Regulatory Peptides 168 (2011) 59–68

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Regulatory Peptides

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Effect of subchronic administration of tachykinin antagonists on response ofguinea-pigs to mild and severe stress

Wayne Anderson, Penny Newson, Maria Skeri, Norma Janssen, Stephen Milne, Jane Yip,Sebastian Jungnickel, Ann Lynch-Frame, Loris A. Chahl ⁎School of Biomedical Sciences and Pharmacy, University of Newcastle, NSW 2308, Australia

⁎ Corresponding author at: School of Biomedical Scieof Newcastle, Callaghan, NSW 2308, Australia. Tel.: +49292461.

E-mail address: loris.chahl@newcastle.edu.au (L.A. C

0167-0115/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.regpep.2011.03.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 July 2010Received in revised form 19 February 2011Accepted 30 March 2011Available online 13 April 2011

Keywords:NK1 antagonistNK3 antagonistFosCentral nervous systemAdrenal glandMorphine withdrawal

The effects of subchronic subcutaneous treatment with tachykinin receptor antagonists over nine days on therepeated mild stress response induced by daily subcutaneous injections and on the severe acute stressinduced by morphine withdrawal were investigated in guinea-pigs. The NK1 receptor antagonist, L733,060,0.25 mg/kg, significantly increased locomotor activity of guinea-pigs compared with animals subjected torepeated injection of the inactive enantiomer, but inhibited Fos-like immunoreactivity (Fos-LI) in thehypothalamus. In animals subjected to the acute severe stress of naltrexone-induced morphine withdrawal,treatment with the NK1 antagonist, L733,060, produced reductions in Fos-LI in the spinal dorsal horn, whereasthose treated with the NK3 antagonist, SSR146,977, 0.3 mg/kg, had reduced Fos-LI in the dorsal horn, adrenalmedulla, nucleus accumbens, ventral tegmental area and periaqueductal grey. Those animals treated withboth NK1 and NK3 antagonists also had reduced Fos-LI in the amygdala and paraventricular nucleus of thethalamus. It was concluded that the NK1 antagonist reduced the hypothalamic response to mild stress but theNK3 antagonist was more effective in reducing the severe stress response to morphine withdrawal.Furthermore, combination of NK1 and NK3 antagonists was more effective than either antagonist in reducingthe Fos-LI response to morphine withdrawal.

nces and Pharmacy, University61 2 49291673; fax: +61 2

hahl).

l rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The mammalian tachykinins, substance P, neurokinin A andneurokinin B, are peptides that are widely distributed in the centraland peripheral nervous systems. Pharmacological and molecularcloning studies have shown that there are three major receptors forthe tachykinins, NK1, NK2 and NK3 [1–3], NK1 and NK3 receptors beingthe most numerous in the central nervous system (CNS) [4,5].Immunohistochemical studies have shown a widespread distributionof both NK1 and NK3 receptors throughout rat, guinea-pig and humanbrain [6–9]. NK1 receptors are widely distributed in the caudateputamen, in diencephalic structures and in the mid and hind brain,whereas NK3 receptors are extensively distributed in deep andsuperficial layers of the cortex where many appeared to be locatedon astrocytes in the glia limitans. The cortical distribution of NK3

receptors is similar in both guinea-pig [9] and human [10] brain butdiffers from that in rat which lacks NK3 receptors in the glia limitans[8].

Preclinical studies in animals indicated that tachykinins, acting viaNK1 receptors, played an important role in nociception [11–13]. In

clinical trials, however, NK1 receptor antagonists failed to produceanalgesia in clinical pain states [14,15]. These results led toquestioning whether animal models of pain are capable of distin-guishing between pain relief and relief of stress [14]. Although theonly clinical use of tachykinin receptor antagonists at present is theuse of NK1 receptor antagonists in the treatment of chemotherapy-induced emesis [16,17], there is considerable preclinical evidence thatantagonists of both NK1 and NK3 receptors are promising therapeutictargets for treatment of psychiatric disorders. Preclinical studies inseveral species e.g. [11,18–23] indicating that NK1 receptor antago-nists had an anxiolytic action suggested that NK1 receptor antagonistsmight be antidepressant in humans [24–26]. The first clinical trialssupported this concept although subsequent trials have beendisappointing [27]. Nevertheless, the findings that NK1 receptorsplay a role in stress and anxiety [28], aggression [29,30], and rewardand positive reinforcement [31–33], and NK3 receptors modulatedopaminergic function and behaviour [34,35] and have antipsychoticactivity [36–38], suggest that the psychotherapeutic potential of theseagents remains to be exploited.

The observation that human tachykinin receptors differed structurallyfrom rat receptors in ways that affected the potency of antagonists, butwere similar to guinea-pig receptors [39], prompted the developmentof suitable guinea-pig models of CNS disorders to facilitate studies ontachykinin receptor antagonists. Previous studies in our laboratoryshowed that naltrexone-induced morphine withdrawal was a stressful

60 W. Anderson et al. / Regulatory Peptides 168 (2011) 59–68

response in guinea-pigs. A marked morphine withdrawal responsecharacterized by behaviours including vocalization, marked locomotoractivity, rearing and digging, occurred following repeated treatment ofguinea-pigs with morphine over three days [40], but a milder responsewas observed following a single dose of morphine [41]. Furthermore,increased levels of catecholamineswere found in the bloodof guinea-pigsfollowingmorphinewithdrawal [42]. Themorphinewithdrawal responsein guinea-pigs was readily quantifiable by behavioural observation of theanimals, and by increased expression of Fos, the protein product of theimmediate-early gene, c-fos [43] in many brain regions [40]. Therefore,the morphine withdrawal response appeared to be a useful model ofstress in the guinea-pig.

