The substance P (NK1) receptor antagonist L-760735 inhibits fear conditioning in gerbils

Neuropharmacology 44 (2003) 516–523 www.elsevier.com/locate/neuropharm

The substance P (NK1) receptor antagonist L-760735 inhibits fear conditioning in gerbils N.M.J. Rupniak ∗, J.K. Webb, A. Fisher, D. Smith, S. Boyce Department of Pharmacology, Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, UK Received 6 August 2001; received in revised form 19 September 2002; accepted 14 November 2002

Abstract The ability of the substance P (NK1 receptor) antagonist (SPA) L-760735 to inhibit conditioned fear was assessed in gerbils using a four plate apparatus. Animals that had been treated with diazepam (3 mg/kg) or L-760735 (3 mg/kg) 30 min before a 3 min conditioning session in the apparatus exhibited a release of plate crossings during the retest session approximately 3 h later. Plate crossings were also increased when animals received diazepam or L-760735 30 min before the retest session. In contrast, fluoxetine and venlafaxine (30 mg/kg) did not exhibit anxiolytic-like effects. During the retest session, gerbils drummed their hind feet on the floor; this behaviour was not observed spontaneously in gerbils that were naı¨ve to the apparatus. Foot drumming was abolished by pretreatment with L-760735 or diazepam (3 mg/kg) but was markedly increased following administration of fluoxetine or venlafaxine (30 mg/kg). Foot drumming elicited by aversive conditioning alone or in combination with fluoxetine was abolished by administration of L-760735 and by amygdala lesions involving the basolateral and lateral nuclei, indicating that this behaviour is an alarm signal or fear response mediated via release of substance P in brain circuits involving the amygdala. The observations provide further evidence for an anxiolytic-like profile of SPAs in preclinical assays and demonstrate a clear difference between the actions of SPAs and established antidepressant drugs.  2003 Elsevier Science Ltd. All rights reserved. Keywords: NK1 receptor; Depression; Anxiety; Foot drumming; Amygdala

1. Introduction Substance P (NK1 receptor) antagonists (SPAs) are a novel class of antidepressant drugs that act by blocking the actions of a neuropeptide rather than through direct pharmacological interactions with monoamine systems (Kramer et al., 1998). Selective serotonin reuptake inhibitors (SSRIs) are being used increasingly to treat anxiety disorders, including panic attack (Lepola et al., 1998) and social phobia (van der Linden et al., 2000), and are recognised as an effective alternative to benzodiazepines, which have the liability to induce sedation, tolerance and dependency. Like SSRIs, SPAs may also be of benefit in the treatment of anxiety disorders. Substance P and its preferred

Corresponding author. Tel.: +1-484-344-4047; fax: +1-484-3442740. E-mail address: [email protected] (N.M.J. Rupniak). ∗

NK1 receptor are expressed in brain regions known to be involved in fear processing (reviewed by LeDoux, 1995). These include the amygdala, septum, hippocampus, hypothalamus, central gray and caudal pontine reticular nucleus (Arai and Emson, 1986; Hokfelt et al., 1987; Mantyh et al., 1984; Pioro et al., 1990). Activation of these pathways by direct central injection of substance P agonists produces a range of fear-related behaviours and defensive cardiovascular changes in animals: conditioned place aversion (Aguiar and Brandao, 1994; Elliott, 1988; Hall et al., 1987); an anxiogenic profile on the elevated plus maze (Aguiar and Brandao, 1996; Teixeira et al., 1996); potentiation of the acoustic startle response (Krase et al., 1994); distress vocalisations and escape behaviour (Kramer et al., 1998; Rupniak et al., 2000); and cardiovascular changes resembling the defence response to threatening stimuli (Culman and Unger, 1995; Unger et al., 1988). There is also evidence from neurochemical studies for changes in the content of substance P in discrete brain regions in response to

0028-3908/03/$ - see front matter  2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0028-3908(03)00023-6

