The Discriminative Stimulus Effects of the Neurotensin NTS 1 Receptor Agonist PD149163 in Rats:...

Discriminative stimulus effects of PD149163 1

RUNNING HEAD: Discriminative stimulus effects of PD149163

The discriminative stimulus effects of the neurotensin NTS1 receptor agonist PD149163 in rats: Stimulus

generalization testing with dopamine D1 and D2 receptor ligands.

Adam J. Prus*, Candice J. Schuck, Kristoffer R. Rusch, and Lawrence M. Carey

Department of Psychology, Northern Michigan University, Marquette, MI

*Corresponding author

Department of Psychology Northern Michigan University 1401 Presque Isle Ave Marquette, MI 49855 Email: [email protected] Phone: 906-227-2941 Fax: 906-227-2954

Funding

Experiment 1 of this study was supported by an NIH grant to AJP (1R15MH083241).

Pages and Figures

Abstract + Intro + Methods + Results + Discussion + References = 18 pages

Figures = 7 figures

Copyright Wiley-Blackwell. DOI: 10.1002/ddr.21171


ABSTRACT

Brain-penetrant neurotensin NTS1 receptor agonists produce antipsychotic drug-like effects in

animal models, including inhibition of conditioned avoidance responding and reversal of psychostimulant-

induced hyperactivity and stereotypy. Allosteric interactions between NTS1 receptors and dopamine D2

receptors may account for some of these antipsychotic effects. In order to determine the role that

dopamine receptors may play in the behavioral effects produced by activation of NTS1 receptors, a drug

discrimination approach was used in rats to evaluate the potential mediation of NTS1 receptor agonist

stimulus effects by dopamine D1 and D2 receptors. Rats were trained to discriminate either the NTS1

receptor agonist PD149163, the D1 receptor agonist SKF81297, or the D2 receptor agonist quinpirole from

vehicle in a two choice drug discrimination task. Full stimulus generalization occurred from PD149163 to

the typical antipsychotic drug and D2 receptor-preferring antagonist haloperidol. However, stimulus

generalization did not occur from SKF81297 or quinpirole to PD149163. The discriminative cue for

SKF91297 and quinpirole was fully blocked the D1 receptor antagonist SCH23390 and the D2/3 receptor

antagonist raclopride, respectively. Cross generalization did not occur between SKF91297 and quinpirole.

Based on these findings, the antipsychotic effects of PD149163 may be mediated, in part, through D2

receptor antagonism, but this may only be evident when PD149163 is used as the training drug.

KEYWORDS: Neurotensin, PD149163, quinpirole, SKF81297, raclopride, SCH23390, antipsychotic,

drug discrimination


INTRODUCTION

Neurotensin is a neuropeptide neurotransmitter that has been evaluated as a pharmacological

target for treating schizophrenia [Tanganelli et al. 2012], anxiety disorders [Prus et al. 2014],

neurocognitive impairment [Azmi et al. 2006; Feifel et al. 1999], and drug addiction [Fredrickson et al.

2005]. The behavioral effects of neurotensin are primarily derived from activation of neurotensin NTS1

receptors, and the emergence of brain-penetrant NTS1 receptor agonists, particularly including NT69L

[Cusack et al. 2000] and PD149163 [Petrie et al. 2004], has stimulated dozens of studies on the putative

therapeutic efficacy of these drugs for mental disorders. NTS1 receptor agonists have consistently

engendered atypical antipsychotic-like effects in animal models, including an ability to attenuate

psychotomimetic-induced deficits in pre-pulse inhibition [Feifel et al. 1999; Shilling et al. 2003], inhibit

conditioned avoidance responding without eliciting catalepsy [Holly et al. 2011a], reverse

psychostimulant-induced hyperactivity and stereotypy [Cusack et al. 2000; Feifel et al. 2008], and

preferentially enhance dopamine concentrations in the prefrontal cortex compared to the nucleus

accumbens [Prus et al. 2007]. There is also emerging evidence that NTS1 receptor agonists have

anxiolytic effects based on the ability of systemically administered PD149163 to reduce fear-potentiated

startle responses [Shilling and Feifel 2008] and footshock-conditioned 22 kHz vocalizations [Prus et al.

2014] in rats, and that NTS1 receptor agonists may treat nicotine addiction, based upon the ability of

NT69L to reduce nicotine self-administration in rats [Boules et al. 2011].