Morphine withdrawal behaviours are similar to those produced bycentrally administered substance P in guinea-pigs [44], and the mildmorphinewithdrawal response following a single dose ofmorphinewasreduced by centrally administered peptide receptor antagonists ofsubstance P [41]. Maldonado et al. [45] and Michaud and Couture [46]also showed that non-peptide NK1 receptor antagonists modified someof the morphine withdrawal signs in rats. However, unpublishedexperiments in our laboratory showed that non-peptide NK1 receptorantagonists produced an apparent reduction in the mild withdrawalbehaviours and Fos-like immunoreactivity (Fos-LI) following a singledose of morphine in guinea-pigs, but produced little change in with-drawal behaviours following the more marked withdrawal responsefollowing repeated treatment with morphine. Thus the possibility wasconsidered that a single dose of antagonistmightnot reveal thepotentialof tachykinin receptor antagonists sincedrugsused to treat human stressconditions are given over several days before an effect becomesapparent. Furthermore, the possibility was considered that an NK1

receptor antagonist alone might not be as effective as a combination ofNK1 and NK3 receptor antagonists. Therefore, in the present study theeffects of the potent, brain penetrant antagonists, L733,060, an NK1

receptor antagonist [47,48], and SSR146977, an NK3 receptor antagonist[49], alone and in combination, given over a nine day period wereinvestigated on the naltrexone-inducedwithdrawal response in guinea-pigs following treatment with morphine administered over the finalthree days of the experiment. The repeated subcutaneous injectionsconstituted a stressor for the animals, albeit a milder stressor than themorphine withdrawal response. Therefore, an experiment was carriedout in separate groups of guinea-pigs to compare the effect of subchronicadministration over ninedays of theNK1 receptor antagonistwith that ofsubchronic administration of its inactive enantiomer to determinewhether theNK1 receptor antagonist reduced themilder stress responseinduced by repeated subcutaneous injection. The behaviours of guinea-pigs as well as Fos-LI immunoreactivity in the brain, spinal cord andadrenal gland were quantified.

2. Materials and methods

2.1. Animals

All experiments were authorized by the Animal Care and EthicsCommittee of the University of Newcastle, an accredited researchestablishment. Young adult outbred tri-colour guinea-pigs, Caviaporcellus, of either sex, weighing 300–650 g, were used. Guinea-pigswere housed in same-sex pairs in plastic cages (73×54×24 cm) withshredded paper bedding and wire mesh covering. They were main-tained at a constant temperature of 23°±1 °C and on a 12–12 h light–dark cycle with lights on at 7:00. Food and water were freely availableand a vitamin C supplementwas added to the drinkingwater daily. Theguinea-pigs were allowed to acclimatize to the environment for sevendays before commencement of experiments. Handling of the animalsoccurred each day for four days before treatment to reduce handlingstress during the experiment. The animals were randomly divided intocontrol and treatment groups.

2.2. Drugs

The drugs used were: NK1 receptor antagonist, L733,060, 3-[[3,5-bis(trifluoromethyl) phenyl] methoxy]-2-phenylpiperidine, and its in-active enantiomer, L733,061 (Sigma-Aldrich Pty Ltd, Australia); NK3

receptor antagonist, SSR146977, ((R)-(N)-[1-[3-[1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]propyl]-4-phenylpiperidin-4-yl]- N-dimethylurea) (kindly donated by Sanofi-Synthélabo Recherche, Mont-pellier, France); morphine sulphate (David Bull Laboratories, Rydal-mere, NSW, Australia); morphine tartrate (David Bull Laboratories);naltrexone hydrochloride (Sigma-Aldrich); sodium pentobarbitone(Lethabarb, Virbac (Australia) Pty Ltd, Peakhurst, NSW, Australia).Stock solutions of 1 mg/ml of L733,060, L733,061 and SSR146977 weremade in 20 mg/ml dimethyl sulphoxide (DMSO) in deionized waterevery three days. Stability of solutions had been tested previously inorgan bath experiments on guinea-pig isolated ileum. Preparedsolutions were stored at 4 °C until time of injection.

2.2.1. Effect of subchronic administration of NK1 receptor antagonist,L733,060, on the behavioural and neuronal stress responses of guinea-pigs to repeated daily subcutaneous injection

Three groups of four guinea-pigs were used. Group 1 animals weretreated subchronically for nine days with L733,060, 0.25 mg/kg(calculated on the weight of the animal on the first day of treatment)subcutaneously (s.c.) once daily each morning. This dose was chosensince it was the ID50 (dose of antagonist that produced 50% inhibition)for inhibition of the formalin paw late phase response in gerbils, acentrallymediated NK1 receptor response [47]. These animalswere alsogiven saline injections twice daily from day 6 to day 9 to parallel thetreatmentof animals givenmorphine (see below), in order to reduce thenumber of animals used. Group 2 animals were treated similarly butwere given the inactive enantiomer, L733,061, 0.25 mg/kg s.c. Theanimals in Group 3 received no treatment, their behaviour was notobserved and they were handled as little as possible during theexperiment. Onday 9, 1 h after thefinal injections of drug and saline, thebehaviours of the guinea-pigs in Groups 1 and 2 were observed andquantified for 90 min as described below under Behavioural observa-tions. All animals were then given a lethal dose of sodium pentobar-bitone (l00 mg/kg, intra-peritoneally (i.p.)) and perfuse-fixed bytranscardial perfusion with heparinized (5000 U/l) phosphate-bufferedsaline (PBS; pH 7.4, 37 °C) followed by 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.4, 37 °C). Brains, adrenal glands and spinal cordswere removed, post-fixed overnight in 4% paraformaldehyde, andtransferred to a cryoprotectant solution of 30% sucrose and 0.5%paraformaldehyde in PBS at 4 °C.

2.2.2. Effect of subchronic administration of NK1 receptor antagonist,L733,060, on the behavioural and neuronal acute severe stress responsesinduced by morphine withdrawal

Two groups of four guinea-pigs were used in this experiment. Onegroup was given L733,060, 0.25 mg/kg s.c. each morning for nine days.The control animals were given the inactive enantiomer L733,061,0.25 mg/kg s.c. From day 6 of treatment the animals were also given s.c.injections of morphine sulphate or morphine tartrate twice daily forthree days, to make the animals dependent. The time between theantagonist or inactive enantiomer andmorphine injectionswas3–5 min.Morphine injections were given between 9 am and 9.30 am andbetween 5.00 pm and 5.30 pm. Due to the development of tolerance,the morphine was given in increasing doses (total dose 410 mg/kgof morphine). On the first day, each animal received doses of 10 and20 mg/kg, on the second day, 40 and 60 mg/kg and on the third day, 80and 100 mg/kg. To compensate for the dehydration effects of repeatedmorphine treatment, the guinea pigs were given a 10 ml injection ofsaline (37 °C) i.p. in themorning and afternoon of the second day and inthe morning of the third day.

