetamine-

78 induced LMA 28. Together these findings all support the concept that NKs, in both the cell body and terminal regions of the mesolimbic pathway, could be important in the control of behaviours associated with this system. In an attempt to investigate this further we have looked at the effect of discrete microinfusion of selective NK agonists on LMA in the rat. Although binding sites for NK~, NK2 and NK 3 receptor ligands have been reported in the rat NAS 23, the NK receptors involved in the increased LMA and DA release have not been identified. The additional aim of this investigation was to characterise the NK receptor(s) linked with DA-mediated behaviours using peptidase-resistant and receptor-selective NK agonists and antagonists. The agonists used were Ava[L-Prog, N M e - L e u l ~ S u b s t a n c e P T - l l (GR73632; N K l ) l~ [Lys 3, GlyS-R-'/-lactam-Leug]NKA3_lo (GR64349; NK2) l~ and senktide (NK3) (Table I). The NK antagonists used were, (2S,3S,2R,3R)-cis-3-[(2-methoxyphenyl)methylamino]-2-benzhydrylquinuclidine ( + CP-96,345; NK1) 24 and its analogue (2S,3S,2R,3R)-cis-3-[(2chlorophenyl) methylamino]-2-benzhydryl quinuclidine (CPQ:NKI) 9, and cyclo[GIn,Trp,Phe,Gly, Leu,Met] (L-659,877: NK2) 18.

Testhzg apparatus Locomotor activity was assessed in clear perspex cages (300 x 405 x 160 mm) equipped with 5 infrared photocell beams placed 35 mm above the floor. Movement of the rats within this apparatus resulted in beam breaks which were recorded automatically onto a computer and were taken to represent LMA. To ensure true motor activity was recorded, rather than stereotyped or non-specific actix,ity, only beam breaks of 3 s - i or less were recorded as LMA.

Behaviottral testing Subsequent to the l-week recovery period, the rats were habituated to the photocell cages for 1.5 h prior to central drug infusion. Drugs and vehicles were administered in 1.0-1d volume over a 1.0 min period using needles which extended 1.0 mm below the guide cannulae, into either the NAS or VTA. A further 1.0 min was allowed for the drug to diffuse away from the needle tip. Co-administration of antagonists with the agonist occurred in a single 1.0-1d volume using the same infusion parameters. Motor activity was recorded for a 1.0-h period following the central drug/vehicle infusion.

Drugs MATERIALS AND METItODS

A nhnals and surgery Male Lister hooded rats (Glaxo, Ware; 220-270 g) underwent stereotaxic surgery to bilaterally implant stainless steel guide cannulae aimed 1.0 mm above the target area within the NAS (incisor bar set at + 5.0 mm; anterior to bregma + 3.4 mm; lateral from midline + 1.5 ram; ventral from skull -6.5 mm) or VTA (incisor bar set at + 5.0 ram; posterior to bregma -3.4 mm; lateral from midline +0.7 ram; ventral from skull -7.6 mm) using a procedure described previously4. Rats were housed in groups of four following the surgery for 1 week.

GR64349, GR73632, + CP-96,345 and CPQ, were all synthesised at Glaxo Group Research. Senktide, L-659,877 and haloperidol were purchased from Peninsula Peptides Inc. (Belmont, CA), Cambridge Research Biochemicals (Norwich, UK) and Sigma (Poole, UK) respectively. All the NK agonists and antagonists were infused in a sterile 0.9% saline solution, except L-659,877 which was dissolved in DMSO. Haloperidol was administered subcutaneously (s.c.) in 2.0~o tartaric acid in water. Appropriate vehicle controls were always used in each study.

Histology Upon conclusion of the study, rats were deeply anaesthetised with sodium pentobarbital, decapitated and their brains removed rapidly. Sectioning (25/am) of the

TABLE I Ambm acid sequences o f the neurokb~h~ (NK) agonists and their selectivity at the three N K receptors in classical in vitro assays

NK~ = rabbit thoracic aorta; NK 2 = guinea-pig trachea; NK 3 = rat portal vein. Numbers represent ECso values in nmol for the agonists. A'ettrokhthz agonist

Ava[ L'Pr~176 Substance P7-11 [ Lysa,GlyS-R-y-lactam-Leug]NeurokininA3_10 Succinyl[Asp6,MePheS]Substance P6-11

Receptor selectivity

[GR73632] [GR64349] [Senktidc]

NK t

NK2

NKj

4.0 4,000 > 30,000

1,000 4.0 > 30,000

> 10,000 !,300 10

79

NAS or VTA was performed on a microtome; the cannulation success rate was I00~o for the NAS and 8590~o for the VTA. Statistics Photocell beam breaks from all studies were analysed using ANOVA and a subsequent post-hoe Dunnett's test. P < 0 . 0 5 was deemed to be statistically significant. Only those animals with correct histologically verified cannulae placements were used in the data analysis.