While neurotensin NTS1 receptors have been found postsynaptically on GABA, serotonin, and

cholinergic neurons, these receptors are particularly located on dopamine neurons, including postsynaptic

localization on ventral tegmental area and substantia nigra dopamine neurons and presynaptic

localization on dopamine terminals in nucleus accumbens, prefrontal cortex, and striatum [Binder et al.

2001; St-Galais et al. 2006]. Neurotensin is also synthesized and released from mesocorticolimbic and

nigrostriatal dopamine neurons. When neurotensin binds to presynaptic NTS1 receptors, these receptors

inhibit D2 autoreceptor functioning through either an allosteric mechanism or by reducing the ability the

receptor to be bound by dopamine [Binder et al. 2001; St-Galais et al. 2006; Tanganelli et al. 2012].

Inhibition of D2 autoreceptors causes increased dopamine efflux and activation of postsynaptic receptors

of the D1 or D2 receptor family [Beaulieu and Gainetdinov 2011]. The findings that the NTS1 receptor is


associated with three different G-proteins (Gq/11, Gi/o, and Gs) and that selective activation of any of these

G-proteins may occur depending on a ligand’s binding site on the receptor provides an additional facet of

complexity for understanding the mediation of behavioral effects by NTS1 receptor agonists [Tanganelli et

al. 2012].

Drug discrimination procedures have long been used to evaluate pharmacological actions that

may mediate a drug’s interoceptive stimulus effects [Porter and Prus 2009]. The procedure has a

remarkable degree of specificity for the stimulus effects elicited by a training drug (i.e., discriminative

stimulus effects, in this context) as indicated by stimulus generalization selectively occurring from the

training drug to compounds that elicit these same effects and by blockade of these discriminative stimulus

effects by compounds with opposing pharmacological actions (e.g., an antagonist). The results from

generalization testing highly correspond to the shared receptor binding affinities of the training and test

compounds.

The present study sought to determine the role that dopamine D1 and D2 receptors may play in

the stimulus effects of a NTS1 receptor agonist by using a two-choice drug discrimination procedure in

rats to assess stimulus generalization occurring between the NTS1 receptor agonist PD149163 and

compounds selective for dopamine D1 or D2 receptors. The first experiment employed PD149163 as the

training drug in one group of rats and the second experiment employed a D1 receptor agonist and a D2

receptor agonist as training drugs in separate groups of rats. The study’s findings provide some tentative

support for mediation of PD149163’s discriminative stimulus effects by D2 receptor antagonism.

METHODS AND MATERIALS

Experiment 1

Subjects

Ten male Sprague Dawley rats (Charles River, Portage, Michigan, USA) obtained at

approximately 90 days old were used and were housed in a temperature and humidity controlled vivarium

under a 12 hour light/dark schedule. Rats were individually housed and with free access to water, but with

restricted access to food in order to maintain 85% of free-feeding weight. All procedures were approved

by the Northern Michigan University Institutional Animal Care and Use Committee and followed the Guide

for the Care and Use of Laboratory Animals [2011].


Apparatus

Six rat operant chambers equipped with two retractable levers, a food receptacle, and 45mg

pellet dispenser were used for all drug discrimination procedures (Med-Associates Inc., St. Albans,

Vermont, USA). A lever was located equidistantly on each side of the food pellet receptacle. The

chambers were housed in sound-attenuating cubicles with fans for ventilation and masking noise. The

chambers were controlled and data were recorded using Med-PC version 4 software (Med-Associates

Inc.).

Drugs

PD149163 (NIMH Chemical Synthesis and Drug Supply Program, National Institute of Mental

Health, Bethesda, Maryland, USA) and haloperidol HCl (Tocris, Ellisville, Missouri, USA) were

administered in 1.0 ml/kg volume. PD149163 and haloperidol were in salt form. PD149163 was

dissolved in sterile water and haloperidol HCl was dissolved in sterile water with a few drops of lactic acid.

The doses were determined based upon behaviorally effective doses used in previous studies [e.g., Feifel

et al. 1999; Holly et al. 2011b; Prus et al. 2004]. PD149163 was injected subcutaneously and haloperidol

was injected intraperitoneally 30 minutes prior to a session.

Drug Discrimination Training

After lever press training to fixed ratio (FR) 20 reinforcement schedule, 5 consecutive training

sessions were conducted with vehicle and with only the vehicle-appropriate lever present. Following this,

consecutive training sessions were conducted with PD149163 and with only the PD149163-lever present.

This initial phase of PD149163 lever training was planned for 5 training sessions, but response inhibition

by PD149163 led to lowering the training dose during this period (see results for experiment 1).