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stressful stimuli. These have shown rapid reductions in substance P content in the mesolimbic system, hippocampus, septum, periaqueductal gray and hypothalamus of rats following inescapable foot shock (Bannon et al., 1986; Siegel et al., 1984) and immobilisation stress (Takamaya et al., 1986). It might be anticipated from these observations that SPAs would exhibit anxiolytic-like activity in animal assays. However, preclinical studies are complicated by marked species variants in NK1 receptor pharmacology (Beresford et al., 1991; Gitter et al., 1991), the majority of compounds having only low affinity for the mouse and rat receptor. Preliminary studies using CGP49823 suggested anxiolytic-like effects of SPAs on social interaction in rats (File, 1997), but this compound does not have high affinity for the rat NK1 receptor and the dose range required to occupy central NK1 receptors in rats was not established. To avoid these difficulties, the social interaction paradigm was established in gerbils, a species with human-like NK1 receptor pharmacology (Beresford et al., 1991). Like the benzodiazepine anxiolytic diazepam, L-760735 increased social interaction at a dose blocking central NK1 receptors in gerbils, whilst its enantiomer that has only low affinity for the NK1 receptor was without effect (Cheeta et al., 2001). Gerbils are of particular interest for examining the role of NK1 receptors in anxiety because central infusion of substance P agonists elicits a vigourous rhythmic drumming or tapping of the hind feet (Bristow and Young, 1994; Graham et al., 1993; Rupniak and Williams, 1994; Vassout et al., 1994), a behaviour recognised as an alarm signal in desert rodents. In feral gerbils and kangaroo rats, foot drumming has been observed when animals are startled (Daly and Daly, 1975), confronted by snakes (Randall and Stevens, 1987), defending their territory (Randall, 1984) and during agonistic encounters (Daly and Daly, 1975). In gerbils studied under laboratory conditions, foot drumming has been elicited by aversive stimuli, including termination of rewarding brain stimulation, foot shock (Kramis and Routtenberg, 1968; Routtenberg and Kramis, 1967) and threatening environmental stimuli (Clark and Galef, 1977). Thus, the ability of substance P agonists to elicit this behaviour appears consistent with other evidence for an involvement of this neuropeptide in stress responses. Recently, Ballard et al. (2001) reported that foot drumming elicited by foot shock and associated environmental cues in gerbils was inhibited by the SPAs MK869 and CP-99,994, consistent with an anxiolytic-like effect. In the present studies we report similar observations made during experiments originally designed to examine the anxiolytic activity of L-760735 in the four plate test in gerbils. We also examined the effect of basolateral amygdala lesions on foot drumming elicited by aversive conditioning as this has been implicated as

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a direct site of action of SPAs in other stress responses (Boyce et al., 2001; Smith et al., 1999).

2. Methods 2.1. Subjects All experiments were conducted in compliance with the Animals (Scientific Procedures) Act, 1986 and its associated guidelines. Male or female Mongolian gerbils (40–60 g; University of Leeds, UK) were housed in groups of four under standardised conditions of temperature and relative humidity and with a 12 h light:dark cycle. The minimum number animals required to yield reliable data were used in each experiment. 2.2. GR73632-induced foot drumming The dose range required to block central NK1 receptors by L-760735 was determined by inhibition of species-specific behaviours elicited by intracerebroventricular (i.c.v.) infusion of the selective NK1 receptor agonist GR73632. Gerbils received an intraperitoneal (i.p.) injection of a range of doses of L-760735 (0.1–3 mg/kg) or its low affinity enantiomer L-781773 (3 mg/kg), 30 min before anaesthesia with isoflurane. An incision was then made in the scalp and GR73632 (d-Ala[L-Pro9,MeLeu8] substance P-(7-11), 10 pmol/5 µl i.c.v.) was administered by vertical insertion of a cuffed 27 gauge needle to a depth of 4.5 mm below bregma as described previously (Rupniak and Williams, 1994). On recovery of the righting reflex, gerbils were placed in a perspex observation box (25 cm × 20 cm × 20 cm) and the duration of foot drumming was recorded for 5 min by direct observation. 2.3. Four plate test The procedure employed a four plate apparatus designed for use with mice as described by Aron et al. (1971). Gerbils were placed in a test chamber with the floor comprised of four metal plates, each approximately 10 × 4 cm, for a 3 min session. During this period, each time the gerbil crossed from one plate to another, it received a mild electric foot shock (0.8 mA for 500 ms). To determine the effect of test compounds on acquisition of fear conditioning, test compounds were administered i.p. 30 min before the first exposure to the apparatus and the number of foot shocks was recorded when the animals were returned to the four plate approximately 3 h later. In experiments examining the effect of test compounds on behaviour conditioned by prior exposure to the apparatus, naı¨ve gerbils were first introduced into the apparatus for 3 min as described above, and were then returned to their home cage for