61W. Anderson et al. / Regulatory Peptides 168 (2011) 59–68

In the morning of day 4 (day 9 of the experiment) all animals weregiven a dose of morphine tartrate, 100 mg/kg. Morphine withdrawalwas induced 1 h later by s.c. injection of naltrexone hydrochloride,15 mg/kg. The dose was calculated on the weight of the animals on day4 of morphine treatment. Animals were observed for 90 min, givensodium pentobarbitone, perfuse-fixed, and brains, adrenal glands andspinal cords were removed and treated as described above.

2.2.3. Effect of subchronic administration of the NK3 receptor antagonist,SSR146977, alone and in combination with the NK1 receptor antagonist, L-773,060, on the behavioural and neuronal acute severe stress responsesinduced by morphine withdrawal

Three groups of four guinea-pigs were used. The first groupreceived the NK3 receptor antagonist, SSR146977, 0.3 mg/kg s.c., dailyin the morning for nine days. This dose was chosen since it was theID50 for guinea-pig turning behaviour, a centrally mediated NK3

receptor response [49]. The second group received an equivalentvolume of the vehicle, 20 mg/ml DMSO, daily for nine days. Injectionof the vehicle s.c. did not cause distress to the animals. The third groupreceived a combination (in separate injections at different skin sites)of the NK1 receptor antagonist, L733,060, 0.25 mg/kg s.c., plus the NK3

receptor antagonist, SSR146977, 0.3 mg/kg s.c. daily for nine days.The animals that received NK3 receptor antagonist or vehicle weregiven an additional injection of vehicle to control for the secondinjection given to the animals treated with the combination ofantagonists. From day 6 to day 9 guinea-pigs in all three groups weregiven morphine to induce tolerance and dependence as describedabove. Morphine withdrawal was induced with naltrexone hydro-chloride, 15 mg/kg s.c. on day 9. Animals were observed for 90 min,given sodium pentobarbitone, perfuse-fixed, and brains, adrenalglands and spinal cords removed as described above.

2.3. Behavioural observations

Guinea-pigs were transferred to activity cages (40 cm×28 cm×15 cm) where they were observed and all behaviours quantified for90 min by a trained observer who was blinded to the treatments. Themorphine withdrawal behaviours included vocalization, rearing,grooming, face washing, digging and head/body shakes. The activitycages were equipped with a single infra-red photocell and detector onthe long axis, located 5 cm above the floor and 14 cm from either endof the cage. Every crossing of the beam at least 1.5 s apart wasrecorded on a digital counter. The time delay was chosen to avoidmeasurements of small body movements. Scores were summed overeach 10 min interval to give an estimate of locomotor activity.

2.4. Localization of Fos-LI

Sections, 50 μm, of brain (coronal), spinal cord (transverse) andadrenal gland (plane of the largest surface), were cut on a cryostat at−20 °C. Free-floating sections were washed twice in PBS, incubatedfor 20 min in 0.3% hydrogen peroxide, washed three times in PBS andincubated for 1 h at room temperature in a blocking solution (PBScontaining 10% normal donkey serum (Sigma-Aldrich), 0.1% sodiumazide (Sigma-Aldrich)). The blocking solution was removed andsections were incubated with polyclonal rabbit Fos antisera (K-25)sc-253 (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of1:2000 (in PBS containing 1% normal donkey serum, 0.1% sodiumazide, 0.075% Triton X-100 (BDH Chemicals Ltd, Poole, England)) for48 h at 4 °C. The sections were washed three times in PBS, incubatedovernight at 4 °C or for 1 h at room temperature, in biotinylateddonkey anti-rabbit IgG (Jackson ImmunoResearch Labs, West Grove,PA, USA) diluted 1:1000 in PBS containing 0.1% normal donkey serum,washed three times in PBS and incubated for 1 h in avidin–biotinhorseradish peroxidase complex (ABC) (Vectastain Elite kit, VectorLaboratories Inc., Burlingame, CA). The sections were then washed

three times in Tris-buffered saline (TBS, pH 7.4, 50 mM Tris–HCl, and150 mMNaCl). Bound antibody was visualized by addition of cold 3, 3′-diaminobenzidine (DAB, Sigma-Aldrich) solution (TBS containing0.033% DAB, 0.03% hydrogen peroxide, 0.004% nickel ammoniumsulphate). The reaction was terminated with excess deionized water.Sections were washed three times in deionised water, mounted ongelatin chrom-alum coated slides, air dried, dehydrated in ascendingconcentrations of ethanol (70%, 95%, and 100%), cleared with Histolene(Fronine, Riverstone, NSW, Australia) and cover-slipped with Ultra-mount (Fronine). Cells in which the nucleus contained a dark brownstain were considered to have Fos-LI.

2.5. Specificity of antibody

The Fos antibody (K-25) sc-253 (Santa Cruz Biotechnology) used inthis studywas raised against amino acids 128–152, a conserved domainof c-Fos p62 of human origin, identical to mouse, rat and chickensequences. This Fos antibody recognizes a conserved region common tomembers of the Fos family, including c-Fos, Fos-B, and Fos-relatedantigens (Fra-1 and Fra-2). This antibody has been extensivelycharacterized in immunohistochemical studies [50]. Negative controlsin the present study comprised sections incubated without primaryantibody or with an inappropriate secondary antibody and thesedeveloped no staining.

2.6. Microscopy

Brain, spinal cord and adrenal sections were examined using a ZeissAxioskop light microscope equipped with a motorised stage and aminiature monitor (Lucivid–Microbrightfield Inc. USA) attached to acamera lucida. Neurolucida 2000 anatomical mapping software (Micro-BrightField Inc, Colchester, VT, USA) was used to trace outlines of thesections, measure areas of particular regions, record the location of cellscontaining Fos-LI, and count neurons using a 2-dimensional countingmethodwithout correction for cell splitting. For determination of Fos-LIin brain regions, closely matched sections from each brain at severallevels were selected by extrapolation from the rat atlas of Paxinos andWatson [51]. Nissl stained reference sections were available. Thenumber of Fos-LI cells was counted and cell densities calculated asnumber of positive cells per unit area (cells/mm2).