Following single-lever training sessions, two-lever drug discrimination training began. Vehicle (V)

and PD149163 (D) training days were assigned based on a single-double alternation pattern (i.e.,

VDVVDVDDV etc.). During two-lever training, every lever press reset the FR20 counter for the opposite

lever. Subjects were tested when the following criteria were met for 5 out of 6 consecutive training

sessions: 1) first FR20 occurring on the condition-appropriate lever, 2) 80% or greater condition-

appropriate responding during the first FR20, 3) 80% or greater condition-appropriate responding for the

total session, and 4) at least 0.1 responses per second for the session.


Before every test session, subjects had to meet the above four criteria for both a vehicle and a

PD149163 training session. Test sessions ended after an FR20 occurred on either lever; otherwise, the

session ended after 20 minutes. No food pellet was delivered during or after a test session. As with

training sessions, every lever press reset the FR20 counter on the opposite lever.

Data Analysis

Percent PD149163-lever responding was expressed as a percentage based on the number of

PD149163-appropriate responses emitted divided by the total number of PD149163- and vehicle-

appropriate responses emitted. In order to be included in the percent PD149163-appropriate responding

calculation for a test session, rats were required to emit at least 10 responses. Percent PD149163-lever

responding and responses per second were reported as means +/- the standard error of the mean (SEM).

Full stimulus generalization from the PD149163 discriminative stimulus was defined as 80% or greater

drug-appropriate responding and partial stimulus generalization was defined as 60-80% drug-appropriate

responding. Statistically significant differences in response rates across doses and vehicle control were

determined using a repeated measures one way analysis of variance (ANOVA). Statistically significant

differences were followed by a Dunnett’s post hoc test for comparison to vehicle control. A nonlinear

regression analysis was used to calculate the ED50 dose (+/- 95% confidence intervals [CI]) for percent

PD149163-appropriate responding for drugs that produced full stimulus generalization. All analyses were

conducted using GraphPad Prism for Windows, version 5.02 (GraphPad Software, San Diego, California,

USA).

Experiment 2

Animals

A total of 20 male Sprague Dawley rats were used in experiment 2, with 10 animals assigned per

group. The housing procedures were identical to those described in experiment. All procedures in

experiment 2 were approved by the IACUC at Northern Michigan University and followed the Guide to the

Care and Use of Laboratory Animals [2011].

Drugs

The drugs assessed in experiment 1 were the D1 receptor agonist SKF81297 (0.15-1.2mg/kg;

generously provided by the NIMH Drug Repository), D2 receptor agonist quinpirole hydrochloride (0.0125-


0.1mg/kg; NIMH Drug Repository), the D1 receptor antagonist SCH23390 (0.001875-0.015mg/kg; Sigma-

Aldrich), the D2 receptor antagonist raclopride (0.2-0.8mg/kg; Sigma-Aldrich), and PD149163 (0.0030125-

0.15mg/kg; NIMH Drug Repository). All drugs were in salt form and every drug was dissolved in saline,

which served as the vehicle in this experiment. Drug injections were given subcutaneously 30 min prior to

training or testing sessions. Doses were chosen based on previous studies [Gleason and Witkin 2006;

Reavill et al. 1993; Weathersby and Appel 1986].

Apparatus

The equipment and data collection procedures were identical to those described for experiment 1.

Drug Discrimination Training and Testing

Two groups of rats (n=10 each) were used for experiment 1, with one group of rats trained to

discriminate the D1 receptor agonist SKF81297 (0.3 mg/kg) from vehicle and the other group of rats

trained to discriminate the D2 receptor agonist quinpirole (0.025 mg/kg) from vehicle. Rats were trained

on a fixed ratio 30 reinforcement schedule, but otherwise, training and testing procedures were identical

to experiment 1.

Data Analysis

In order to be included in the percent drug-lever responding calculation for a test session, rats were

required to emit at least 15 responses. Otherwise these data analysis procedures were identical to

experiment 1.

RESULTS

Experiment 1

Training

The initial training dose of PD149163, 0.5mg/kg, completely abolished responding during single

lever PD149163 training sessions. A subsequent reduction to 0.25 mg/kg was less disruptive but still

precluded adequate responding for training. These first failed attempts to identify a non-disruptive

PD149163 training dose led to a short series of dose and route adjustments until a 0.0625 mg/kg doses,

administered, was utilized. Six of the ten rats met the training criteria for the 0.0625 mg/kg training dose.