~k 1200 -

1000

g 800

iiiiiiiiiiil iiiiiiiiii!l

o

0

0

!iiiiiii~ii~J 0.3

VE~CtE

0.0b

0,3

GR73632

0 C5

HALOPERIDOL (rr~1~ -$c I

(nmol}

RESULTS

lntra- VTA admhfistration of GR 73632 and NK~ antagonists Infusion of GR73632 (0.003-1.0 nmol) into the VTA increased LMA in a dose-dependent and statistically significant manner (Fig. 1). Infusion of either NKI antagonist into the VTA up to 30 nmol had no effect on basal LMA (Photocell counts: mean + S.E.M.: Veh: 412+72; CP-96,345 (30nmol): 515+63; CPQ (30 nmol): 529 + 107; n = 5-12). Furthermore, neither of the NK~ antagonists (30 nmol) reduced the motor response o f a submaximal dose of GR73632 (0.3 nmol) when they were co-infused into the VTA (Photocell counts: mean+S.E.M.: Veh: 240+85; GR73632: 1209+ 196; GR73632 + CP-96,345: 1040+316; GR73632 + CPQ: 1025 + 56; n = 6-13). However, pretreatment with the DA antagonist, haloperidol (0.05 mg.kg- i; s.c.), attenuated the LMA induced by GR73632 (0.3 nmol) without altering basal motor activity (Fig. 2).

Fig. 2. Effect of subcutaneous haloperidol on the locomotor activity induced by the NK 1 agonist, GR73632, after infusion into the ventral tegmental area of rats. * represents P < 0 . 0 5 wrt vehicle-treated group. ~ wrt agonist-treatcd group. All values represent mean _+S.E.M. (n = 8-10).

httra-NA S admhdstration of NK agonists The NK] agonist, GR73632 (0.03-10nmol), increased LMA in a dose-dependent and significant manner after infusion into the NAS (Fig. 3). Administration of the NK 2 agonist, GR64349 (0.3-3.0 nmol), or the N K 3 agonist, senktide (0.1-3.0 nmol), into the NAS did not elevate basal LMA (Photocell counts: mean + S.E.M.: Veh: 1000 + 229; GR64349 (3.0 nmol): 954 + 126; senktide (3.0 nmol): 959 + 78; n = 10). Subcutaneous administration ofhaloperidol (0.1 mg.kg- ]),

1400

1200

2000 1000

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750

~

500

600

o

250

400

N VEHICLE

200

0~3

001

0.03

0.1

1.0

GR73632 (nmol}

Fig. 1. Locomotor activity induced by the NK I agonist, GR73632, after infusion into the ventral tegmental area in rats. *represents P < 0 . 0 5 w r t vehicle-treated group. All values represent mean_+ S.E.M. ( n = 7-11).

Veh'

0.03 0.3 1.0

Fig. 3. Locomotor activity induced after infusion into the nucleus sents P < 0 . 0 5 w r t vehicle-treated mean +_S.E.M.

3.0 10.0

GR73632 (nmol)

by the NK I agonist, GR73632, accumbens in rats. *repregroup. All values represent Ol = 8-16).

80 1600

nist, + CP-96,345 (30 nmol), did not (Fig. 5). Neither of the NK~ antagonists (30 nmol) has any effect on basal motor activity (photocell counts: mean + S.E.M.: Veh: 144+21; CP-96,345: 205+78; CPQ: 225+63; n = 6-8). Finally, the NK2 antagonist, L-659,877 (up to 10nmol), had no effect on the LMA induced by GR73632 (Photocell counts: mean+S.E.M.: Veh: 204 + 52; GR73632:783 + 91; GR73632 + L-659,877 (10 nmol): 692 + 183; n = 6-7).

1400

1200

~

I000

o.

