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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Behav Pharmacol. Author manuscript; available in PMC 2010 October 12.
Published in final edited form as:
PMCID: PMC2946835

Anxiolytic-like Effects of the Neurokinin 1 Receptor Antagonist GR-205171 in the Elevated Plus-Maze and Contextual Fear-Potentiated Startle Model of Anxiety in Gerbils


Gerbils show a NK1 receptor pharmacological profile which is similar to that seen in humans and thus have become a commonly used species to test efficacy of NK1 receptor antagonists. The aim of the present study was to determine whether systemic administration of the NK1 receptor antagonist GR-205171 produced anxiolytic-like effects in the elevated plus maze and in a novel contextual conditioned fear test using fear-potentiated startle (FPS). On the elevated plus maze, treatment with GR-205171 at 0, 0.3, 1.0 and 5.0 mg/kg doses 30 min before testing produced anxiolytic-like effects in an increasing dose-response fashion as measured by the percentage of open arm time and percentage of open arm entries. For contextual fear conditioning, gerbils were given 10 unsignaled footshocks (0.6 mA) at a 2 min variable interstimulus interval in a distinctive training context. Twenty-four hours after training gerbils received treatment of GR-205171 at 0, 0.3, 1.0 and 5.0 mg/kg doses, 30 min before testing in which startle was elicited in the same context in which they were trained. Contextual FPS was defined as an increase in startle over pre-training baseline values. All drug doses levels (0.3, 1.0 and 5.0 mg/kg) significantly attenuated contextual FPS when compared to the vehicle control group. A control group which received testing in a different context, showed little FPS. These findings support other evidence for anxiolytic activity of NK1 receptor antagonists and provide a novel conditioned fear test that may be an appropriate procedure to test other NK1 antagonists for preclinical anxiolytic activity in gerbils.

Keywords: Anxiety, Substance P, context conditioning, plus maze, gerbil


The mammalian tachykinins include substance P (SP), neurokinin (NK) A, and NKB, the effects of which are preferentially mediated by the G protein-coupled receptors NK1, NK2, and NK3, respectively (Pennefather et al. 2004; Regoli et al. 1994). In the nervous system, tachykinins operate as neurotransmitters and neuromodulators and have historically been implicated in a wide variety of biological actions including pain transmission, inflammation, smooth muscle contraction, vasodilation, gland secretion, and activation of the immune system (Kramer et al. 1998; Quartara and Maggi 1998; Severini et al. 2002). More recent evidence suggests that the central SP-NK1 system is also involved in various stress-related pathologies, including anxiety and depression (for review see,Ebner and Singewald 2006). In mammals, SP is the most abundant tachykinin in the central nervous system, where, along with NK1, it is widely distributed in brain regions involved in the regulation of affective behavior and mediation of stress responses, such as the amygdala, septum, hippocampus, hypothalamus, and periaqueductal gray (Barbaresi 1998; Commons and Valentino 2002; Hietala et al. 2005; Hokfelt et al. 1985; Maeno et al. 1993; Nagano et al. 2006; Rigby et al. 2005; Szeidemann et al. 1995).

Previous animal studies have shown that exposure to a variety of aversive and stressful situations alter SP transmission in various brain regions (Bannon et al. 1986; Brodin et al. 1994; Ebner et al. 2004; Kramer et al. 1998; Rosen et al. 1992; Siegel et al. 1987). In addition, both systemic and central injections of SP agonists elicit a variety of anxiety-like behaviors in animals, including conditioned place aversion (Aguiar and Brandao 1994; Elliott 1988), more time in the closed arms of the elevated plus-maze (Aguiar and Brandao 1996; Bassi et al. 2007; De Araujo et al. 2001; Duarte et al. 2004; Teixeira et al. 1996) and enhanced inhibitory avoidance learning (Hasenohrl et al. 1990; Pelleymounter et al. 1986). In contrast, pharmacological blockade of SP with NK1 receptor antagonists reduced depressant- and anxiety-like behaviors in different animal species (Dableh et al. 2005; Kramer et al. 1998; Rupniak et al. 2001, 2003b; Varty et al. 2002a; Woolley et al. 2006). NK1 antagonists also have a robust anxiolytic effect in various conditions of the social interaction test of anxiety (Cheeta et al. 2001; File 1997; Gentsch et al. 2002).

These findings have led pharmaceutical companies to develop a variety of selective and potent NK1 antagonists to offer new options for the treatment of anxiety and depression (for recent review see Quartara and Altamura 2006). Unfortunately, assessing the preclinical efficacy of these NK1 antagonists has been complicated by marked differences in the pharmacology between human and rat or mouse NK1 receptors. Most NK1 receptor antagonists that display high affinity at human receptors show low affinity, selectivity, and brain penetration in rats and mice, due to differences in the amino acid sequence of the NK1 receptor (Beresford et al. 1991; Fong et al. 1992), thus necessitating the administration of high doses that typically results in unspecific pharmacological effects in these rodents (Rupniak et al. 2001, 2003a;; Rupniak and Jackson 1994; Smith et al. 1994). The NK1 receptor antagonist GR-205171 has high binding selectivity for rat NK1 receptors; however, as seen with other NK1 receptor antagonists, the ability of GR-205171 to inhibit SP binding in rat NK1 receptors is low, with Ki values approximately 40 fold higher than those obtained using human NK1 receptors (Gitter et al. 1991; Saria 1999a). Furthermore, GR-205171 causes only a moderate rightward shift in SP-induced inositol-1-phosphate accumulation and acidification rate in cells expressing rat NK1 receptors relative to human NK1 receptors.

In contrast to mice and rats, gerbils show a NK1 receptor pharmacological profile which is very similar to that seen in humans (Griffante et al. 2006; Saria 1999b), and thus the gerbil has become a commonly used preclinical model to test efficacy of NK1 receptor antagonists. In vivo studies reveal that the doses of GR-205171 needed for central NK1 receptor occupancy are several hundred-fold higher for rats than gerbils. For example, in gerbils the ID50 for inhibition of NK1 agonist-induced foot drumming is 0.02 mg/kg; however, in rats, 10–30 mg/kg of GR-205171 are needed to significantly reduce SP-induced sniffing and agonist-induced hypertension (Rupniak et al. 2003a).

In clinical trials, several studies have revealed that NK1 antagonists, including GR-205171, show promise as treatment options for individuals suffering with pathological anxiety (Kramer et al. 2003). For example, as a treatment for symptoms of social phobia, the efficacy of GR-205171 is reportedly similar to that of citalopram (Furmark et al. 2005). The NK1 receptor antagonist GW597599 is effective against CO2-induced panic attack with an efficacy similar to alprazolam (McLean 2005). In a more recent study, George, et al. (2008) have shown that the NK1 receptor antagonist LY686017 suppresses spontaneous alcohol cravings and blunts craving and cortisol responses induced by alcohol-cue challenges in alcohol-dependent subjects with high trait anxiety. Currently, phase II clinical trials are also evaluating the effectiveness of GR205171 in decreasing symptoms of post-traumatic stress disorder.

At present, relatively few studies have examined the effects of NK1 antagonists on conditioned fear. The fear-potentiated startle (FPS) paradigm may be particularly useful for examining conditioned fear in gerbils, because fear induces foot drumming in this species, which inhibits the expression of conditioned freezing - a behavior used to index levels of fear and anxiety in many conditioning paradigms (Rupniak et al. 2003a; Woolley et al. 2006). Furthermore, because much is know about the neurocircuits mediating startle and the augmentation of startle by states of fear and anxiety, the FPS paradigm may be particularly useful in future studies design to identify central mechanisms responsible for anxiolytic-like actions of NK1 antagonists. Thus, the purpose of the current study was to determine whether the NK1 receptor antagonist GR-205171 inhibits contextual conditioned fear using the FPS paradigm. Because past research has shown that a number of NK1 receptor antagonists produce an anxiolytic-like profile in gerbils as assessed by the elevated plus maze (Varty et al. 2002a), we also examined whether GR-205171 produces anxiolytic-like effects in the elevated plus maze.