In total, 121 mean training sessions were required to meet the training criteria. Poorly maintained

performance at the training criteria limited stimulus generalization testing to only PD149163 and


haloperidol. Three of the four remaining rats were subsequently trained using a 0.03125 mg/kg dose and

met training criteria after 212 mean training sessions. However, these three rats failed to maintain the

training criteria, and therefore stimulus generalization data are unavailable for these subjects.

PD149163 testing in PD149163 trained rats

PD149163 produced full stimulus generalization to itself at the 0.0625 mg/kg training dose as well

as the 0.125 mg/kg dose (ED50= 0.04 mg/kg, 95% confidence-interval (CI)= 0.03- 0.05 mg/kg; figure 1a).

Full stimulus generalization did not occur to the 0.25 mg/kg dose, but this was largely influenced by one

rat (out of only three responding at this dose) that selected the vehicle lever. A statistically significant

reduction in response rates was shown (F[5,25]= 7.63, p<0.001; figure 1b). Multiple comparisons testing

revealed that these reductions occurred at the 0.0625, 0.125, and 0.25 mg/kg doses compared to vehicle.

Haloperidol testing in PD149163 trained rats

Full stimulus generalization occurred to the 0.1 mg/kg dose of haloperidol (ED50 = 0.05 mg/kg,

95% CI= 0.03-0.08 mg/kg; figure 2a). Haloperidol significantly reduced response rates (F[4,16]= 6.41,

p<0.01; figure 2b) at the 0.05, 0.1, and 0.2 mg/kg doses compared to vehicle. Due to rate suppression,

two of five rats emitted enough responses to be included in the percent PD149163-appropriate

responding calculation for the 0.1 mg/kg dose and three of five rats were included in the calculation for

the 0.2 mg/kg dose.

Experiment 2

Training

The mean number of training sessions needed to meeting the training criteria was 42.6 sessions

in the group of rats trained to discriminate SKF81297 from vehicle and 26.0 sessions in the group of rats

trained to discriminate quinpirole from vehicle. Four rats from each group failed to meet the training

criteria within 200 training sessions and were removed from the study, leaving six rats each group.

SKF81297 testing in SKF81297 trained rats

SKF81297 (0.15 to 1.2 mg/kg) led to full stimulus generalization to a 0.3 and 0.6 mg/kg dose in

rats trained to discriminate a 0.3 mg/kg dose of SKF81297 from vehicle, while partial stimulus

generalization occurred to a 1.2 mg/kg dose (ED50 = 0.2 mg/kg, 95% confidence interval [CI] = 0.12 to

0.34 mg/kg; figure 3a). SKF81297 also produced a statistically significantly reduction in response rates


(F[4,20]=7.02, p<0.01), which was found for a 0.3, 0.6, and 1.2 mg/kg dose compared to vehicle (figure

3c).

Quinpirole testing in quinpirole trained rats

Quinpirole (0.0125 – 0.1 mg/kg) produced full stimulus generalization to a 0.025 and 0.05 mg/kg

dose (ED50=0.007mg/kg, 95% CI = 0.002 to 0.018 mg/kg) and produced partial stimulus generalization to

a 0.1 mg/kg dose, although 4 out of 6 of the rats produced greater than 90% responding on the quinpirole

lever (figure 3b). Quinpirole also produced a significant decrease in response rates (F[4,20]=3.72,

p<0.05), which occurred at the 0.1 mg/kg dose compared to vehicle (figure 3d).

PD149163 testing in SKF81297 trained rats

PD149163 did not produce stimulus generalization from SKF81297, with the highest level of

generalized responding (32.15%) occurring to a 0.1 mg/kg dose (figure 4a). No animals produced full

generalized responding on the SKF81297 lever for any of the doses tested. PD149163 significantly

reduced response rates (F[4,20]=9.37, p<0.0001), which occurred at a 0.075, 0.1 and 0.15 mg/kg dose

compared to vehicle (figure 4c).

PD149163 testing in quinpirole trained rats

PD149163 neither engendered partial nor full generalized responding from quinpirole with the

highest level of generalized responding (42.24%) occurring to a 0.075 mg/kg dose (figure 4b). Two of the

five rats that responded at the 0.075 mg/kg dose emitted full generalized responding on the quinpirole

lever. PD149163 significantly reduced response rates (F[4,20]=8.82, p<0.0001), which occurred at a

0.075, 0.1 and 0.15 mg/kg dose compared to vehicle (figure 4d).