For determination of Fos-LI in the spinal cord, Rexed's laminae I, II–III, IV–V of the dorsal horn, and lamina X, were identified at thethoracic, cervical and lumbar levels by comparison to an atlas of therat spinal cord [51]. The number of cells in the different laminae wascounted in several matched sections at the same spinal cord level anda mean number was calculated for each lamina for each animal. Forlaminae I–V and X, results from sections at each spinal cord level werepooled. For the intermediolateral cell column (lateral horn), resultswere obtained only from the thoracic level, the site of origin of most ofthe preganglionic sympathetic innervation to the adrenal gland [52].

For determination of Fos-LI in the adrenal medulla and cortex,areas were outlined, care being taken to distinguish between corticaland medullary regions. Stained cells were marked, and the density ofFos-LI cells was calculated using the Neurolucida software. Tovisualise the structure of the adrenal gland and distinguish betweencortex and medulla at the microscopic level, additional sections werestained with haematoxylin for 5 min and washed with water for5 min. These sections were dehydrated in ethanol (70%, 95%, and100%), cleared with Histolene and coverslipped.

2.7. Statistical analysis

GraphPad Prism Version 3.0 or 4.0 (GraphPad Software Inc., SanDiego, CA, USA)was used for statistical analyses. Bodyweight datawereanalysed using two-way analysis of variance for repeatedmeasures andlinear regression to compare the rate of growth between groups. Two-

A. BASELINE ACTIVITY – NK1 ANTAGONIST

B. MORPHINE WITHDRAWAL – NK1 ANTAGONIST

C. MORPHINE WITHDRAWAL – NK1 and NK3 ANTAGONIST

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Fig. 1. Behaviour scores over 90 min for guinea-pigs treated for nine days with: A. theNK1 receptor antagonist L733,060, 0.25 mg/g, or the inactive enantiomer, L733,061;B. the NK1 receptor antagonist, L733,060 or the inactive enantiomer, L733,061 and withmorphine from days 6 to 9 and withdrawn with naltrexone on day 9; C. vehicle(DMSO), or the NK3 receptor antagonist, SSR146977, or a combination of both the NK1

receptor antagonist L733,060 and the NK3 receptor antagonist, SSR146977 and withmorphine (M) from days 6 to 9 and withdrawn with naltrexone on day 9. Bar graphsrepresent means and standard errors of the means from four animals. Asterisksrepresent significant differences from control ** Pb0.01; * Pb0.05. Loco, Locomotoractivity; Vocal, Vocalization; Rear, Rearing; Groom, Grooming; Face wash, Facewashing; Dig, Digging; and H/B shake, Head/Body shakes.

62 W. Anderson et al. / Regulatory Peptides 168 (2011) 59–68

way analysis of variance for repeated measures, and Bonferroni post-tests were used to compare the behavioural data between animalgroups. The densities or numbers (spinal cord) of Fos-LI cells werecompared using unpaired t-tests (comparison of two groups) or one-way analysis of variance followed by Bonferroni post-tests (comparisonof three groups). Results were expressed as means and standard errorsof the means. A value of Pb0.05 was considered significant.

3. Results

3.1. Effect of subchronic administration of NK1 receptor antagonist,L733,060, on the behavioural and neuronal stress responses of guinea-pigsto repeated daily subcutaneous injection

3.1.1. Body weightThere was no significant difference between the mean body weight

of guinea-pigs treated with the NK1 receptor antagonist and that ofanimals treated with the inactive enantiomer.

3.1.2. BehaviourGuinea-pigs treated subchronically with the NK1 receptor antago-

nist, L733,060, exhibited increased locomotor activity compared withanimals treated with the inactive enantiomer (repeated measuresanalysis of variance, F1,48=12.91; Pb0.05) (Fig. 1A). Although therewas a trend for increased vocalization and rearing in the antagonisttreated group these did not reach significance. There was no sign ofsedation or motor impairment in animals receiving either treatment.

3.1.3. Fos-LIThe effects of subchronic treatment with the NK1 receptor

antagonist, L733,060, or its inactive enantiomer, L733,061, comparedwith no treatment of guinea-pigs, on expression of Fos-LI are shown inTable 1. Data for areas of the brain and for the adrenal medulla areexpressed as densities of Fos-LI cells (cells/mm2), and for the spinal cordas absolute numbers. It may be seen that untreated (control) guinea-pigs had lower Fos-LI densities in all regions than either of the other twogroups. Bonferroni comparisons following one way analysis of varianceshowed that repeated injections of the inactive enantiomer produced asignificant increase in Fos-LI cells in the cingulate cortex (Pb0.05), thebasomedial nucleus of the amygdala (Pb0.01), the arcuate, supraopticand paraventricular nuclei of the hypothalamus, Rexed's laminae I andXof the spinal cord, and the adrenal medulla (all Pb0.05). The NK1

receptor antagonist significantly reduced the density of Fos-LI cells inthe supraoptic (Pb0.01) and paraventricular (Pb0.05) (Fig. 2) nuclei ofthe hypothalamus compared with the inactive enantiomer. Thedensities of Fos-LI cells in these two regions in animals treated withthe NK1 receptor antagonist were not significantly different from thosein untreated control animals indicating that theNK1 receptor antagonistabolished the effect of repeated injection stress in these brain regions.However, the densities of Fos-LI cells in the basolateral and basomedialnuclei of the amygdala and the arcuate nucleus of the hypothalamus ofanimals treated with the NK1 antagonist were significantly higher thanthose in untreated animals indicating that the antagonist did not inhibitthe effect of repeated injection stress in these brain regions.

3.2. Effect of subchronic administration of NK1 receptor antagonist,L733,060, on the behavioural and neuronal acute severe stress responsesinduced by morphine withdrawal

3.2.1. Body weightThere was no significant difference between the weights of animals

treated with the tachykinin receptor antagonist, L733,060, and thosetreated with the inactive enantiomer, L733,061, either before or aftermorphine treatment . Despite rehydration both groups of animals lostapproximately 15% of their body weight during morphine treatment.

3.2.2. Morphine withdrawal behaviourDuring the naltrexone-induced morphine withdrawal, guinea-pigs

showed typical signs ofwithdrawal reported inprevious studies [40,53],such as increased locomotor activity, vocalization, rearing, grooming,facewashing, digging andhead/body shakes. The behavioural responsesof the two groups of animals did not differ significantly. Themean sumsand standard errors for each behaviour over the 90 min period ofwithdrawal for control and treated groups are shown in Fig. 1B.