Male Mongolian gerbils (Charles River, USA) weighing between 60 and 75 g at the onset of testing were used in all experiments. Gerbils were housed 4 per cage with food and water freely available in a room maintained under constant temperature (22°C). Gerbils were allowed 2 weeks to acclimate to the housing conditions prior to the start of experiments. All testing was done in the light phase of a 12 h light/dark cycle (lights on: 07:00h). All experimental procedures used in the present investigation were carried out in accordance with the National Institute of Health ‘Guide for the Care and Use of Laboratory Animals’ and approved by our Institutional Protocol Approval Committee in accordance with Yerkes Primate Research Center Regulations.

Startle Apparatus

Four SR-LAB startle response systems (SR-LAB, San Diego Instruments, San Diego, California, USA) were used for training and testing. Each startle device consisted of a clear Plexiglas cylinder (8.8 cm in diameter and 20.5 cm in length) mounted on a Plexiglas base and placed in a ventilated, sound- and vibration-attenuated chamber. Each chamber was equipped with a horn Radio Shack speaker mounted 24 cm above each cylinder, which was used to present a background white-noise stimulus and 50-ms noise-burst startle stimuli at intensities ranging from 90–120 dB. A 15-W light bulb attached 24 cm above each cylinder was used to provide the light stimulus. The footshock unconditioned stimulus (US) was delivered through a removable stainless steel grid floor using one of four LeHigh Valley shock generators (SGS-004; LeHigh Valley, Beltsville, MD) located outside the sound-attenuating chamber. Movements within the cylinder created changes in voltage as detected by a piezoelectric accelerometer attached to the Plexiglas base. Voltage output signals were rectified, amplified, and digitized on a 0–4096 unit scale. Startle amplitude was defined as the peak accelerometer voltage that occurred during a 200-ms period beginning at the onset of the startle stimulus. Data acquisition and stimuli deliveries were controlled by a computer using SR-LAB software designed by San Diego Instruments.

To establish a second context used to evaluate the context-specificity of FPS, the SR-LAB startle response system was modified in terms of odor, somatosensory, auditory and visual cues. The stainless steel grid floors were removed and 2 chains of 2 cm in length were hung from the top of each cylinder to provide distinctive somatosensory environments. A jar containing a lavender odor was placed inside the sound -attenuated chamber to provide a distinctive olfactory environment. To further differentiate environments, the chamber was supplied with an ambient white-noise stimulus that raised the overall ambient background to 70 dB. In addition, testing and/or training were performed in the chamber illuminated with a light bulb (15 W) located inside the sound-attenuated chamber.

Elevated plus-maze

The elevated plus maze consisted of two open arms (50 × 6.5 cm) and two closed arms with a wall (50 × 6.5 × 15 cm) attached to a common central platform (6.5 × 6.5 cm) to form a cross. The maze was elevated 65 cm above the floor. Test sessions were conducted under standard room lighting (100 lux) where behaviors were continuously videotaped by a video camera placed over the apparatus. Before each test, the plus maze was cleaned with Quatricide (Pharmacal, Waterbury, CT).

Handling and animal assignment

Gerbils used in all experiments were accustomed to repeated handling prior to initiating experimental testing. On each of 3 days prior to testing, animals were transported to an experimental room and each animal was handled for approximately 1 min until they appeared to show reduced indicators of stress when handled (e.g., absence of vocalization and struggling). Following this handling period, each animal was weighed and the tail was inked with permanent marker to designate subject number before returning to colony housing.

Gerbils used to determine the floor and ceiling effects to various startle intensities were also used to examine the effects of GR-205171 on baseline startle responses. Naïve animals were used to characterize contextual FPS, evaluate of the effects of GR-205171 contextual FPS, and evaluate of the effects of GR-205171 on elevated plus-maze testing.

Evaluation of the effects of GR-205171 on baseline startle responses

For two consecutive days, each gerbil was acclimated to the startle cylinder for 20 min during which time no startle stimuli were presented and then they were returned to their home cage. The following day gerbils were placed in the cylinder and after 5 min given 10 startle stimuli at each of four different startle stimulus intensities (90, 100, 110, 120 dB) with an interstimulus interval (ISI) of 30 s. All startle stimuli were presented in a pseudorandom order with the constraint that each stimulus intensity occurred only once in each consecutive four-trial block. Ten blocks were presented for a total of 40 trials. This 40-trial startle-stimulus session was used to determine the floor and ceiling effects to various startle intensities, to enable selection of startle intensities which produced reliable yet submaximal startle responses for subsequent testing (i.e. 110- and 115-dB).

Gerbils were subsequently matched into 4 groups with similar mean startle amplitudes to test the effects of GR-205171 on startle responses. The mean startle amplitudes were calculated by averaging the startle amplitude across all previous 40 test trials. The following day gerbils were injected i.p. with GR-205171 (0, 0.3, 1.0 or 5.0 mg/kg) 30 min before receiving a drug startle test. During the drug startle test, animals received a total of 24 startle stimuli at two different intensities (110- and 115-dB) starting after a 5-min acclimation period. The interstimulus interval was 30 s and the test session was 20 min in duration.

Characterization of contextual FPS in gerbils

The characterization of contextual FPS in gerbils was conducted with a separate cohort of animals using a modified procedure originally designed to validate contextual FPS in rats as described by McNish et al. (1997). After handling, 16 animals received a pre-training startle session and were subsequently matched into groups with similar baseline startle amplitudes. During the pre-training startle session, animals received a total of 24 startle stimuli at two different intensities (110- and 115-dB) starting after a 5-min acclimation period (identical to the drug startle test session described above). The interstimulus interval was 30 s and the test session was 20 min in duration. Half of the animals were tested in chamber A, the other half in chamber B.

Animals in the “Same Group” (A–A and B–B) were trained and tested in the same chamber, whereas animals in the “Different Group” (A–B and B–A) were trained and tested in a different chamber. The assignment of animals to groups and chambers (context A or B) was counterbalanced so that 4 animals were represented in each of the four possible training and testing conditions. In the training session, gerbils were placed in the startle cylinder and after 5 min received the first of 10 unsignaled foot shocks (0.6 mA) at a 2-min variable interstimulus interval (range, 1–3 min). The training session was 25 min in duration, after which gerbils were returned to their home cages. Twenty-four hours after training, animals received a post-training test that was identical to the pre-training startle session.

Evaluation of the effects of GR-205171 contextual FPS in gerbils

After handling, naïve gerbils were first given a pre-training startle session in Context A and matched into groups with similar startle amplitudes. One day later gerbils were given unsignaled footshock training sessions on each of two consecutive days. Two days of training sessions were given to increase levels of FPS. The following day gerbils were injected i.p. with GR-205171 (0, 0.3, 1.0 or 5.0 mg/kg) 30 min before they were tested for startle in Context A. This test was identical to the pre-training startle session.

Evaluation of the effects of GR-205171 on elevated plus-maze testing

After handling, naïve gerbils were injected i.p. with GR-205171 (0, 0.3, 1.0 or 5.0 mg/kg) 30 min before elevated plus-maze testing. At the start of each session, one gerbil was placed at the distal end of a closed arm with their heads facing the wall - rather than the central portion of the maze - to prevent possible ambiguous or biased arm entries. Animals were allowed to explore freely for 5 min. The percentage of open arm entries [open arm/(open + closed arm) entries] x 100 and percentage time in open arms [time in open arms/(time in open + closed arms)] x 100 were computed. Both of these parameters are indicators of anxiolytic-like activity (Hogg 1996; Pellow and File 1986). The total number of closed arm entries was used as an indicator of locomotor activity (Rodgers and Dalvi 1997). Arm entry was considered complete if all four paws entered a closed or open arm from the central platform. In addition to these standard parameters, we also measured the number of entries and amount of time spent by the animals in the center platform. Center time and entries accumulated when least one paw was placed out of an arm.