PD149163 + SKF81297 testing in SKF81297 trained rats

PD149163 did not produce partial or full blockade of the SKF81297 discriminative stimulus (figure

5a). Response rates did differ across the dose combinations of PD149163 and SKF81297 (figure 5c).

PD149163 + quinpirole testing in quinpirole trained rats

PD149163 did not fully block the quinpirole discriminative stimulus, although a 0.0125 mg/kg

dose of PD149163 did reduce quinpirole lever responding (78.6%) to slightly below the 80% criterion for

full stimulus generalization (figure 5b). Combination testing with these compounds did not lead to

statistically significant changes in response rates (figure 5d).


SCH22390+SKF81297 testing in SKF81297 trained rats

The D1 receptor antagonist SCH23390 (0.001875 – 0.015 mg/kg) produced full blockade of the

SKF81297 discriminative cue, which occurred after administration of a 0.00375, 0.075, and 0.015 mg/kg

dose (figure 6a). A statistically significant increase in response rates occurred (F[4,20], =5.90, p<0.05),

which was found at a 0.001875 mg/kg and 0.015 mg/kg dose compared to vehicle (figure 6c).

Raclopride + quinpirole testing in quinpirole trained rats

The D2 receptor antagonist raclopride (0.2 – 0.8 mg/kg) produced full blockade of the quinpirole

discriminative cue, which occurred after administration of a 0.4 and 0.8 mg/kg dose of raclopride (figure

6b). A statistically significant decrease in response rates occurred (F[3,15] =4.34, p<0.05), which was

found at a 0.8 mg/kg dose compared to vehicle (figure 6d).

Quinpirole testing in SKF81297 trained rats

Quinpirole (0.0125 – 0.05 mg/kg) did not produce stimulus generalization from the SKF81297

discriminative cue (figure 7a). A significant reduction in response rates was found during quinpirole

testing (F[3,9]=6.75, p<0.05), which occurred at the 0.05 mg/kg dose compared to vehicle (figure 7c).

SKF81297 testing in quinpirole trained rats

SKF81297 (0.15-0.6 mg/kg) did not produce stimulus generalization from the quinpirole

discriminative cue (figure 7b). A significant reduction in rates occurred during SKF81297 testing

(F[3,9]=4.77, p<0.05), although a post hoc test failed to reveal a dose that produced statistically

significant differences in response rates compared to vehicle (figure 7d). The inability to find statistical

differences using a post hoc test is most likely due to the low sample size (N=4) that was available at this

later part of the study.

DISCUSSION

This is the first known report on the discriminative stimulus effects of a NTS1 receptor ligand.

Experiment 1 demonstrated that the NTS1 receptor agonist PD149163 could be established as a

discriminative stimulus in a drug discrimination procedure. Full stimulus generalization occurred from the

training dose of PD149163 to PD149163 and to the D2 receptor-preferring antagonist and typical

antipsychotic drug haloperidol. In experiment 2, PD149163 was neither found to produce stimulus


generalization from the D1 receptor agonist SKF81297 nor the D2 receptor agonist quinpirole. The

selectivity of the discriminative stimulus effects of SKF81297 and quinpirole for D1 and D2 receptors,

respectively, was demonstrated by full stimulus blockade by antagonists for these receptors and by a lack

of cross generalization between these training drugs.

While PD149163 was established as a discriminative stimulus in experiment 1, these stimulus

effects were not robust. Only 6 of the 10 rats met the training criteria after a lengthy number of sessions,

and the discriminative stimulus effects of PD149163 could not be maintained after stimulus generalization

testing with PD149163 and haloperidol was completed. As reviewed in the introduction, NTS1 receptor

agonists likely functionally antagonize D2 receptors, and therefore the discriminative stimulus effects of a

NTS1 receptor agonist may in part be mediated by D2 receptor antagonism, which would account for

stimulus generalization to haloperidol. D2 receptor antagonism is a central pharmacological mechanism

for antipsychotic drugs, and therefore, this is a potential mechanism through which NTS1 receptor

agonists may engender antipsychotic-like effects in preclinical models. However, D2 receptor antagonism

may only mediate the stimulus properties of PD149163 when this compound is used as a training drug,

since D2 receptor antagonism was not apparent when PD149163 was tested in the quinpirole-trained rats.

However, response suppression caused by PD149163 precluded higher testing doses that were found to

produce full stimulus generalization from PD149163 in experiment 1. Thus, the lack of stimulus blockade

of quinpirole by PD149163 may be due to testing doses that were too low to adequately counteract these

stimulus effects.