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approximately 3 h before being placed in the apparatus for a further 3 min retest session, and the number of plate crossings was recorded. Drug treatments were administered 30 min prior to the retest. During these experiments, it was noticed that gerbils drummed their feet when they were returned to the apparatus, but quantification of the behaviour was difficult since it was disrupted if the animals received further foot shocks during the retest session. Therefore, in order to quantify the conditioned foot drumming, separate experiments were conducted in which foot shocks were not delivered during the retest session, so that the duration of foot drumming could be recorded. Drug treatments were administered 30 min prior to the retest. 2.4. Ibotenic acid lesions of the basolateral amygdala Gerbils were exposed to the four plate apparatus for 3 min as described above prior to surgery. Animals were then anaesthetised with isoflurane/oxygen and placed in a stereotaxic frame. Bilateral excitotoxic lesions aimed at the basal and lateral nuclei of the amygdala were produced by infusion of ibotenic acid (5 µg in 0.5 µl per side; injection co-ordinates were: AP ⫺0.8 mm from bregma, ML ± 4.0 mm, DV ⫺5.4 mm from the dura). The lesion extended from the site of injection in both a rostral and caudal direction for a distance of approximately 0.5 mm to give a final lesion volume of approximately 1.5 mm3. This extension of the lesion included the basal and lateral amygdala in all animals tested. Sham-operated animals received an infusion of sterile distilled water. Following surgery, the animals were allowed to recover for 7 days before the retest session. Lesions were confirmed by histological examination; only those animals with confirmed lesions of the basal and lateral amygdala were included in the data analysis.

2.6. Statistical analysis Unless otherwise stated, data were subjected to oneway analysis of variance (ANOVA), followed by Dunnett’s multiple comparison t-tests using bmdp statistical software (BBN Software Products Corporation, USA). Data from the amygdalectomy experiment were analysed by two-way ANOVA followed by Student–Newman– Keuls tests. ID50 values were calculated by non-linear least squares regression analysis of mean data using GraFit (Erithacus Software, UK).

3. Results 3.1. GR73632-induced foot drumming Pretreatment of gerbils with L-760735 (0.03–3 mg/kg i.p.), 30 min prior to central agonist challenge, produced a dose-dependent inhibition of GR73632-induced foot drumming, with full blockade observed at doses of 1 and 3 mg/kg (ID50 0.3 mg/kg i.p.) [F(6,24) = 47.01, P ⬍ 0.001, n = 3 - 6]. In contrast, the low affinity enantiomer L-781773 failed to inhibit foot drumming when administered at 3 mg/kg i.p. (Fig. 1). 3.2. Four plate crossings Gerbils that had received vehicle 30 min before the conditioning exposure to the four plate apparatus exhibited little exploratory activity and so received few foot shocks when they were retested approximately 3 h later.

2.5. Preparation of test compounds Test compounds were administered by the i.p. route using an injection volume of 10 ml/kg for gerbils. L760735 ((2-(R)-(1-(R)-3,5-bis(trifluoromethyl)phenyl) ethoxy-4-(5-(dimethylamino-methyl)-1,2,3-triazol-4-yl) methyl-3-(5)-phenyl)morpholine), and its low affinity enantiomer L-781773, were synthesised by the Department of Medicinal Chemistry, MSD, Harlow and were prepared as hydrochloride salts that were freely soluble in 0.9% saline. Reference drugs were the clinically used anxiolytic benzodiazepine diazepam, the SSRI fluoxetine and the SNRI venlafaxine (Sigma), which were suspended in 0.5% methocel. Doses were based on those that caused maximal inhibition of neonatal vocalisations in guinea-pig pups (Kramer et al., 1998; Rupniak et al., 2000). Between 9 and 25 animals received each test compound or vehicle.