Drug preparation

The NK1 antagonist GR-205171 (GlaxoSmithKline Pharmaceuticals) was administered intraperitoneally (i.p.) in phosphate buffered saline (PBS) at a volume of 5 ml/kg 30 min before testing. Past research has shown GR-205171 to have high affinity and selectivity for NK1 receptors (Gardner et al. 1996). Doses and pretreatment times were based on data from autoradiography and behavioral experiments demonstrating that the doses selected for the current study, 0.3, 1.0, and 5.0 mg/kg, are sufficient to linearly inhibit 125I-SP binding approximately 25–75% in striatal homogenates of gerbils (IC50=1.25mg/kg) and significantly inhibit NK1 agonist-induced foot drumming in gerbils (Duffy et al. 2002).

Data reduction and analyses

Mean startle response elicited at increasing noise-burst intensities were calculated by averaging the startle amplitude at each intensity (90, 100, 110, 120 dB). Mean startle amplitudes for the pre-training and post-training test sessions were calculated by averaging the startle amplitude across all test trials. In cases where analyses were conducted on the first and/or second halves of the post-training test, mean startle responses were calculated by averaging the startle amplitude across Trials 1–12 or Trials 13–24, respectively. Block values represented in figures denote the mean startle response of two sequential test trials of different intensity (110, 115 dB). Fear-potentiated startle was detected by comparing pre-training mean startle amplitudes to post-training mean startle amplitudes. Significant fear-potentiated startle was defined as a reliable increase from pre- to post-training startle. Therefore, group differences were examined using repeated-measures ANOVAs, with session (pre-training, post-training) as the within-subject variable, or paired t-tests. Group differences were also evaluated with simple effects at each level of session. Each elevated plus-maze dependent measure was examined using a one-way ANOVA. Significant mean groups differences were detected by use of using Bonferroni t-tests to control the experimentwise error rate at α = 0.05.


Effects of GR-205171 on baseline startle response

Figure 1A displays the mean startle responses elicited at increasing noise-burst intensities in gerbils. Startle amplitudes increased as a function of stimulus intensity, as confirmed by a repeated measures ANOVA which revealed a significant intensity effect, F(4, 95)=20.78, p<0.001. Bonferroni t-tests revealed significant differences among all intensities (ps<0.001), and a trend analysis indicated that the linear component accounted for a largest and significant proportion of the variance, η2=0.73, p<0.001. As seen in Figure 1B, none of the doses of GR-205171 altered mean baseline startle responses when gerbils were subsequently given a test in which startle was elicited with two different noise-burst intensities (110- and 115-dB). The lack of drug effect on startle was verified by a one-way ANOVA which yielded no significant main effect of drug dose, F(3, 19)=0.04, NS.

Figure 1
A) Mean startle responses elicited at increasing noise-burst intensities in gerbils (N=20). Startle responses in gerbils increased as a function of stimulus intensity (90, 100, 110, 120 dB). B) Effects of the NK1 antagonist GR-205171 (0, 0.3, 1.0 or 5.0 ...

Contextual FPS in gerbils

Figure 2A displays the mean pre-training and post-training startle response of gerbils in the same (A–A and B–B) and different (A–B and B–A) context groups. Animals trained and tested in the same context displayed greater mean startle responses after training relative to before training, indicating significant FPS to the context; whereas, gerbils trained and tested in different contexts showed no FPS. Figure 2B displays the mean startle responses of gerbils across blocks during the post-training test. The significant difference in startle responses between groups gradually declined across the test session, and comparatively little differences in startle amplitudes could be detected during the second half of the test session. Together, these findings suggest that gerbils tested in the same context, but not in a different context, displayed significant contextual FPS, and within-session extinction of contextual FPS.

Figure 2
A) Mean pre-training and post-training startle response of gerbils trained and tested in the same (SAME) context (n=8; A–A and B–B) or different (DIFF) contexts (n=8; A–B and B–A). B) Mean startle responses of gerbils across ...

An overall ANOVA using group (same, different) as a between-subjects factor and session (pre-training, post-training) as a within-subjects factor revealed a significant main effect of group, F(1,14)=4.73; p<0.05, and a significant Group by Session interaction, F(1,14)=10.96; p<0.01. Subsequent paired t-tests with Bonferroni corrections indicated that the same-context group displayed greater mean startle amplitudes after training relative to before training, t(7)=2.99; p<0.02, whereas the different context group showed no differences in startle between sessions, t(7)=1.69; NS. Same- and different-context groups displayed similar startle amplitude during the pre-training startle sessions, t(14)=0.92; NS.

A closer examination of the post-training test session revealed that during the first half of the test session (Blocks 1–6), the gerbils in the same-context group showed greater mean startle responses, t(14)=5.07; p<0.05 (Figure 2B inset). However, during the second half of the test session (Blocks 7–12), no reliable differences in mean startle responses were detected, t(14)=0.32; NS. Thus, while the same-context group showed a greater overall FPS than the different-context group, this effect was mainly mediated by differences during the first half of the post-training test session.

Effects of GR-205171 on contextual FPS

The effects of administration of GR-205171 (0, 0.3, 1.0 or 5.0 mg/kg, i.p) on contextual FPS are shown in Figure 3. Because greater contextual FPS was seen during the first half of the post-training test session, we evaluated the effects of GR-205171 during the first half of the test session, in addition to an analysis of the post-training session as a whole. Figure 3 displays the mean startle response of groups before training, as well as their mean startle responses during the first and second halves of the post-training test (the inset displays post-training session as a whole). Overall, all groups displayed greater mean startle responses during the post-training test session relative to pre-training session, indicating contextual FPS in all groups. However, relative to the vehicle control group (0 mg/kg), groups of animals that received pretreatment of GR-205171 (0.3, 1.0 or 5.0 mg/kg) prior to the post-training test displayed less FPS. The reduction of contextual FPS was greatest in the group treated with 5 mg/kg of GR-205171.

Figure 3
Effects of the NK1 antagonist GR-205171 on contextual FPS. Gerbils were administered GR-205171 (0, 0.3, 1.0 or 5.0 mg/kg, i.p) 30 min before the post-training test session. The bar graph presents the mean startle response of groups before training as ...

An ANOVA using group (0, 0.3, 1.0 or 5.0 mg/kg) and session (pre-training, post-training) as factors yielded significant main effects of dose, F(3,23)=4.05; p<0.05, and session, F(1,23)=151.80; p<0.01. More importantly, there was a significant Dose x Session interaction, F(3,23)=5.45; p<0.01, signifying that test performance was differentially affected by drug dose. Simple one-way ANOVAs at each session level revealed a reliable post-training dose effect, F(3,23)=5.46; p<0.01, but no pre-training dose effect, F(3,23)=0.64; p<0.05. Follow-up Bonferroni t-tests revealed that FPS in the group treated with 5 mg/kg of GR-205171 was significantly less (p<0.01) than in the vehicle control group (0 mg/kg; Figure 3, inset). No other t-test was statistically significant.

The planned analysis of FPS levels during the first half of the test session (Blocks 1–6) also yielded a reliable post-training dose effect, F(3,23)=8.06; p<0.01. Follow-up Bonferroni t-tests indicated that all groups that received GR-205171 (0.3, 1.0 and 5.0 mg/kg) displayed less FPS than the vehicle control group (ps<0.05; Figure 3). Thus, while the highest dose of GR-205171 (5mg/kg) reduced overall FPS (Blocks 1–12), lower doses (0.3 and 1.0mg/kg) also induced a reduction of FPS, which could be detected during the first half of the post-training test session when FPS expression is greatest (Blocks 1–6).