Antipsychotic drugs have been thoroughly evaluated in drug discrimination procedures.

Generally, atypical antipsychotic drugs, which exhibit an extensive binding profile for monoaminergic and

cholinergic receptors, are readily established and robustly maintained as discriminative stimuli (i.e., as

training drugs in drug discrimination procedures). For example, the atypical antipsychotic drugs clozapine

[Porter et al. 2005; Prus et al. 2004; Prus et al. 2005], olanzapine [Porter and Strong 1996], and

quetiapine [Smith and Goudie 2002] have been established as discriminative stimuli in rats. On the other

hand, typical antipsychotic drugs, which engender a more selective receptor profile that primarily involves

D2 receptor antagonism, are less readily established as discriminative stimuli in rats.


While the typical antipsychotic drug chlorpromazine, which exhibits a more general

monoaminergic receptor binding profile than most other typical antipsychotic drugs [Kroeze et al. 2003],

can be readily established as a discriminative stimulus in rats [Goas and Boston 1978; Porter et al. 1998],

haloperidol establishes a weaker discriminative stimulus. Colpaert [1976] was one of the first to establish

a haloperidol discriminative stimulus in rats but did so with difficulty, requiring a dose reduction from a

0.04 mg/kg to 0.02 mg/kg training dose. Subsequently, a haloperidol discriminative stimulus was

established after 80 mean training sessions. McElroy et al. [1989] later trained rats to discriminate

haloperidol 0.04 mg/kg from vehicle in a mean of 45 sessions and found that full stimulus generalization

occurred to chlorpromazine and that the haloperidol discriminative stimulus was blocked by cocaine and

d-amphetamine. Wiley and Porter [1993] also attempted to train rats to discriminate haloperidol from

clozapine but found that pharmacologically dissimilar drugs produced stimulus generalization from

haloperidol. These authors concluded that haloperidol was treated as the “other” condition (similar to a

vehicle condition in most two-lever drug discrimination studies) versus clozapine, rather than an

independent discriminative stimulus. If a PD149163 discriminative stimulus were mediated in a significant

way by D2 receptor antagonism, then this may account for the difficulty in establishing and maintaining

these stimulus effects.

The training data for SKF81297 and quinpirole confirm previous studies that these compounds

can be established as discriminative stimuli. Reavill et al. [1993] first established SKF81297, at a 0.1 and

0.2 mg/kg dose, as a discriminative stimulus in rats and reported full stimulus to SKF91297 up to a 0.4

mg/kg dose. In the present study, the 0.3 mg/kg training dose of SKF81297 produced full stimulus

generalization to a 0.3 and 0.6 mg/kg dose and partial stimulus generalization to a 1.2 mg/kg dose. The

lack of full stimulus generalization to a 1.2 mg/kg dose of SKF81297 was likely due to rate disruptive

effects or other nonspecific behavioral effects (e.g., impaired working memory [Zahrt et al. 1997]) of this

dose. The discriminative stimulus effects of quinpirole have been established in previous studies [Baladi

et al. 2010; Weathersby and Appel 1986] and appear to be primarily mediated by agonism of both D2 and

D3 receptors [Baladi et al. 2010]. In the present study, full stimulus generalization occurred from the 0.025

mg/kg training dose of quinpirole to a 0.025 and 0.05 mg/kg dose and partial generalization occurred to a

0.1 mg/kg dose. The reduction to partial generalization at a 0.2 mg/kg dose of quinpirole may be have


been due to rate disruptive effects of this dose. The selectivity of the discriminative stimulus effects of

SKF81297 and quinpirole for D1 and D2 receptors, respectively, was supported by full stimulus blockade

by antagonists for these receptors and by a lack of cross generalization between SKF81297 and

quinpirole.

While generalization from a D2 receptor agonist to PD149163 was not considered likely based on

past molecular and behavioral studies, the disinhibition of dopamine release caused by D2 autoreceptor

inhibition was thought to possibly lead to postsynaptic D1 receptor binding [Binder et al. 2001]. Stimulus

generalization testing with PD149163 indicated a lack of D1 receptor or D2 receptor agonism for the

stimulus effects of PD149163. PD149163 significantly disrupted responding in both the SKF81297- and

quinpirole-trained rats, although fewer animals responded to higher doses of PD149163 in the

SKF81297-trained rats than in the quinpirole-trained rats. A 0.0125 mg/kg dose of PD149163 reduced

responding on the quinpirole lever to below the full stimulus generalization criterion. These effects might

be reflective of D2 receptor antagonism by PD149163, but if this is true, the effects are less pronounced

than were found in experiment 1, in which PD149163 was used as a discriminative stimulus.