Fig. 1. Inhibition of GR73632-induced foot drumming by L-760735 in gerbils. Animals received L-760735 (0.03–3 mg/kg i.p.) or its low affinity enantiomer L-781773 (3 mg/kg i.p.) 30 min before i.c.v. infusion of GR73632 (10 pmol/5 µl i.c.v.). The duration of foot drumming was recorded for 5 min.∗P ⬍ 0.05 compared with vehicle-treated animals, one-way ANOVA followed by Dunnett’s t-test.

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Animals that had received diazepam (3 mg/kg i.p.) or L-760735 (3 mg/kg i.p.), but not its less active enantiomer L-781773 (3 mg/kg i.p.), prior to conditioning explored the apparatus more freely and received approximately twice the number of foot shocks compared with control animals [F(3,28) = 8.94, P ⬍ 0.001, n = 8] (Fig. 2(a)). Similarly, gerbils that had received vehicle after the conditioning exposure to the four plate apparatus and 30 min before retest received few foot shocks. Administration of diazepam or L-760735, but not its less active enantiomer L-781773 (all at 3 mg/kg i.p.) 30 min before retest again increased the number of plate crossings, although this effect of L-760735 was modest by comparison with diazepam [F(5,107) = 38.50, P ⬍ 0.001, n = 11 - 36]. Administration of the antidepressant drugs fluoxetine or venlafaxine (30 mg/kg) had no effect on the number of foot shocks received compared with vehicle treatment (Fig. 2(b)).

tration of fluoxetine or venlafaxine (30 mg/kg) before retest [F(5,82) = 23.51, P ⬍ 0.001, n = 8 - 26] (Fig. 3(a)). Fluoxetine did not elicit foot drumming in naı¨ve gerbils that had not been previously exposed to the four plate apparatus. The potentiation of conditioned foot drumming by fluoxetine was dose-related, reaching significance at 30 mg/kg i.p. [F(3,66) = 33.62, P ⬍ 0.001, n = 17 - 18] (Fig. 3(b)). At this dose of fluoxetine, foot drumming was dose-dependently and completely inhibited by co-administration of L-760735 (0.3–3 mg/kg i.p.; ID50 0.4 mg/kg) [F(5,50) = 6.60, P ⬍ 0.001, n = 7 - 10]. In contrast, the low affinity enantiomer L-781773 (3 mg/kg i.p.) did not inhibit fluoxetineinduced foot drumming (Fig. 3(c)).

3.3. Conditioned foot drumming

Gerbils that had undergone amygdala lesions after conditioning exposure to the four plates explored the apparatus freely and received a larger number of foot shocks than sham-operated animals when returned to the four plates 7 days later. Sham lesioned animals exhibited foot drumming when they were returned to the apparatus 7 days after the initial exposure that was of a similar duration to that observed previously in un-operated gerbils tested with a 3 h inter-trial interval. Spontaneous and fluoxetine-induced foot drumming in the four plate apparatus was also abolished by bilateral amygdala lesions [Treatment: F(1,30) = 12.87, P = 0.001; Lesion: F(1,30) = 30.79, P ⬍ 0.001; Interaction: F(1,30) = 5.26, P = 0.029, n = 4 - 15] (Fig. 4).

3.4. Effect of amygdala lesions on behaviour in the four plate apparatus

When vehicle-treated gerbils were retested in the four plate apparatus they were observed to periodically drum their hind feet on the floor. Foot drumming occurred in approximately 50% of the gerbils of either sex and was never seen spontaneously in animals that were naı¨ve to the apparatus. In order to quantify this behaviour, separate animals were retested in the apparatus without receiving further foot shocks. Foot drumming was completely abolished by pretreatment with diazepam or L760735, but not L-781773, (all at 3 mg/kg) 30 min before the retest session. In contrast, foot drumming was markedly induced in every subject after acute adminis-

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Fig. 2. Number of foot shocks received by gerbils exploring the four plate apparatus. To determine the effect of test compounds on acquisition of fear conditioning (a), test compounds were administered i.p. 30 min before the first exposure to the apparatus and the number of foot shocks was recorded when the animals were returned to the four plate approximately 3 h later. To examine the effect of test compounds on behaviour conditioned by prior exposure to the apparatus (b), naı¨ve gerbils were introduced to the four plate for 3 min as described above, and then returned to their home cage for approximately 3 h before being placed in the apparatus for a further 3 min retest session, and the number of plate crossings was recorded. Drug treatments were administered 30 min prior to the retest.Test compounds were diazepam (Diaz), L-760735 (735), L-781773 (773), fluoxetine (Fluox) and venlafaxine (Venl).∗P ⬍ 0.05 compared with vehicle-treated animals, one-way ANOVA followed by Dunnett’s t-test.