The effects of GR-205171 on elevated plus-maze testing

An evaluation of the percentage time in open arms revealed a significant treatment effect F(3,30)=5.14, p<0.01. As seen in Figure 4A, there was a significant relationship between drug dose and percentage time in open arms, F(1,30)=14.78, p<0.01. Individual contrasts revealed that treatment with 5 mg/kg of GR-205171 increased percentage time in open arms, t(19)=3.86, p<0.01. The comparison between vehicle (0 mg/kg) and 1.0 mg/kg groups was marginally nonsignificant, t(19)=1.79, p=0.08, and the difference between vehicle and 0.3 mg/kg groups was nonsignificant, t(19)=1.01, NS.

Figure 4
Effects of the NK1 antagonist GR-205171 on elevated plus-maze performance. Gerbils were administered GR-205171 (0, 0.3, 1.0 or 5.0 mg/kg, i.p) 30 min before testing. A) Percentage of open arm time. B) Number of closed arm entries. C) Percentage of open ...

Assessment of total closed arm entries indicated significant differences in locomotor activity among treatments groups, F(3,30)=8.33, p<0.01. Individual contrasts comparing the vehicle group to each GR-205171 treatment group indicated that gerbils treated with the high dose of GR-205171 (5.0mg/kg) made significantly more closed arm entries than the vehicle group, indicating a drug-induced increase in exploratory/motor-stimulant activity, t(19)=4.39, p<0.01. No significant differences in number of closed arm entries were observed between the vehicle, 0.3 mg/kg, and 1.0 mg/kg treatment groups (all NS, Figure 4B). To factor out this increase in locomotor activity, the data were analyzed using percentage open arm entries. At doses of 0.3, 1.0, and 5.0 mg/kg, GR-205171 caused a significant anxiolytic-like effect as revealed by significant increases in the percentage open arm entries. As seen in Figure 4C, all three treatment levels of GR-205171 reliably increased the percentage open arm entries as revealed by a significant one-way ANOVA, F(3,30)=8.45, p<0.01, and individual contrasts comparing the vehicle group to subsequent treatments groups were all significant (ps< 0.01). No other t-test was statistically significant. Group means for the number of open, total, and center entries, as well as center arm time are presented in Table 1.

Effects of GR-205171 (0, 0.3, 1.0 or 5.0 mg/kg) on the behavioral measures from the elevated plus-maze.


The present study sought to determine whether systemic administration of the NK1 receptor antagonist GR-205171 in gerbils produced anxiolytic-like effects in the elevated plus maze and a novel contextual conditioned fear test using the FPS paradigm. In the contextual FPS test, gerbils displayed reliable increases in startle when tested in a context previously associated with unsignaled footshocks. In contrast, previous unsignaled footshock training did not augment startle if gerbils were tested in a different context, indicating context-specific conditioning. When gerbils were tested for FPS after GR-205171 administration, all drug doses (0.3, 1.0 and 5.0 mg/kg) significantly attenuated contextual FPS when compared to the vehicle control group.

Data from previous studies conducted with laboratory mice and rat demonstrate that reference anxiolytics, such as benzodiazepine agonists, effectively reduce the expression of both cue-specific (Guscott et al. 2000; Risbrough et al. 2003) and contextual FPS (Guscott et al. 2000; Joordens et al. 1997; Young et al. 1991), thus supporting the use of the FPS paradigm to investigate anxiolytic properties of drugs. Independent of the anxiolytic-like effects, benzodiazepine agonists also attenuate baseline startle amplitudes in a dose-dependent manner (Joordens et al. 1998). In contrast, none of the doses of GR-205171 tested altered baseline startle amplitude in the present study. Future studies will need to explore the behavioral profile of anxiolytics such as benzodiazepines, serotonin agonists, and other NK1 antagonists in gerbils using the FPS paradigm.

In the elevated plus maze, GR-205171 produced an anxiolytic-like effect as indexed by an increase in the percent time spent in the open arms and percent of open arms entries. In the case of percentage open arms entries, all 3 doses of GR-205171 produced significant increases as compared with the control vehicle group. GR-205171 administration increased percentage open arm time in a dose-dependent manner, causing a significant increase above the control group at the highest dose (5.0 mg/kg). The highest dose of GR-205171, however, also increased locomotor activity as reflected by the total number of closed arms entries (Cruz et al. 1994; Rodgers et al. 1997; Rodgers and Johnson 1995). It is often assumed that increases in open arm indices reflect a specific effect on fear/anxiety, provided there is no simultaneous change in locomotor activity. The increase in closed arm entries could reflect a non-specific arousal/locomotor stimulation resulting in potential “false positives.” However, the increase in open arms entries seen at lower doses levels (0.3 and 1.0 mg/kg) were not accompanied by significant locomotor effects. Furthermore, no group differences were detected in center hub behaviors, which have been used as indices of exploratory motivation (Lee and Rodgers 1991; Rodgers et al. 1992). Thus, whether changes in open arm time reflect an anxiolytic-like effect or some other processes independent of the fear, such as general arousal or an enhanced motivation to explore is unclear. However, reference anxiolytics such as benzodiazepine agonists, which robustly increase open arm indices, also increase locomotion in the elevated plus-maze (Dawson et al. 1995; Derrien et al. 1994; File and Aranko 1988; Lister 1987; Moser 1989; Rodgers et al. 1992; Varty et al. 2002b). In contrast, locomotor stimulants, such as amphetamine and caffeine have inconsistent effects on locomotion in the elevated plus-maze (Pellow et al. 1985). Such evidence leads one to question the rationale for excluding compounds that increase closed-area entries.

Varty et al. (2002a) have previously demonstrated that a number of NK1 receptor antagonists produce anxiolytic-like effects in a novel gerbil elevated plus maze. In that study, investigators reported that antagonists produced no enhanced locomotor activity. Additionally, open arm indices in vehicle-treated control animals were higher than the present study. Because the plus-maze is very sensitive to methodological factors, it is likely that differences in maze construction and test procedures account for the observed discrepancies. For example, testing in our study was carried out on a plus-maze elevated higher(65 cm vs. 35 cm) and with narrower arms (6.5 cm vs. 8 cm). Furthermore, the maze used by in the study of Varty et al (2002a) had clear closed arms to allow for constant illumination in all parts of the maze and holes incorporated into the Plexiglas floor, to allow gerbils to grip the surface. That study also used female gerbils as opposed to the males gerbils used in the current study. In addition to the above factors, animals in our study were tested under higher light conditions (100-Lux vs. 5-Lux), which have been shown to reduce open arm entries in gerbils (Varty et al. 2002b). It is possible that any one or combination of these variables could account for the baseline differences observed in our vehicle-treated animals.

In recent years a number of different behavioral models have been used to assess the anxiolytic-like properties of NK1 receptor antagonists in gerbils. Varty et al. (2002b) demonstrated that the elevated plus-maze induces an anxiety-like profile and has predictive validity for anxiolytics like benzodiazepines in gerbils. Past research indicates that the NK1 antagonists MK-869, L-742,694, L-733,060, CP-99,994, and CP-122,721 produced anxiolytic-like effects in the gerbil elevated plus-maze (Varty et al. 2002a). Consistent with these findings, we have shown that the NK1 antagonist GR-205171 also induces an anxiolytic-like profile in the elevated plus-maze without accompanying sedative effects often observed with anxiolytic drugs like benzodiazepines (Rupniak et al., 2001). NK1 antagonists have also been tested against behavioral conditioning paradigms that induce foot drumming, which is seen as a species-specific alarm or fear response in gerbils (Randall 2001). Ballard et al. (2001) have shown that pairing a light-tone conditioned stimulus (CS) with a footshock US produces a robust foot-drumming response during both the conditioning period and following presentation of the conditioned stimulus in a retest 24 h later. Moreover, gerbils treated with the NK1 antagonist MK-869 prior to conditioning show less shock-induced foot drumming and a significant inhibition of CS-induced foot drumming during subsequent testing. In the same study, Ballard et al. (2001) showed that pretreatment with the NK1 antagonist CP-99,994 also produced a significant reduction in foot drumming induced by a 2-mA footshock US. Similarly, exposure to the aversive four-plate fear conditioning apparatus induced foot drumming in gerbils, which could be abolished by either diazepam and the NK1 receptor antagonist L-760735 given before testing (Rupniak 2003).