In conclusion, the present study provided tentative evidence for the mediation of the

discriminative stimulus effects of PD149163 by dopamine D2 receptor antagonism, although these effects

were most pronounced when PD149163 was used as the training drug. These findings are consistent

with the putative antipsychotic effects found in preclinical models, although the atypical antipsychotic

features of NTS1 receptor agonists suggest that they may involve more than antagonism of D2 receptors.

Currently available NTS1 receptor antagonists (i.e., SR14297 and SR48692) have poor solubility and

therefore precluded their systemic use in this study to determine if NTS1 receptor antagonism may block

the discriminative stimulus effects of PD149163. Moreover, other NTS1 receptor agonists, which could be

used in stimulus generalization testing, are not commercially available. Further elucidation of the

discriminative stimulus effects of NTS1 receptor agonists will be improved upon the availability of other

brain penetrant NTS1 receptor ligands and with stimulus generalization testing conducted with ligands for

other receptors (e.g., receptors important for atypical antipsychotic drugs, such as 5-HT2A receptors) that

may be important for the behavioral effects of neurotensin.


ACKNOWLEDGEMENT

PD149163 was generously provided by the NIMH Chemical Synthesis and Drug Supply Program.

Experiment 1 of this study was supported by an NIH grant to AJP (1R15MH083241). The authors thank

Alexis Bench and Casey Cook for their technical assistance.

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FIGURES

Figure 1. The neurotensin NT1 receptor agonist PD149163 was tested for stimulus generalization in rats

trained to discriminate PD149163, 0.0625 mg/kg, from vehicle (VEH) in a two-choice drug discrimination

task. Mean (+/- SEM) percent drug lever responding is shown in panel A and the mean (+/-) responses

per second is shown in panel B. The sample size is shown in parentheses; otherwise, N=6. The dashed

line indicates the full stimulus generalization criterion. *P<0.05, **P<0.01, ***P<0.001 versus VEH

control.

Figure 2. Stimulus generalization results for the typical antipsychotic drug and D2-receptor preferring

antagonist haloperidol (N=5). See figure 1 for further details.

Figure 3. Stimulus generalization testing with the D1 receptor agonist SKF81297 in rats trained

discriminate SKF81297 (0.3 mg/kg) from VEH (panels A and C) and with the D2 receptor agonist

quinpirole in rats trained to discriminate quinpirole (0.025 mg/kg) from VEH (panels B and D) in a two

choice drug discrimination task. Mean percent (+/- SEM) lever responding for SKF81297 and quinpirole

are shown in panels A and B, respectively, and the mean responses per second (+/- SEM) for SKF81297

and quinpirole are shown in panel C and D, respectively. **P<0.01 and ***P<0.001 versus VEH control.

Figure 4. Stimulus generalization testing with PD149163 in SKF81297- (panel A, percent SKF81297-

appropriate responding; panel C, responses per minute) and quinpirole-trained rats (panel B, quinpirole-

appropriate responding; panel D, responses per minute). *P<0.05, **P<0.01, ***P<0.001, and

****P<0.0001 versus VEH control. See figure 3 for further details.

Figure 5. Generalization testing with PD149163 in combination with SKF81297 (0.3 mg/kg) in

SKF81297-trained rats (A & C) and in combination with quinpirole (0.025 mg/kg) in quinpirole-trained rats

(B & D). See figure 3 for further details.


Figure 6. Generalization testing with either the dopamine D1 receptor antagonist SCH23390 in

combination with SKF81297 (0.3 mg/kg) in SKF81297-trained rats (A & C) and the dopamine D2/D3

receptor antagonist raclopride in combination with quinpirole (0.025 mg/kg) in quinpirole-trained rats (B &

D). See figure 3 for further details.


Figure 1

VEH 0.016 0.031 0.0625 0.13 0.25

0

20

40

60

80

100

Dose (mg/kg)

% P

D14

9163

Lev

er R

espo

ndin

g

(3)

N=6 (5)

VEH 0.016 0.031 0.0625 0.13 0.25

0.0

0.5

1.0

1.5

2.0

Dose (mg/kg)

Res

pons

es P

er S

econ

d

** ****

PD149163A.

B.