3.2.3. Fos-LIThe results for Fos-LI expression in the CNS and adrenal medulla

following naltrexone-induced morphine withdrawal are shown inTable 2. Morphine withdrawal induced marked increases in Fos-LI

Table 1Effect of subchronic treatment with the NK1 receptor antagonist, L733,060 and itsinactive enantiomer, L733,061, 0.25 mg/kg, on Fos-LI expression in regions of guinea-pig CNS and adrenal medulla, compared with control (no treatment).

Region Control(no treatment)

L733,061 Inactiveenantiomer

L733, 060 NK1antagonist

CortexCingulate 3.01±1.41 59.71±10.70 # 36.97±15.90Piriform 23.96±10.89 75.75±15.86 39.42±11.29

AmygdalaCentral n. 3.04±2.61 93.32±37.91 122.40±43.62Medial n. 31.30±10.71 127.60±38.43 184.31±68.37Basolateral n. 4.86±2.43 122.37±23.52 155.65±46.96 †

Basomedial n. 4.70±3.22 78.17±12.99 ## 75.49±16.53 ††

HypothalamusArcuate n. 158.26±44.45 467.85±62.64 # 566.42±89.43 ††

Supraoptic n. 410.96±58.43 1250.63±261.77 # 332.51±68.82 **Paraventricular n. 155.67±32.33 625.42±161.30 # 115.13±7.86 *

ThalamusParaventricular n. 43.61±5.94 113.92±46.65 119.37±27.30

Spinal cordLamina I 1.01±0.15 2.66±0.57 # 2.04±0.17Laminae II–III 3.73±0.40 5.44±0.59 5.77±0.67Laminae IV–V 0.79±0.26 1.30±0.26 1.42±0.21Lamina X 0.45±0.09 1.26±0.24 # 1.18±0.21Lateral horn 0.05±0.02 0.20±0.08 0.08±0.05

Adrenal glandAdrenal medulla 99±47 612±189 # 391±85

Values for all areas except the spinal cord are mean densities (cells/mm2) of Fos-LI cells±standard errors of the means obtained from 4 animals. Values for the spinal cord areabsolute counts. Asterisks indicate significance levels obtained from comparisons betweeninactive enantiomer and NK1 antagonist treatment groups by Bonferroni post testsfollowing one way analysis of variance, * Pb0.05; ** Pb0.01. Hatches indicate significancelevels obtained from comparisons between control (no treatment) group and the inactiveenantiomer group, # Pb0.05; ## Pb0.01. Daggers indicate significance levels obtainedfrom comparisons between control and NK1 antagonist treatment groups, † Pb0.05; ††Pb0.01. n.; nucleus.

Fig. 2. Photomicrographs of Fos-LI in the paraventricular nucleus of the hypothalamus(PVN) following subchronic administration of the inactive enantiomer (A) or thetachykinin NK1 receptor antagonist (B). Scale bar=1000 μm.

63W. Anderson et al. / Regulatory Peptides 168 (2011) 59–68

neurons in most regions. Comparison of results for animals treatedwith the inactive enantiomer, L733,061, in Table 1 with those treatedwith the inactive enantiomer and subjected to morphine withdrawalin Table 2, showed that morphine withdrawal produced significantincreases in the cingulate and piriform cortices, central amygdala (allPb0.01), basomedial nucleus of the amygdala and paraventricularnucleus of the thalamus (both Pb0.001), as well as in laminae II–III(Pb0.05), laminae IV–V and the lateral horn (both Pb0.001) of thespinal cord (unpaired t tests). The results for the adrenal medullashowed that morphine withdrawal in the presence of the inactiveenantiomer produced a marked increase in Fos-LI cells (Table 2), butbecause of high variance the increase did not reach statisticalsignificance when compared with results from animals treated withthe inactive enantiomer, L733,061 without morphine withdrawal(Table 1).

In animals withdrawn frommorphine, the NK1 receptor antagonist,L733,060 produced a trend towards decreased expression of Fos-LIneurons in some brain regions but no significant differences in thedensities of Fos-LI neurons were found between the results for the twogroups of animals in anyof the brain regions studied (Table 2).However,therewas a significant reduction innumber of Fos-LI neurons in laminaeII–III (Pb0.01), IV–V (Pb0.001) and X (Pb0.05) of the spinal cord inanimals treated with L733,060. In these laminae the numbers of Fos-LIcells in the antagonist treated group were reduced to values similar tothose in animals that had not been subjected tomorphinewithdrawal. Asignificant increase in Fos-LI neurons in the central amygdala in animalstreated with the NK1 receptor antagonist was found (Pb0.05).

3.3. Effect of subchronic administration of the NK3 receptor antagonist,SSR146977, alone and in combination with the NK1 receptor antagonist,L-773,060, on the behavioural and neuronal acute severe stressresponses induced by morphine withdrawal

3.3.1. Body weightThere was no significant difference between themean body weights

of guinea-pigs treatedwithvehicle (DMSO), theNK3 receptor antagonist,SSR146977, or the combination of both NK1 receptor antagonist,L733,060, and NK3 receptor antagonist, SSR146977, either prior to, orfollowing the commencement of morphine injections (Day 6). Animalsin these groups also lost weight during treatment with morphine.

3.3.2. Morphine withdrawal behaviourAnalysis of the effects of treatments on each of the morphine

withdrawal behavioural responses using two-way analysis of variancewith time as repeated measure followed by Bonferroni post-tests,showed that rearing was significantly reduced in animals treated withthe NK3 antagonist (F 2,72=13.48; Pb0.01) compared with vehiclecontrols. Bonferroni post-tests showed that the significant reductionin rearing occurred in the first 20 min of the observation period(Pb0.05). Grooming behaviour was significantly reduced in animalstreated with a combination of NK1 and NK3 receptor antagonistscompared with controls, in the first 10 min time period (Pb0.05). Themean sums and standard errors for each behaviour over the 90 minperiod of withdrawal, for control and treated groups are shown inFig. 1C.

3.3.3. Fos-LITable 3 shows the results for Fos-LI expression in the CNS and

adrenal medulla and cortex following naltrexone-induced morphinewithdrawal in animals treated subchronically with vehicle (control),the NK3 receptor antagonist, SSR146977, or with a combination of

Table 2Effect of NK1 receptor antagonist, L733,060, on Fos-LI expression in areas of the guinea-pigCNS and adrenal gland 90 min after naltrexone-induced morphine withdrawal. Fos-LIexpression was measured in animals treated with the inactive enantiomer L733,061,0.25 mg/kg, and the NK1 receptor antagonist, L733,060, 0.25 mg/kg.