Our findings support evidence from other laboratories for anxiolytic activity of NK1 receptor antagonists and provide a novel Pavlovian conditioned fear test that may be an appropriate procedure to test other NK1 antagonists for preclinical anxiolytic activity in gerbils. The FPS paradigm is widely recognized as a valid measure of fear and anxiety because: (1) humans demonstrate FPS (Ameli et al. 2001; Grillon and Davis 1997; Grillon et al. 1999), (2) anxiolytic drugs decrease FPS (Davis 1979; Grillon et al. 2006; Patrick et al. 1996; Riba et al. 2001; Winslow et al. 2007), and (3) lesions of brain structures critical for the expression of fear eliminate the expression of FPS (Campeau and Davis 1995; Falls and Davis 1995; Heldt et al. 2000; Sananes and Davis 1992). Fear-potentiated startle may be particularly useful for examining conditioned fear in gerbils because fear-induced foot drumming can inhibit the expression of conditioned freezing (Woolley et al. 2006), which many conditioning paradigms use as a behavioral response to index levels of fear and anxiety. It is worth noting, however, that conditioning procedures that induce foot drumming often use higher footshock US intensity than used in the current study. Although not quantified, we did not provoke noticeable foot drumming, presumably due to the relatively low US intensity (i.e. 0.6mA).

A growing line of evidence indicates that the amygdala is a potential site of action for NK1 receptor antagonists in anxiety. Autoradiographic experiments with NK1 radioligands performed in brain slices of gerbils reveal a distribution profile that is highly homologous to human, and in both species NK1 receptors are widely distributed in brain regions involved in the regulation of affective behavior and the mediation of stress responses, including various nuclei of the amygdala (Caberlotto et al. 2003; Griffante et al. 2006; Nagano et al. 2006; Rigby et al. 2005). Previous studies have also shown that exposure to a variety of aversive stressors such as immobilization and forced swim stress increase substance P release in the amygdala, as indexed by microdialysis and corresponding NK1 receptor internalization (Ebner et al. 2004; Ebner and Singewald 2005; Smith et al. 1999). In contrast, microinjection of NK1 antagonists into the amygdala blocks stress-induced anxiogenic effects (Ebner et al. 2004). Furthermore, the effect of amygdala lesions resembled that seen after administration of the NK1 antagonists. For example, gerbils that had undergone basolateral and lateral amygdala lesions after aversive four-plate fear conditioning exhibited a release of plate crossings and reduced foot drumming when they were returned to the apparatus 7 days after the initial exposure, resembling effects seen after administration of the SPA, L-760735 (Rupniak et al. 2003b). Bilateral amygdala lesions also blocked footshock-induced immobility (Woolley et al. 2006), and drug-induced foot drumming (Rupniak et al. 2003b).

At present, much less is known about the efficacy of NK1 receptor antagonists as a treatment option for individuals suffering with pathological anxiety; however, several studies have revealed that NK1 antagonists, including GR-205171, show promise as anxiolytic and antidepressant drugs in clinical trials (Furmark et al. 2005; Kramer et al. 2003). Recent clinical findings have also shown that SP concentrations are elevated both during and after the symptom-provoking stimulus in the CSF of PTSD patients (Geracioti et al. 2006). Similarly, in a recent PET study using a radioactive analog of GR205171, Michelgard et al. (2007) found that patients viewing phobia-inducing pictures had reduced uptake of the labeled GR-205171 in the amygdala during symptom provocation. This reduction reflected a fear-induced increase in the release of endogenous SP and corresponding reduction in NK1 receptor availability. More recently, Fujimoura et al. (2009) have shown that compared with healthy subjects, patients with panic disorder have widespread reduction of NK1 receptor binding in brain. Together, these findings support the notion that the SP-NK1 receptor system might be an important neurochemical target for the development of selective drugs designed to control pathological anxiety.

Within the context of the current study, extensive evidence indicates that the amygdala is critically involved in the acquisition and retrieval of contextual fear conditioning. Amygdala lesions or inactivation block conditioned fear responses to both the context and the explicit cue (Davis 1992; Kapp et al. 1984; LeDoux 1994). Studies using freezing as an index of fear have shown that hippocampal lesions made either before training (Phillips and LeDoux 1992) or after training (Kim and Fanselow 1992) also disrupted fear conditioning to the context but not to a explicit cue. However, as assessed by the FPS, the hippocampus may not be as critical as the amygdala for contextual learning (Gewirtz et al. 2000; McNish et al. 1997). Lastly, there is published evidence supporting the possibility that augmented startle responses associated with FPS may result from the influence of SP on downstream targets of amygdala. Microinjections of SP into the caudal pontine reticular nucleus (PnC), an important part of the primary acoustic startle circuit, increases the amplitude of the startle response (Krase et al. 1994). This effect can be antagonized by local pretreatment with the NK1 antagonist CP-96,345. Furthermore, sensitization of the startle response by footshocks can also be blocked by local microinjections of CP-96,345 into the PnC (Krase et al. 1994).

In conclusion, the current study demonstrates that the NK1 receptor antagonist GR-205171 induced an anxiolytic-like response in the elevated plus maze test. In addition, we have shown that gerbils can display contextual FPS, which can be reduced by pretreatment of GR-205171.These findings support evidence from other laboratories for anxiolytic activity of NK1 antagonists and provide a novel Pavlovian conditioned fear test, which may be an appropriate procedure to test other NK1 antagonists for preclinical anxiolytic activity in gerbils.


This research was supported by National Institute of Mental Health grants MH069056, MH47840, MH57250 and MH59906, NIH (DA-019624 and F32 MH073389-01), NARSAD, Burroughs Wellcome Foundation, and the Science and Technology Center (The Center for Behavioral Neuroscience of the National Science Foundation under Agreement No. IBN-9876754) and the Yerkes Base Grant.