:~ 6oo DISCUSSION

o i~iiiii~ iii.:.i'i

o

200

iiiiii.--:,z Veh

I 0

3 0

0 0.1

1 0.1

3 GR73632 (nrnol) 0.1 Halopefidol (m~l~g;s.c.)

Fig. 4. Effect of subcutaneous, haloperidol, on the locomotor activity induced by the N K l agonist, GR73632, after infusion into the nucleus accumbens in rats. * represents P < 0 . 0 5 wrt vehicle-treated group and o P < 0 . 0 5 ~xt agonist-treated group. All values represent mean + S.E.M. (n = 10-12).

30 min prior to the central infusion of submaximal doses of GR73632 (i.0 and 3.0 nmol), inhibited the agonistinduced LMA (Fig. 4). Simultaneous intra-NAS infusion of CPQ (30 nmol) abolished the LMA response of a sub-maximal dose of GR73632 (1.0 nmol), whereas the other NK1 antago-

1500

1250

5 oo 75o

250

VEHICLE

1.0

3.0

1.0

tO

30

GR73632 (nmol) CP 96.345 (ninon)

30

CPQ

(nmoI)

Fig. 5. Effect of co-infusion of the NK1 antagonists, + CP-96,345 and CPQ, on the locomotor activity induced by the NK~ agonist, GR73632, after infusion into the nucleus accumbens in rats. * represents P < 0 . 0 5 wrt vehicle-treated group and o P < 0 . 0 5 wrt agonisttreated group. All values represent mean_+ S.E.M. (n = 5-29)~

The present study has confirmed our previous report s that infusion of the selective NK~ agonist, GR73632, into the cell body region of the mesolimbic DA pathway, the VTA, increases LMA. GR73632, but not GR64349 (NK2) or senktide (NK3), also increased LMA after infusion into the NAS, the cell terminal region of the mesolimbic DA pathway. These effects of GR73632, in the VTA and NAS, are likely to be mediated by the release of DA since they were inhibited by haloperidol. From these results it would appear that activation of NK~ receptors, at either end of the mesolimbic DA system, might be important in the modulation ofbehaviours and/or disease states with which this pathway is associated. The SP-eontaining afferents in the VTA have been shown to have direct contact with DA cells and their dendrites26. Furthermore, infusion of naturally occurring NKs and their analogues into the VTA have been previously shown to increase LMA and the subsequent release of DA in the areas innervated by DA cells in the A10 group a'6"s. Recently it has been shown that, in anaesthetised rats, the firing rate of A10 DA cells can be selectively increased by local application of the NK~ agonist, GR73632 z~ The present data is in agreement with the hypothesis that NKs and DA interact within the VTA and that the DA cells which project to the NAS can be influenced by GR73632, probably acting at NK~ receptors. Both SP and NKA, along with their preprotachykinin (PPT) mRNA, have been shown to have a heterogeneous distribution within the rat NAStL The highest concentrations of these NKs are found in the medial and ventral portions of the N A S z9 where a majority of the DA receptors ~ and terminals2~ are located. In fact NKA/SP immunoreactive terminals are known to be in close proximity to DA containing axons 22. Based on this anatomical information it was proposed that the local NK neurones within the NAS, influence the control of DA release and function in this nucleus. The stimulatory effect of GR73632 observed in this study is