Region L733, 061 Inactiveenantiomer

L733, 060 NK1antagonist

CortexCingulate 291.75±56.12 235.62±38.28Piriform 422.91±63.32 260.01±12.01

Septal areaNucleus accumbens-core 426.89±54.48 344.46±61.93Nucleus accumbens-shell 261.40±46.97 229.59±52.54Lateral septum 417.42±102.74 410.59±70.83

AmygdalaCentral n. 917.38±148.48 1494.29±56.59*Medial n. 148.78±11.07 166.04±5.90Basolateral n. 268.86±89.78 324.95±53.95Basomedial n. 349.21±32.16 351.28±27.46

HypothalamusArcuate n. 481.96±110.38 773.02±72.45Supraoptic n. 1521.34±271.42 1813.80±217.35Paraventricular n. 1177.26±279.25 1303.59±253.50

Thalamic areaParaventricular n. 708.24±74.18 658.13±144.91Lateral habenula 52.44±2.06 49.53±7.28

MidbrainPeriaqueductal grey—dorsal 251.30±61.93 127.88±10.47Periaqueductal grey—lateral 178.48±36.15 123.87±19.73Ventral tegmental area 211.74±65.38 165.57±45.71Interpeduncular n. 465.52±53.31 304.23±41.43

Spinal cordLamina I 4.66±1.03 3.43±0.29Laminae II–III 13.26±2.37 6.75±0.74**Laminae IV–V 5.87±0.52 2.11±0.29***Lamina X 3.35±0.90 1.44±0.36*Lateral horn 1.30±0.16 0.99±0.16

Adrenal glandAdrenal medulla 4005±2029 2952±1496Adrenal cortex Not available Not available

Values for all areas except the spinal cord are mean densities (cells/mm2) of Fos-LI cells±standard errors of the means obtained from 4 animals. Values for the spinal cord areabsolute counts. Asterisks represent significant differences from inactive enantiomer* Pb0.05; ** Pb0.01; *** Pb0.001. n., nucleus.

Table 3Effect of tachykinin antagonists on Fos-LI expression in areas of the guinea-pig CNS andadrenal gland 90 min after naltrexone-inducedmorphinewithdrawal. Fos-LI expressionwas measured in animals treated with vehicle (control), the NK3 receptor antagonist,SSR146977, 0.3 mg/kg, or with a combination of both the NK1 receptor antagonist,L733,060, and the NK3 receptor antagonist, SSR146977.

Region Vehicle(control)

SSR146977 NK3

antagonistNK1+NK3

antagonist

CortexCingulate 278.50±54.33 251.80±18.79 245.8±32.68Piriform 248.8±40.45 103.80±14.09 206.3±46.47

Septal areaNucleus accumbens-core 192.8±5.38 130.00±21.47* 80.75±6.84***Nucleus accumbens-shell 110.5±4.50 105.30±22.32 99.25±6.98Lateral septum 85.50±3.97 55.00±7.08 56.75±7.04

AmygdalaCentral n. 622.0±35.11 493.80±25.56 471.5±70.81Medial n. 268.3±55.00 141.0±25.16 129.5±39.45Basolateral n. 157.5±35.11 99.25±5.81 44.75±7.36*Basomedial n. 128.8±16.91 79.00±10.66 64.25±16.45*

HypothalamusArcuate n. 68.25±6.84 69.75±5.94 74.00±16.05Supraoptic n. 267.0±58.01 135.3±18.01 170.8±32.81Paraventricular n. 280.30±66.73 200.80±23.24 190.00±21.32

Thalamic areaParaventricular n. 142.80±22.28 133.80±15.50 70.50±10.81*Lateral habenula 16.25±1.93 16.50±1.56 16.50±2.63

MidbrainPeriaqueductal grey—dorsal 64.00 ±1.78 31.00±6.61 30.00±12.62*Periaqueductal grey—lateral 74.00±2.35 44.75±2.56* 41.00±10.07*Ventral tegmental area 54.75±1.70 19.25±3.95*** 25.75±3.88***Interpeduncular n. 98.00±6.79 86.00 ±8.07 45.25±6.85*

Spinal cordLamina I 13.75±1.03 4.25±0.48*** 4.50±0.96***Laminae II–III 24.50±1.26 11.50±1.50** 13.25±2.75**Laminae IV–V 22.25±2.25 7.25±1.60*** 5.50±1.19***Lamina X 21.25±1.55 4.25±1.03*** 4.50±0.87***Lateral horn 2.50±0.50 2.00±0.00 1.75±0.25

Adrenal glandAdrenal medulla 101.80±14.39 39.00±8.04** 53.50±7.46*Adrenal cortex 111.50±18.59 32.75±8.06** 38.75±9.76**

Values for all areas except the spinal cord are mean densities (cells/mm2) of Fos-LI cells±standard errors of the means obtained from 4 animals. Values for the spinal cord areabsolute counts. Asterisks represent significant differences from vehicle (control)* Pb0.05; ** Pb0.01; *** Pb0.001. n., nucleus.

64 W. Anderson et al. / Regulatory Peptides 168 (2011) 59–68

both the NK1 receptor antagonist, L733,060, plus the NK3 receptorantagonist, SSR146977.

In the higher CNS, treatmentwith the NK3 receptor antagonist aloneproduced significant reductions in the density of Fos-LI cells in thenucleus accumbens-core (Pb0.05), periaqueductal grey lateral region(Pb0.05) and ventral tegmental area (Pb0.001). However, treatmentwith both the NK1 and NK3 receptor antagonists had a greater effectproducing significant reductions in the density of Fos-LI cells in thenucleus accumbens-core (Pb0.001) (Fig. 3), basolateral and basomedialnuclei of the amygdala (both Pb0.05), paraventricular thalamic nucleus(Pb0.05), periaqueductal grey dorsal and lateral regions (both Pb0.05),ventral tegmental area (Pb0.001) and interpeduncular nucleus(Pb0.05). In all laminae of the dorsal horn of the spinal cord, guinea-pigs treatedwith either theNK3 receptor antagonist, or the combinationof NK1 and NK3 receptor antagonists, showed highly significantreductions in the numbers of Fos-LI cells (lamina I, Pb0.001; laminaeII–III, Pb0.01; laminae IV–V, Pb0.0001, lamina X, Pb0.0001). Nosignificant effect was seen in the lateral horn of the spinal cord witheither the NK3 receptor antagonist, or the combination of NK1 and NK3

receptor antagonists. In the adrenal medulla significant reductions inthe densities of Fos-LI cellswere found in the group treatedwith theNK3

receptor antagonist (Pb0.01) and in the group treated with thecombination NK1 and NK3 receptor antagonists (Pb0.05). Fos-LI in theadrenal cortex was also measured in this experiment. In the adrenalcortex there were also significant reductions in the densities of Fos-LIcells in both the group treated with NK3 receptor antagonist (Pb0.01),and the group treated with a combination of NK1 and NK3 receptorantagonists (Pb0.01) compared with the vehicle control group.