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  • Aguiar MS, Brandao ML. Conditioned place aversion produced by microinjections of substance P into the periaqueductal gray of rats. Behav Pharmacol. 1994;5:369–373. [PubMed]
  • Aguiar MS, Brandao ML. Effects of microinjections of the neuropeptide substance P in the dorsal periaqueductal gray on the behaviour of rats in the plus-maze test. Physiol Behav. 1996;60:1183–1186. [PubMed]
  • Ameli R, Ip C, Grillon C. Contextual fear-potentiated startle conditioning in humans: replication and extension. Psychophysiology. 2001;38:383–390. [PubMed]
  • Ballard TM, Sanger S, Higgins GA. Inhibition of shock-induced foot tapping behaviour in the gerbil by a tachykinin NK1 receptor antagonist. Eur J Pharmacol. 2001;412:255–264. [PubMed]
  • Bannon MJ, Deutch AY, Tam SY, Zamir N, Eskay RL, Lee JM, Maggio JE, Roth RH. Mild footshock stress dissociates substance P from substance K and dynorphin from Met- and Leu-enkephalin. Brain Res. 1986;381:393–396. [PubMed]
  • Barbaresi P. Immunocytochemical localization of substance P receptor in rat periaqueductal gray matter: a light and electron microscopic study. J Comp Neurol. 1998;398:473–490. [PubMed]
  • Bassi GS, Nobre MJ, Carvalho MC, Brandao ML. Substance P injected into the dorsal periaqueductal gray causes anxiogenic effects similar to the long-term isolation as assessed by ultrasound vocalizations measurements. Behav Brain Res. 2007;182:301–307. [PubMed]
  • Beresford IJ, Birch PJ, Hagan RM, Ireland SJ. Investigation into species variants in tachykinin NK1 receptors by use of the non-peptide antagonist, CP-96,345. Br J Pharmacol. 1991;104:292–293. [PMC free article] [PubMed]
  • Brodin E, Rosen A, Schott E, Brodin K. Effects of sequential removal of rats from a group cage, and of individual housing of rats, on substance P, cholecystokinin and somatostatin levels in the periaqueductal grey and limbic regions. Neuropeptides. 1994;26:253–260. [PubMed]
  • Caberlotto L, Hurd YL, Murdock P, Wahlin JP, Melotto S, Corsi M, Carletti R. Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain. European Journal of Neuroscience. 2003;17:1736–1746. [PubMed]
  • Campeau S, Davis M. Involvement of the central nucleus and basolateral complex of the amygdala in fear conditioning measured with fear-potentiated startle in rats trained concurrently with auditory and visual conditioned stimuli. Journal of Neuroscience. 1995;15:2301–2312. [PubMed]
  • Cheeta S, Tucci S, Sandhu J, Williams AR, Rupniak NM, File SE. Anxiolytic actions of the substance P (NK1) receptor antagonist L-760735 and the 5-HT1A agonist 8-OH-DPAT in the social interaction test in gerbils. Brain Res. 2001;915:170–175. [PubMed]
  • Commons KG, Valentino RJ. Cellular basis for the effects of substance P in the periaqueductal gray and dorsal raphe nucleus. J Comp Neurol. 2002;447:82–97. [PubMed]
  • Cruz AP, Frei F, Graeff FG. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav. 1994;49:171–176. [PubMed]
  • Dableh LJ, Yashpal K, Rochford J, Henry JL. Antidepressant-like effects of neurokinin receptor antagonists in the forced swim test in the rat. Eur J Pharmacol. 2005;507:99–105. [PubMed]
  • Davis M. Diazepam and flurazepam: effects on conditioned fear as measured with the potentiated startle paradigm. Psychopharmacology (Berl) 1979;62:1–7. [PubMed]
  • Davis M. Analysis of aversive memories using the fear-potentiated startle paradigm. In: Squire LR, Butters N, editors. Neuropsychology of Memory. New York, NY: The Guilford Press; 1992. pp. 35–41.
  • Dawson GR, Crawford SP, Collinson N, Iversen SD, Tricklebank MD. Evidence that the anxiolytic-like effects of chlordiazepoxide on the elevated plus maze are confounded by increases in locomotor activity. Psychopharmacology (Berl) 1995;118:316–323. [PubMed]
  • De Araujo JE, Huston JP, Brandao ML. Opposite effects of substance P fragments C (anxiogenic) and N (anxiolytic) injected into dorsal periaqueductal gray. Eur J Pharmacol. 2001;432:43–51. [PubMed]
  • Derrien M, McCort-Tranchepain I, Ducos B, Roques BP, Durieux C. Heterogeneity of CCK-B receptors involved in animal models of anxiety. Pharmacol Biochem Behav. 1994;49:133–141. [PubMed]
  • Duarte FS, Testolin R, De Lima TC. Further evidence on the anxiogenic-like effect of substance P evaluated in the elevated plus-maze in rats. Behav Brain Res. 2004;154:501–510. [PubMed]
  • Duffy RA, Varty GB, Morgan CA, Lachowicz JE. Correlation of neurokinin (NK) 1 receptor occupancy in gerbil striatum with behavioral effects of NK1 antagonists. J Pharmacol Exp Ther. 2002;301:536–542. [PubMed]
  • Ebner K, Rupniak NM, Saria A, Singewald N. Substance P in the medial amygdala: emotional stress-sensitive release and modulation of anxiety-related behavior in rats. Proc Natl Acad Sci U S A. 2004;101:4280–4285. [PubMed]
  • Ebner K, Singewald N. Effects of NK1 receptor antagonist administration on basal and stress-induced substance P release in the amygdala. Journal of Neurochemistry. 2005;94:183.
  • Ebner K, Singewald N. The role of substance P in stress and anxiety responses. Amino Acids. 2006;31:251–272. [PubMed]
  • Elliott PJ. Place aversion induced by the substance P analogue, dimethyl-C7, is not state dependent: implication of substance P in aversion. Exp Brain Res. 1988;73:354–356. [PubMed]
  • Falls WA, Davis M. Lesions of the central nucleus of the amygdala block conditioned excitation, but not conditioned inhibition of fear as measured with the fear-potentiated startle effect. Behavioral Neuroscience. 1995;109:379–387. [PubMed]
  • File SE. Anxiolytic action of a neurokinin1 receptor antagonist in the social interaction test. Pharmacol Biochem Behav. 1997;58:747–752. [PubMed]
  • File SE, Aranko K. Sodium valproate and chlordiazepoxide in the elevated plus-maze test of anxiety in the rat. Neuropsychobiology. 1988;20:82–86. [PubMed]
  • Fong TM, Yu H, Strader CD. The extracellular domain of substance P (NK1) receptor comprises part of the ligand binding site. Biophys J. 1992;62:59–60. [PubMed]
  • Fujimura Y, Yasuno F, Farris A, Liow J-S, Geraci M, Drevets W, Pine DS, Ghose S, Lerner A, Hargreaves R, Burns HD, Morse C, Pike VW, Innis RB. Decreased Neurokinin-1 (Substance P) Receptor Binding in Patients with Panic Disorder: Positron Emission Tomographic Study with [18F]SPA-RQ. Biological Psychiatry. 2009 in press. [PMC free article] [PubMed]
  • Furmark T, Appel L, Michelgård Å, Wahlstedt K, Åhs F, Zancan S, Jacobsson E, Flyckt K, Grohp M, Bergström M, Pich EM, Nilsson L-G, Bani M, Långström B, Fredrikson M. Cerebral Blood Flow Changes After Treatment of Social Phobia with the Neurokinin-1 Antagonist GR205171, Citalopram, or Placebo. Biological Psychiatry. 2005;58:132–142. [PubMed]
  • Gardner CJ, Armour DR, Beattie DT, Gale JD, Hawcock AB, Kilpatrick GJ, Twissell DJ, Ward P. GR205171: A novel antagonist with high affinity for the tachykinin NK1 receptor, and potent broad-spectrum anti-emetic activity. Regulatory Peptides. 1996;65:45–53. [PubMed]