81 consistent with the idea that NK-modulation of DA activity occurs via NKt receptors. Previously, it has been reported that N K A administration into the NAS enhances the LMA response of amphetamine zS. Furthermore, amphetamine increases the release not only of DA but also N K A 17 in the N A S . Thus, the increase in LMA observed after infusion of SP into the NAS would be expected to occur from an increase in DA release acting upon DA receptors. Indeed, Kalivas and Miller ~4 showed that intra-NAS infusion of SP increases DA release in the same area, and amphetamine-induced DA release in the NAS can be reduced with SP antibodies 7. The blockade of the GR73632-induced LMA response, by haloperidol, further supports the idea that this behavioural effect is mediated via release of DA onto DA receptors in the NAS, and supports a functional relationship between DA and the NKs in this area. The picture emerging from studies undertaken with the NKI antagonists is not as clear. Of the two NK~ antagonists tested, only CPQ antagonised the LMA response of GR73632; and only in the NAS, not the VTA. The poor antagonist activity of these compounds in both mesolimbic structures was unexpected and is, to date, unexplained. The failure of the parent nonpeptide NK~ antagonist, + CP-96,345, to inhibit the LMA response of GR73632 in the VTA or NAS cannot be explained by its lack of potency compared to CPQ since it has a higher potency in NK1 binding assays, conducted in a number of species 9. One key factor in the poor effect of these compounds may be their lower potency at rat CNS NK~ receptors, which is seen in both binding and functional studies 2. A further explanation is the possible existence of additional NK1 subtypes in the rat CNS, which are resistant to blockade by these compounds. Finally, these compounds may be rapidly re-distributed after their central administration and thus are not accessing the receptors at which GR73632 produces its effect. Interestingly CPQ, but not + CP-96,345, has recently been shown to inhibit the LMA induced by GR73632 after intracerebroventricular administration in guinea pigs 19 and after intrathecal injection +CP-96,345 does not inhibit the caudally-directed scratching and biting response in mice produced by the NK~ selective agonist, SP-methyl ester 3. The lack of effect of the N K 2 agonist to increase LMA and the failure of the NK2 ant~igonist, L-659,877, to inhibit the LMA effect of GR73632 in the NAS, implies that N K z receptors are unlikely to be involved with the LMA response of GR73632. This antagonist has previously been shown to antagonise contralateral rotations induced by a N K 2 selective agonist given into the substantia

nigra, which is believed to be a NK2 receptor-mediated event 8. In conclusion, based on data with selective agonists, NK~ but not N K z or N K 3 receptors appear to be important in the excitatory influence of SP on mesolimbic DA neurotransmission. However, results obtained with NK~ antagonists were inconclusive and a definitive view of the nature of the N K receptors involved awaits further studies. These hopefully will be undertaken when a wider variety of NK~ receptor antagonists become available. At such time it would be of great interest to investigate the activity of these compounds for the treatment of disorders associated with hyperactivity of the mesolimbic DA pathway, including schizophrenia.

ACKNOWLEDGEMENTS The authors would like to thank Mr. P. Smith and Mr. K. Mantle for their help with the figures.

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82 investigating neurokinin function, Neuropeptides, 19 (1991) 127135. 11 Harlan, R.E., Garcia, M.M. and Krause, J.E., Cellular localization of substance P- and neurokinin A-encoding preprotachykinin mRNA in the female rat brain, J. Comp. Neurol., 287 (1989) 179-212. 12 Iversen, S.D., Brain dopamine systems and behaviour. In L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.) Handbook of Psychophannacology, VoL 8: Dntgs, Neurotransmitters and Behaviours, Plenum, New York, 1977, pp. 333-384. 13 Kalivas, P.W., Evidence for interactions of endogenous peptides with the mesolimbic dopamine system, Psychopharmacol. Bull., 20 (1984) 354-357. 14 Kalivas, P.W. and Miller, J.S., Substance P modulation of dopamine in the nucleus accumbens, NeuroscL Lett., 48 (1984) 55-59. 15 Kanazawa, I., Ogawa, T., Kimura, S. and Munekata, E., Regional distribution of substance P, neurokinin A and neurokinin B in rat central nervous system, NeuroscL Res., 2 (1984) 111-120. 16 Langer, D.H., Brown, G.L. and Docherty, J.P., Dopamine receptor supersensitivity and schizophrenia: a review, Schizophr. Bull., 7 (1981) 208-224. 17 Lindefors, N., Brodin, E. and Ungerstedt, U., Amphetamine facilitates the in vivo release of neurokinin A in the nucleus accumbens of the rat, Eur. J. PhannacoL, 160 (1989) 417-420. 18 Maguire, J.J., Elliott, N.J., Varney, M.A., McKnight, A.T., Williams, B.J., Foster, A.C. and Tridgett R., Pharmacological specificity of synthetic peptides as antagonists of tachykinin receptors, Br. J. PhannacoL, 96 (1989) 124P. 19 Mason, G.S., Graham, E.A. and Elliott, P.J., Neurokinin NK-1 receptors in the guinea-pig mediate locomotor hyperactivity, Br. J. PhannacoL, 105 (1992) 251P. 20 Overton, P., Elliott, P.J., Hagan, R.M. and Clark, D., Neurokinin agonists differentially affect A9 and A10 dopamine cells in rat, Eur. J. PhannacoL, 213 (1992) 165-166.

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