4. Discussion

The repeated mild stress used in the present study comprised aregimen of repeated daily injections over nine days of the inactiveenantiomer of the NK1 receptor antagonist and saline injections onthe last three days. Compared with the levels found in animals thatwere not injected and were minimally handled, the repeated mildstress produced significant increases in Fos-LI expression in severalbrain areas that have been implicated in mild ‘emotional’ stress andanxiety in other species, including the paraventricular [54,55],supraoptic [55] and arcuate [56] nuclei of the hypothalamus. Thecingulate cortex, which is a region of interaction of pain and emotion

Fig. 3. Photomicrographs of Fos-LI in the nucleus accumbens core (dotted outline)following subchronic administration of vehicle (A), or the combination of NK1 and NK3

receptor antagonists (B). LV, lateral ventricle. Scale bar=1000 μm.

65W. Anderson et al. / Regulatory Peptides 168 (2011) 59–68

[57] was also activated. Although the density of Fos-LI neurons in allsubregions of the amygdala was increased, only the basomedialnucleus, an area involved in linking emotion and memory [58], wassignificantly increased. In the spinal cord, neurons in laminae I and X,regions that have been shown to be involved in nociception [59,60]were activated, presumably because the repeated mild stress used inthis study was repeated subcutaneous injections. Cells in the adrenalmedulla were activated which indicated that the sympatho-adrenalsystem was activated by the repeated mild stress. However, althoughthere was some increase in the number of Fos-LI cells in the lateralhorn, this did not reach significance.

Guinea-pigs subjected to repeated mild stress and treated sub-chronically with the NK1 receptor antagonist, L733,060, exhibitedsignificantly increased locomotor activity compared with animalstreated with the inactive enantiomer. This was a surprising resultsince there is considerable evidence that substance P is released duringstress and produces behavioural activation which is inhibited by acuteadministration of tachykinin receptor antagonists [61]. However, thefinding that subchronic blockade of NK1 receptors produces hyperac-tivity complements thefinding that somestrains ofmice lacking theNK1

receptor exhibit hyperactivity [62]. Thus subchronic loss of NK1 receptorfunctionmight produce different effects fromacute inhibition of functioneither as a result of adaptive changes in the CNS or recruitment of otherpathways. There is evidence that acute and chronic NK1 receptor

antagonist treatment have different effects on locus coeruleus neurons,in that an increase in burst activity, thought to be correlated with anenhancement of noradrenaline release in terminal areas, occurredfollowing chronic treatment [63]. Enhanced restraint stress-induced c-Fos expression in the locus coeruleus has also been reported followingacute treatment with NK1 receptor antagonists [64]. Nevertheless,despite behavioural activation, NK1 receptor antagonist treatment inthe present study produced a marked reduction in Fos-LI in theparaventricular and supraoptic nuclei of the hypothalamus of guinea-pigs subjected to repeated mild stress compared with treatment withthe inactive enantiomer. This finding indicates that the NK1 receptorantagonist reduced the hypothalamic component of the repeated mildstress response. However, Fos-LI in other regions was not significantlyreduced. Theeffects of acute andchronicNK1 receptorblockadeandbrainregional differences in response to these antagonists have yet to be fullyexplored. Experiments using behavioural tests of anxiety and stress suchas the elevated plus maze test would be a useful extension to the study.

As previously reported [40,65], naltrexone-induced morphinewithdrawal in guinea-pigs produced marked behavioural activationand increase in Fos-LI in many brain regions and in the spinal cord. Themorphine withdrawal response is muchmore than a stress response. Itis a complex response involving algesia and responses such as aversionand cravingmediated by themesolimbic system. Therefore, the changesin Fos-LI expression following morphine withdrawal would result fromactivation of regions involved in these responses as well as in the stressresponse. Following morphine withdrawal, increases in Fos-LI weregreater than found following repeatedmild stress in areas including thecortex, central amygdala, paraventricular nucleus of the thalamus, anddorsal and lateral horn of the spinal cord. The NK1 receptor antagonistproduced no significant change in morphine withdrawal behavioursand no significant reduction in Fos-LI in any brain region. However, theNK1 receptor antagonist significantly reduced numbers of Fos-LI cellsin laminae II–III, IV–V and X of the dorsal horn of the spinal cord. Thusthe NK1 receptor antagonist appeared to be ineffective in reducing theacute response tomorphinewithdrawal in thebrain but the reductionofFos-LI neurons in the dorsal horn indicated that it might have beeneffective in reducing the nociceptive response induced by withdrawal.

There is considerable evidence that substance P acting via NK1

receptors is involved in morphine reward. In particular, NK1 receptorknock-outmice exhibit attenuatedmorphine reward [31]. Furthermore,there is increasing evidence that a major site of action of NK1 receptormediated reward is the amygdala [32,33]. Morphine withdrawal is anaversive stimulus which might be expected to produce changes inreward regions. In the present study an increase in Fos-LI in the centraland basomedial nuclei of the amygdala was found following morphinewithdrawal. Surprisingly, the NK1 receptor antagonist did not reducethe effect of morphine withdrawal in the amygdala and in fact, asignificant increase in Fos-LI in the central amygdala was found. Thismight be explained by the findings of Maubach et al. [66] who showedthat activation of NK1 receptors by substance P stimulated inhibitorytransmission in the guinea-pig basolateral amygdala since NK1 re-ceptors were present mainly on GABA neurons. It is therefore possiblethat an NK1 receptor antagonist might cause an increase in Fos-LI inexcitatory neurons due to release of inhibition. Interestingly, Ebner et al.[54] also found that NK1 receptor antagonists did not inhibit stress-induced Fos expression in the amygdala, although the amygdala hasbeen proposed to be a site of action for the antidepressant and anxiolyticaction of NK1 receptor antagonists [67]. The failure of an NK1 receptormediated self-regulatory substance P release mechanism in states ofstress such as suggested by the results of Singewald et al. [68]might alsooccur.