  • Gentsch C, Cutler M, Vassout A, Veenstra S, Brugger F. Anxiolytic effect of NKP608, a NK1-receptor antagonist, in the social investigation test in gerbils. Behav Brain Res. 2002;133:363–368. [PubMed]
  • George DT, Gilman J, Hersh J, Thorsell A, Herion D, Geyer C, Peng X, Kielbasa W, Rawlings R, Brandt JE, Gehlert DR, Tauscher JT, Hunt SP, Hommer D, Heilig M. Neurokinin 1 Receptor Antagonism as a Possible Therapy for Alcoholism. Science. 2008;319:1536–1539. [PubMed]
  • Geracioti TD, Jr, Carpenter LL, Owens MJ, Baker DG, Ekhator NN, Horn PS, Strawn JR, Sanacora G, Kinkead B, Price LH, Nemeroff CB. Elevated Cerebrospinal Fluid Substance P Concentrations in Posttraumatic Stress Disorder and Major Depression. Am J Psychiatry. 2006;163:637–643. [PubMed]
  • Gewirtz JC, McNish KA, Davis M. Is the hippocampus necessary for contextual fear conditioning? Behav Brain Res. 2000;110:83–95. [PubMed]
  • Gitter BD, Waters DC, Bruns RF, Mason NR, Nixon JA, Howbert JJ. Species differences in affinities of non-peptide antagonists for substance p receptors. European Journal of Pharmacology. 1991;197:237–238. [PubMed]
  • Griffante C, Carletti R, Andreetta F, Corsi M. [3H]GR205171 displays similar NK1 receptor binding profile in gerbil and human brain. Br J Pharmacol. 2006;148:39–45. [PMC free article] [PubMed]
  • Grillon C, Baas JMP, Pine DS, Lissek S, Lawley M, Ellis V, Levine J. The Benzodiazepine Alprazolam Dissociates Contextual Fear from Cued Fear in Humans as Assessed by Fear-potentiated Startle. Biological Psychiatry. 2006;60:760–766. [PubMed]
  • Grillon C, Davis M. Fear-potentiated startle conditioning in humans: explicit and contextual cue conditioning following paired versus unpaired training. Psychophysiology. 1997;34:451–458. [PubMed]
  • Grillon C, Merikangas KR, Dierker L, Snidman N, Arriaga RI, Kagan J, Donzella B, Dikel T, Nelson C. Startle potentiation by threat of aversive stimuli and darkness in adolescents: A multi-site study. International Journal of Psychophysiology. 1999;32:63–73. [PubMed]
  • Guscott MR, Cook GP, Bristow LJ. Contextual fear conditioning and baseline startle responses in the rat fear-potentiated startle test: a comparison of benzodiazepine/gamma-aminobutyric acid-A receptor agonists. Behav Pharmacol. 2000;11:495–504. [PubMed]
  • Hasenohrl RU, Gerhardt P, Huston JP. Substance P enhancement of inhibitory avoidance learning: Mediation by the N-terminal sequence. Peptides. 1990;11:163–167. [PubMed]
  • Heldt S, Sundin V, Willott JF, Falls WA. Posttraining lesions of the amygdala interfere with fear-potentiated startle to both visual and auditory conditioned stimuli in C57BL/6J mice. Behavioral Neuroscience. 2000;114:749–759. [PubMed]
  • Hietala J, Nyman MJ, Eskola O, Laakso A, Gronroos T, Oikonen V, Bergman J, Haaparanta M, Forsback S, Marjamaki P, Lehikoinen P, Goldberg M, Burns D, Hamill T, Eng WS, Coimbra A, Hargreaves R, Solin O. Visualization and quantification of neurokinin-1 (NK1) receptors in the human brain. Mol Imaging Biol. 2005;7:262–272. [PubMed]
  • Hogg S. A review of the validity and variability of the Elevated Plus-Maze as an animal model of anxiety. Pharmacology Biochemistry and Behavior Anxiety, Stress and Depression. 1996;54:21–30. [PubMed]
  • Hokfelt T, Skirboll L, Everitt B, Meister B, Brownstein M, Jacobs T, Faden A, Kuga S, Goldstein M, Markstein R, et al. Distribution of cholecystokinin-like immunoreactivity in the nervous system. Co-existence with classical neurotransmitters and other neuropeptides. Ann N Y Acad Sci. 1985;448:255–274. [PubMed]
  • Joordens RJ, Hijzen TH, Olivier B. The anxiolytic effect on the fear-potentiated startle is not due to a non-specific disruption. Life Sci. 1998;63:2227–2232. [PubMed]
  • Joordens RJ, Hijzen TH, Peeters BW, Olivier B. Control conditions in the fear-potentiated startle response paradigm. Neuroreport. 1997;8:1031–1034. [PubMed]
  • Kapp BS, Pascoe JP, Bixler MA. The amygdala: a neuroanatomical systems approach to its contribution to aversive conditioning. In: Butlers N, Squire LS, editors. The Neuropsychology of Memory. New York: The Guilford Press; 1984. pp. 473–488.
  • Kim JJ, Fanselow MS. Modality specific retrograde amnesia of fear. Science. 1992;256:675–677. [PubMed]
  • Kramer MS, Cutler N, Feighner J, Shrivastava R, Carman J, Sramek JJ, Reines SA, Liu G, Snavely D, Wyatt-Knowles E, Hale JJ, Mills SG, MacCoss M, Swain CJ, Harrison T, Hill RG, Hefti F, Scolnick EM, Cascieri MA, Chicchi GG, Sadowski S, Williams AR, Hewson L, Smith D, Carlson EJ, Hargreaves RJ, Rupniak NM. Distinct mechanism for antidepressant activity by blockade of central substance P receptors. Science. 1998;281:1640–1645. [PubMed]
  • Kramer MS, Winokur A, Kelsey J, Preskorn SH, Rothschild AJ, Snavely D, Ghosh K, Ball WA, Reines SA, Munjack D, Apter JT, Cunningham L, Kling M, Bari M, Getson A, Lee Y. Demonstration of the Efficacy and Safety of a Novel Substance P (NK1) Receptor Antagonist in Major Depression. Neuropsychopharmacology. 2003;29:385–392. [PubMed]
  • Krase W, Koch M, Schnitzler HU. Substance P is involved in the sensitization of the acoustic startle response by footshocks in rats. Behav Brain Res. 1994;63:81–88. [PubMed]
  • LeDoux JE. Emotion, memory and the brain. Scientific American. 1994;6:50–57. [PubMed]
  • Lee C, Rodgers RJ. Effects of benzodiazepine receptor antagonist, flumazenil, on antinociceptive and behavioural responses to the elevated plus-maze in mice. Neuropharmacology. 1991;30:1263–1267. [PubMed]
  • Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology (Berl) 1987;92:180–185. [PubMed]
  • Maeno H, Kiyama H, Tohyama M. Distribution of the substance P receptor (NK-1 receptor) in the central nervous system. Brain Res Mol Brain Res. 1993;18:43–58. [PubMed]
  • McLean S. Do substance P and the NK1 receptor have a role in depression and anxiety? Curr Pharm Des. 2005;11:1529–1547. [PubMed]
  • McNish KA, Gewirtz JC, Davis M. Evidence of contextual fear after lesions of the hippocampus: a disruption of freezing but not fear-potentiated startle. J Neurosci. 1997;17:9353–9360. [PubMed]
  • Michelgård Å, Appel L, Pissiota A, Frans Ö, Långström B, Bergström M, Fredrikson M. Symptom Provocation in Specific Phobia Affects the Substance P Neurokinin-1 Receptor System. Biological Psychiatry. 2007;61:1002–1006. [PubMed]
  • Moser PC. An evaluation of the elevated plus-maze test using the novel anxiolytic buspirone. Psychopharmacology (Berl) 1989;99:48–53. [PubMed]
  • Nagano M, Saitow F, Haneda E, Konishi S, Hayashi M, Suzuki H. Distribution and pharmacological characterization of primate NK-1 and NK-3 tachykinin receptors in the central nervous system of the rhesus monkey. Br J Pharmacol. 2006;147:316–323. [PMC free article] [PubMed]
  • Patrick CJ, Berthot BD, Moore JD. Diazepam blocks fear-potentiated startle in humans. Journal of Abnormal Psychology. 1996:1. [PubMed]