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Fig. 3. Duration of foot drumming in gerbils in the four plate apparatus. Naı¨ve gerbils were placed in the four plate apparatus for 3 min and then returned to their home cage for approximately 3 h before being retested in the apparatus for a further 3 min, during which the duration of foot drumming was recorded. (a) Effect of administration of test compounds i.p. 30 min prior to the retest. Compounds were diazepam (Diaz), L760735 (735), L-781773 (773), fluoxetine (Fluox) and venlafaxine (Venl); (b) Dose-dependent induction of foot drumming by fluoxetine (3–30 mg/kg i.p.); (c) Effect of co-administration of L-760735 (0.3–3 mg/kg i.p.; 735) or L-781773 (3 mg/kg i.p.) with fluoxetine (30 mg/kg i.p.; Fluox). ∗P ⬍ 0.05 compared with vehicle-treated animals, one-way ANOVA followed by Dunnett’s t-test. + P ⬍ 0.05 compared with fluoxetine-treated animals, one-way ANOVA followed by Dunnett’s t-test.

Fig. 4. Effect of amygdala lesions on plate crossing and foot drumming in the four plate apparatus. Animals were reintroduced into the apparatus for 3 min approximately 7 days after an initial exposure, followed by amygdala lesions. Fluoxetine (30 mg/kg) or vehicle were administered i.p. 30 min prior to the retest. ∗P ⬍ 0.05 versus vehicle-treated gerbils, one-way ANOVA followed by Dunnett’s t-test.

4. Discussion The present studies describe a fear conditioning paradigm using a four plate apparatus in gerbils, a suitable species in which to examine the anxiolytic activity of L760735, a close analogue of MK-869 that has nanomolar affinity for the human and gerbil NK1 receptor (IC50 for inhibition of [125I]substance P binding = 0.3 and 0.5 nM, respectively; Kramer et al., 1998). Treatment of naı¨ve animals with diazepam or L-760735 (3 mg/kg) prior to a conditioning exposure to the four plate apparatus inhibited the acquisition of fear as evidenced by

release of plate crossings compared with vehicle-treated animals during the retest session. This was not observed with the low affinity enantiomer L-781773 (IC50 for inhibition of [125I]substance P binding = 545 nM; Cheeta et al., 2001), confirming the NK1 receptor specificity of this effect. L-760735 also exhibited enantioselective anxiolytic activity when it was administered 30 min before the retest session, although this was modest by comparison with diazepam. The effects of L-760735 cannot be attributed to an acute analgesic action since SPAs do not inhibit spinal nociceptive reflexes (Rupniak and Hill, 1999). These findings support other evidence for anxi-