TheNK3 receptor antagonist significantly inhibited rearingbehaviourinduced by morphine withdrawal. In rats, motor activities such asrearing have been associated with dopaminergic mechanisms in thenucleus accumbens shell [69]. The nucleus accumbens shell whichprojects primarily to limbic structures such as the ventral tegmental

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area, lateral hypothalamus and ventromedial ventral pallidum, containsgreater concentrations of dopamine and substance P than the nucleusaccumbens core, which projects to motor-related structures such as theglobus pallidus and the dorsolateral ventral pallidum [69]. Furthermore,in terms of dopamine release, the nucleus accumbens shell has beenshown to be more responsive to stress [70] and to drugs of abuse [71]than the nucleus accumbens core. However more recent data from ratsgiven repeated doses of morphine have shown greater Fos increases inthe nucleus accumbens core than the shell [72]. The functions ofsubregions of the nucleus accumbens in the guinea-pig have not been asextensively studied as those of the rat. It has been shown that acuteintracerebral injection of morphine produced greater Fos-LI in thenucleus accumbens shell than the core [73] but the effects of repeatedmorphine administration have not been explored. The results from thepresent study would suggest that the core region of the nucleusaccumbens might play a role in withdrawal behaviours such as rearingfollowing repeated morphine administration.

The mesolimbic dopaminergic pathway from the ventral tegmentalarea to the nucleus accumbens is involved in reward, reinforcement andmotivation [74]. Substance P levels in the nucleus accumbens have beendemonstrated to increase following morphine withdrawal in rats [75].NK3 receptors are present in both the ventral tegmental area and thenucleus accumbens core but not the shell [7]. Asmight be expected fromthedistributionofNK3 receptors, theNK3antagonist in thepresent studyinhibited Fos-LI in the ventral tegmental area and nucleus accumbenscore. Results from studies using cocaine as a dopamine releasing agent,have demonstrated that NK3 receptors in the mesolimbic systemare involved in cocaine-induced behavioural hyperactivity but not inconditioned place preference, a test of reward [34]. However, NK3

receptors in the VTA have recently been found to be more abundant inmesocortical projection neurons [76] afinding that hasbeenproposed toexplain the proposed ‘atypical’ antipsychotic activity of NK3 receptorantagonists [38,76]. The highly significant reduction in Fos-LI in theventral tegmental area found in the present study supports an action ofNK3 receptor antagonists in modulation of the mesocortical system.

The periaqueductal grey plays a central role in the descendingantinociception system.Recent studieshave shown thatNK1 receptors inthe periaqueductal grey mediate antinociception [77] and anxiogenesis[78],whereasNK3 receptor stimulation produces nociception [79]. In thepresent study theNK3 receptor antagonist significantly reduced Fos-LI inthe lateral periaqueductal grey which might indicate a supraspinalantinociceptive effect of NK3 receptor antagonists. The NK3 receptorantagonist also reduced Fos-LI in all laminae of the dorsal horn andwas more effective in the spinal cord than the NK1 receptor antagonist,producing a reduction in Fos-LI in lamina I as well as reductionsin laminae II–III, IV–V and X. Therefore, the results from theseexperiments suggest that NK3 receptor antagonists might be moreeffective antinociceptive agents than NK1 receptor antagonists sincethe spinal antinociceptive action may not be opposed by a supraspinalpro-nociceptive action.

The NK3 receptor antagonist also reduced Fos-LI in the adrenalmedulla and cortex following morphine withdrawal. The reductionof Fos-LI in the adrenal medulla and cortex indicate that both thesympathetic component, as well as the hypothalamic–pituitary–adrenal response to the stress of morphine withdrawal, was reducedby the NK3 receptor antagonist.

Although the combination of the NK1 receptor antagonist and theNK3 receptor antagonist did not produce a reduction in morphinewithdrawal behaviours, the combination of antagonists was moreeffective overall in reducing the central neuronal response to morphinewithdrawal than either antagonist alone. The combination producedreduction in Fos-LI in both the dorsal and lateral periaqueductal grey aswell as in basolateral and basomedial nuclei of the amygdala, areas thatare associated with stress and anxiety. Fos-LI expression was alsoreduced in the paraventricular nucleus of the thalamus, an area whichhas been proposed to limit responses to chronic stress [80]. Although

Fos-LI in other areas associatedwith stress, such as themedial amygdalaand paraventricular nucleus of the hypothalamus were reduced, theeffects did not reach significance.

There were several limitations to this study. Firstly, the doses oftachykinin antagonists selected for the studywere chosen on the basisof the best information available. Interpretation of the results must bemade with caution since only a single dose of each drug has beentested. Furthermore, drug half-lives are not known, although bothdrugs have been reported to have pharmacological effects for greaterthan 6 h [47,49]. It was not feasible in this study tomeasure brain drugconcentrations and receptor occupancy. It is possible that once dailydosing was, in effect, repeated acute dosing rather than subchronictreatment. Nevertheless, the results from the study showed thatchanges occurred in animals treatedwith the drugs indicating that thedoses of drugs used were effective. Another limitation of the studywas that the three experiments were conducted sequentially ratherthan concurrently. Although conditions were standardized this mighthave contributed to variability in the results between experiments. Toreduce the effect of this limitation comparisons have been madewithin experiments only.

In conclusion, the results from the present study indicate thatsubchronic treatment with an NK1 receptor antagonist was effective inreducing the central response to repeatedmild stress butwas ineffectivein reducing the acute response to the more severe stress of morphinewithdrawal. On the other hand, the NK3 receptor antagonist mightreduce the stress and aversion associated with morphine withdrawal.The results from the spinal cord data suggest that both antagonistswould be effective antinociceptive agents in morphine withdrawal.Furthermore, the combination of anNK1 receptor antagonist and anNK3

receptor antagonist was apparently more effective than either antago-nist alone. It is concluded that further investigation of the effect oftachykinin receptor antagonists in combination is warranted.

Acknowledgement

The project was supported by a grant from the Research Committeeof the University of Newcastle.

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