Figure 2

VEH 0.025 0.05 0.1 0.2

0

20

40

60

80

100

Dose (mg/kg)

% P

D14

9163

Lev

er R

espo

ndin

g

(3)

N=5 (2)

VEH 0.025 0.05 0.1 0.2

0.0

0.5

1.0

1.5

2.0

Dose (mg/kg)

Res

pons

es P

er S

econ

d

*

** **

HaloperidolA.

B.


Figure 3

VEH 0.15 0.3 0.6 1.2

0

20

40

60

80

100

Dose (mg/kg)

% S

KF8

1297

Lev

er R

espo

ndin

gN=6

VEH 0.0125 0.025 0.05 0.1

0

20

40

60

80

100

% Q

uinp

irole

Lev

er R

espo

ndin

g

Dose (mg/kg)

N=6

VEH 0.15 0.3 0.6 1.2

0.0

0.5

1.0

1.5

2.0

Res

pons

es P

er S

econ

d

Dose (mg/kg)

****

***

VEH 0.0125 0.025 0.05 0.1

0.0

0.5

1.0

1.5

2.0

Res

pons

es P

er S

econ

d

Dose (mg/kg)

**

SKF81297 QuinpiroleA. B.

C. D.


Figure 4

Dose (mg/kg)

% S

KF8

1297

Lev

er R

espo

ndin

g

VEH 0.05 0.075 0.1 0.15

0

20

40

60

80

100

N=6

(5) (5)

(2) (2)

VEH 0.05 0.075 0.1 0.15

0

20

40

60

80

100

Dose (mg/kg)

% Q

uinp

irole

Lev

er R

espo

ndin

g

N=6

(5) (5)

(1)

VEH 0.05 0.075 0.1 0.15

0.0

0.5

1.0

1.5

2.0

Dose (mg/kg)

Mea

n R

espo

nses

Per

Sec

ond

**

*******

VEH 0.05 0.075 0.1 0.15

0.0

0.5

1.0

1.5

2.0

Dose (mg/kg)

Mea

n R

espo

nses

Per

Sec

ond

** **

***

PD149163 PD149163A. B.

C. D.


Figure 5

VEH 0.025 0.05 0.075

0

20

40

60

80

100

Dose (mg/kg)

% S

KF8

1297

Lev

er R

espo

ndin

g

N=6

(4)(3)

VEH 0.0030125 0.006125 0.0125

0

20

40

60

80

100

Dose (mg/kg)

% Q

uinp

irole

Lev

er R

espo

ndin

g

N=6

(5) (4)

(4)

VEH 0.025 0.05 0.075

0.0

0.5

1.0

1.5

2.0

Dose (mg/kg)

Mea

n R

espo

nses

Per

Sec

ond

VEH 0.0030125 0.006125 0.0125

0.0

0.5

1.0

1.5

2.0M

ean

Res

pons

es P

er S

econ

d

Dose (mg/kg)

PD149163+ 0.3mg/kg SKF81297 PD149163+ 0.025 mg/kg QuinpiroleA. B.

C. D.


Figure 6.

VEH 0.001875 0.00375 0.0075 0.015

0

20

40

60

80

100

Dose (mg/kg)

% S

KF8

1297

Lev

er R

espo

ndin

gN=6

VEH 0.2 0.4 0.8

0

20

40

60

80

100

% Q

uinp

irole

Lev

er R

espo

ndin

g

Dose (mg/kg)

(2) (1)

N=6

VEH 0.001875 0.00375 0.0075 0.015

0.0

0.5

1.0

1.5

2.0

Mea

n R

espo

nses

Per

Sec

ond

Dose (mg/kg)

*

*

*

VEH 0.2 0.4 0.8

0.0

0.5

1.0

1.5

2.0

Mea

n R

espo

nses

Per

Sec

ond

Dose (mg/kg)

SCH23390 + 0.3 mg/kg SKF81297 Raclopride + 0.025 mg/kg QuinpiroleA. B.

C. D.


Figure 7

VEH 0.0125 0.025 0.05

0

20

40

60

80

100

Dose (mg/kg)

% S

KF8

1297

Lev

er R

espo

ndin

g

VEH 0.15 0.3 0.6

0

20

40

60

80

100

Dose (mg/kg)

% Q

uinp

irole

Lev

er R

espo

ndin

g

VEH 0.0125 0.025 0.05

0.0

0.5

1.0

1.5

2.0

Dose (mg/kg)

Res

pons

es P

er S

econ

d

**

VEH 0.15 0.3 0.6

0.0

0.5

1.0

1.5

2.0

Dose (mg/kg)

Res

pons

es P

er S

econ

d

Quinpirole SKF81297A. B.

C. D.

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