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olytic activity of SPAs in gerbils using the social interaction test (Cheeta et al., 2001) and the elevated plus maze (Varty et al., 2002). Unlike L-760735 and diazepam, the antidepressant drugs fluoxetine and venlafaxine did not release plate crossings in the retest session. Consistent with the present study, anxiolytic activity was not observed using the SSRI fluoxetine in the gerbil social interaction or elevated plus maze tests (Cheeta et al., 2001; Varty et al., 2002), a finding that distinguishes the preclinical profile of SPAs from that of established antidepressant drugs. The four plate test was originally designed as a simple behavioural assay of unconditioned fear for benzodiazepine anxiolytics in mice (Aron et al., 1971; Bourin et al., 1991) and was recently reported to also detect anxiolytic-like effects of certain antidepressant drugs, but not fluoxetine (Hascoet et al., 2000). The effect of test compounds on unconditioned fear was not examined in the present studies. Repeated exposure to the aversive four plate apparatus (a form of contextual fear conditioning) was found to induce foot drumming in gerbils that was blocked by diazepam and L-760735, and markedly enhanced by fluoxetine and venlafaxine. These findings are strikingly similar to those recently reported by Ballard et al. (2001), who exposed gerbils to foot shocks signalled by a light-tone conditioned stimulus. It is likely that the induction of foot drumming by aversive conditioning and its exacerbation after acute antidepressant drug administration is a fear or alarm signal since antidepressant drugs exhibit acute anxiogenic activity in several preclinical assays (Cheeta et al., 2001; Dekeyne et al., 2000; File et al., 1999; Silva and Brandao, 2000). Acute worsening of anxiety is also a common side-effect of SSRIs in clinical use (Spigset, 1999) that necessitates gradual dose titration. This interpretation is supported by evidence from ethological studies showing that foot drumming in gerbils and related species is an alarm signal (Clark and Galef, 1977; Daly and Daly, 1975; Randall and Stevens, 1987; Routtenberg and Kramis, 1967). Since foot drumming in gerbils is known to be elicited by selective stimulation of central NK1 receptors (Bristow and Young, 1994; Graham et al., 1993; Rupniak and Williams, 1994; Vassout et al., 1994), it is possible that fluoxetine and venlafaxine-induced foot drumming reflects an increase in the stress-induced release of substance P. There is no direct neurochemical evidence for this, but previous studies have reported a decrease in the content of substance P in discrete brain regions, including the amygdala, after subchronic administration of SSRIs (Brodin et al., 1987; Shirayama et al., 1996). Consistent with this proposal, co-administration of L760735, but not its low affinity enantiomer L-781773, abolished the enhancement of conditioned foot drumming by fluoxetine in the present studies. The ID50 for inhibition of fluoxetine-induced foot drumming by L-

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760735 was similar to that needed to inhibit NK1 agonist-induced foot drumming (0.4 and 0.3 mg/kg i.p., respectively), consistent with blockade of central NK1 receptors. These findings indicate that fear conditioning causes activation of substance P pathways and that this activation is enhanced by acute administration of an SSRI. These studies highlight important differences in the mechanism of action of SPAs and established antidepressant drugs. If this interpretation is correct, it would be anticipated that SPAs might have the advantage of not worsening acute anxiety in depressed and anxious patients. Another striking difference between the preclinical profile of SPAs and established antidepressant and anxiolytic drugs is the absence of sedation or motor impairment (Rupniak et al., 2001); this is consistent with the distinct side-effect profile observed for MK-869 and paroxetine in patients with major depression (Kramer et al., 1998). Therefore, whilst there is growing evidence that NK1 receptor blockade or deletion produces alterations in 5-HT (Conley et al., 2002; Froger et al., 2001; Santarelli et al., 2001) and norepinephrine neuronal function (Haddjeri and Blier, 2000; Maubach et al., 2002) that are likely to contribute to the antidepressant efficacy of SPAs, there are also distinguishing features that underline the unique mechanism of action of this new pharmacological class. In order to further define whether foot drumming elicited by aversive conditioning represented an alarm or fear response, we examined the effects of amygdala lesions on behaviour. The lateral, basal and central nuclei of the amygdala are involved in contextual and auditory fear conditioning as assessed by freezing behaviour in rats (Goosens and Maren, 2001; Nader et al., 2001). In the present studies, the effect of lesions encompassing the basal and lateral amygdala were examined because of evidence that substance P is released in this region following maternal separation of guinea-pig pups (Boyce et al., 2001; Kramer et al., 1998) and immobilisation stress in gerbils (Smith et al., 1999). Animals with amygdala lesions received an increased number of foot shocks in the four plate apparatus and, unlike sham operated animals, did not exhibit foot drumming on being retested in the apparatus, even following administration of fluoxetine. Thus, the effect of amygdala lesions on fear conditioning resembled that seen after administration of the SPA, L-760735. These studies provide further evidence that the amygdala and its associated output pathways are potential sites where SPAs may act to attenuate stress responses. References Aguiar, M.S., Brandao, M.L., 1994. Conditioned place aversion produced by microinjections of substance P into the periaqueductal gray of rats. Behavioural Pharmacology 5, 369–373.

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