  • Pelleymounter MA, Fisher Q, Schlesinger K, Hall M, Dearmey P, Stewart J. The effect of substance P and its fragments on passive avoidance retention and brain monoamine activity. Behavioural Brain Research. 1986;21:119–127. [PubMed]
  • Pellow S, Chopin P, File SE, Briley M. Validation of open : closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. Journal of Neuroscience Methods. 1985;14:149–167. [PubMed]
  • Pellow S, File SE. Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: A novel test of anxiety in the rat. Pharmacology Biochemistry and Behavior. 1986;24:525–529. [PubMed]
  • Pennefather JN, Lecci A, Candenas ML, Patak E, Pinto FM, Maggi CA. Tachykinins and tachykinin receptors: a growing family. Life Sciences. 2004;74:1445–1463. [PubMed]
  • Phillips RG, LeDoux JE. Differential contributions of amygdala and hippocampus to cued and contextual fear conditioning. Behavioral Neuroscience. 1992;106:274–285. [PubMed]
  • Quartara L, Altamura M. Tachykinin receptors antagonists: from research to clinic. Curr Drug Targets. 2006;7:975–992. [PubMed]
  • Quartara L, Maggi CA. The tachykinin NK1 receptor. Part II: Distribution and pathophysiological roles. Neuropeptides. 1998;32:1–49. [PubMed]
  • Randall JA. Evolution and Function of Drumming as Communication in Mammals1. American Zoologist. 2001;41:1143–1156.
  • Regoli D, Boudon A, Fauchere JL. Receptors and antagonists for substance P and related peptides. Pharmacol Rev. 1994;46:551–599. [PubMed]
  • Riba J, Rodriguez-Fornells A, Urbano G, Morte A, Antonijoan R, Barbanoj MJ. Differential effects of alprazolam on the baseline and fear-potentiated startle reflex in humans: A dose-response study. Psychopharmacology (Berl) 2001;157:358–367. [PubMed]
  • Rigby M, O'Donnell R, Rupniak NM. Species differences in tachykinin receptor distribution: further evidence that the substance P (NK1) receptor predominates in human brain. J Comp Neurol. 2005;490:335–353. [PubMed]
  • Risbrough VB, Brodkin JD, Geyer MA. GABA-A and 5-HT1A receptor agonists block expression of fear-potentiated startle in mice. Neuropsychopharmacology. 2003;28:654–663. [PubMed]
  • Rodgers RJ, Cao BJ, Dalvi A, Holmes A. Animal models of anxiety: an ethological perspective. Braz J Med Biol Res. 1997;30:289–304. [PubMed]
  • Rodgers RJ, Dalvi A. Anxiety, defence and the elevated plus-maze. Neuroscience & Biobehavioral Reviews. 1997;21:801–810. [PubMed]
  • Rodgers RJ, Johnson NJ. Factor analysis of spatiotemporal and ethological measures in the murine elevated plus-maze test of anxiety. Pharmacol Biochem Behav. 1995;52:297–303. [PubMed]
  • Rodgers RJ, Lee C, Shepherd JK. Effects of diazepam on behavioural and antinociceptive responses to the elevated plus-maze in male mice depend upon treatment regimen and prior maze experience. Psychopharmacology (Berl) 1992;106:102–110. [PubMed]
  • Rosen A, Brodin K, Eneroth P, Brodin E. Short-term restraint stress and s.c. saline injection alter the tissue levels of substance P and cholecystokinin in the peri-aqueductal grey and limbic regions of rat brain. Acta Physiol Scand. 1992;146:341–348. [PubMed]
  • Rupniak NM, Carlson EJ, Shepheard S, Bentley G, Williams AR, Hill A, Swain C, Mills SG, Di Salvo J, Kilburn R, Cascieri MA, Kurtz MM, Tsao KL, Gould SL, Chicchi GG. Comparison of the functional blockade of rat substance P (NK1) receptors by GR205171, RP67580, SR140333 and NKP-608. Neuropharmacology. 2003a;45:231–241. [PubMed]
  • Rupniak NM, Carlson EJ, Webb JK, Harrison T, Porsolt RD, Roux S, de Felipe C, Hunt SP, Oates B, Wheeldon A. Comparison of the phenotype of NK1R-/- mice with pharmacological blockade of the substance P (NK1 ) receptor in assays for antidepressant and anxiolytic drugs. Behav Pharmacol. 2001;12:497–508. [PubMed]
  • Rupniak NM, Jackson A. Non-specific inhibition of dopamine receptor agonist-induced behaviour by the tachykinin NK1 receptor antagonist CP-99,994 in guinea-pigs. Eur J Pharmacol. 1994;262:171–175. [PubMed]
  • Rupniak NM, Webb JK, Fisher A, Smith D, Boyce S. The substance P (NK1) receptor antagonist L-760735 inhibits fear conditioning in gerbils. Neuropharmacology. 2003b;44:516–523. [PubMed]
  • Sananes CB, Davis M. N-methyl-D-aspartate lesions of the lateral and basolateral nuclei of the amygdala block fear-potentiated startle and shock sensitization of startle. Behav Neurosci. 1992;106:72–80. [PubMed]
  • Saria A. The tachykinin NK1 receptor in the brain: pharmacology and putative functions. European Journal of Pharmacology. 1999a;375:51–60. [PubMed]
  • Saria A. The tachykinin NK1 receptor in the brain: pharmacology and putative functions. Eur J Pharmacol. 1999b;375:51–60. [PubMed]
  • Severini C, Improta G, Falconieri-Erspamer G, Salvadori S, Erspamer V. The tachykinin peptide family. Pharmacol Rev. 2002;54:285–322. [PubMed]
  • Siegel RA, Duker EM, Pahnke U, Wuttke W. Stress-induced changes in cholecystokinin and substance P concentrations in discrete regions of the rat hypothalamus. Neuroendocrinology. 1987;46:75–81. [PubMed]
  • Smith DW, Hewson L, Fuller P, Williams AR, Wheeldon A, Rupniak NMJ. The substance P antagonist L-760,735 inhibits stress-induced NK1 receptor internalisation in the basolateral amygdala. Brain Research. 1999;848:90–95. [PubMed]
  • Smith G, Harrison S, Bowers J, Wiseman J, Birch P. Non-specific effects of the tachykinin NK1 receptor antagonist, CP-99,994, in antinociceptive tests in rat, mouse and gerbil. Eur J Pharmacol. 1994;271:481–487. [PubMed]
  • Szeidemann Z, Jakab RL, Shanabrough M, Leranth C. Extrinsic and intrinsic substance P innervation of the rat lateral septal area calbindin cells. Neuroscience. 1995;69:1205–1221. [PubMed]
  • Teixeira RM, Santos AR, Ribeiro SJ, Calixto JB, Rae GA, De Lima TC. Effects of central administration of tachykinin receptor agonists and antagonists on plus-maze behavior in mice. Eur J Pharmacol. 1996;311:7–14. [PubMed]
  • Varty GB, Cohen-Williams ME, Morgan CA, Pylak U, Duffy RA, Lachowicz JE, Carey GJ, Coffin VL. The gerbil elevated plus-maze II: anxiolytic-like effects of selective neurokinin NK1 receptor antagonists. Neuropsychopharmacology. 2002a;27:371–379. [PubMed]
  • Varty GB, Morgan CA, Cohen-Williams ME, Coffin VL, Carey GJ. The gerbil elevated plus-maze I: behavioral characterization and pharmacological validation. Neuropsychopharmacology. 2002b;27:357–370. [PubMed]
  • Winslow JT, Noble PL, Davis M. Modulation of Fear-Potentiated Startle and Vocalizations in Juvenile Rhesus Monkeys by Morphine, Diazepam, and Buspirone. Biological Psychiatry. 2007;61:389–395. [PubMed]
  • Woolley ML, Haman M, Higgins GA, Ballard TM. Investigating the effect of bilateral amygdala lesions on fear conditioning and social interaction in the male Mongolian gerbil. Brain Research. 2006;1078:151–158. [PubMed]
  • Young BJ, Helmstetter FJ, Rabchenuk SA, Leaton RN. Effects of systemic and intra-amygdaloid diazepam on long-term habituation of acoustic startle in rats. Pharmacol Biochem Behav. 1991;39:903–909. [PubMed]