The NCDDG proposes to ascertain the extent to which compounds that differentiallydisplay functional selectivity in a range of in vitro assays can be differentiated in a battery of in vivo tests thatpredict antipsychotic drug-like efficacy.The central role of dopamine in the pathophysiology of schizophrenia and in the modes of action ofclinically efficacious anti-psychotics has led to the development and common use of rodent behavioral modelsthat are highly predictive of anti-psychotic drug efficacy primarily as a result of blockade of dopamine D2receptors. Several behavioral animal models that are predictive of antipsychotic drug efficacy will be employedto profile the functionally selective compounds identified by the NCDDG, including conditioned avoidanceresponding, antagonism of apomorphine-induced climbing and stereotypy (AICS), blockade ofPCP/amphetamine induced locomotor activity, prepulse inhibition (PPI) of the acoustic startle response.Measurements of drug-induced catalepsy will be performed to assess the potential for extrapyramidal motorside-effects liability. Where appropriate, routine pharmacokinetic measurements will be employed in order toensure that the compound exposure is sufficient. Selective 5-HT2A antagonists such as M100907 have shownactivity in some, but not all, of these models. While M100907 is reported to reduce amphetamine and PCPstimulatedlocomotor activity (Sorensen et al., 1993; Maurel-Remy et al., 1995; Gleason and Shannon, 1997)and attenuate MK-801 induced prepulse inhibition deficits (Varty et al., 1999), the compound does not blockapomorphine-induced climbing (Sorensen et al., 1993) and there are no reports of efficacy in the conditionedavoidance response assay, despite reports of M100907 enhancing the efficacy of typical antipsychotics likehaloperidol (Wadenberg et al., 2001). These data underscore the potential for the selected behavioral assaysto further extend and contrast the antipsychotic profiles of compounds varying in effects on 5-HT2A receptorinternalization identified as part of Project 3. In addition, as many dopamine D2 partial agonists show activityin the preclinical models, these assays will be used to profile compounds with D2 functional selectivity profilesidentified in Project 1. These studies are proposed to complement the mechanistic studies proposed in Project2 that will employ transgenic mouse models of NMDA receptor hypofunction and dopamine hyperactivity.Conditioned avoidance responding. The conditioned avoidance responding model has been shown tobe highly predictive of anti-psychotic drug efficacy (Wadenburg and Hicks, 1999). The model is performedusing Shuttlebox test chambers (MED Associates, St Albans, VT) divided into two compartments by anarchway. Each chamber floor half is composed of thirteen 3/16' diameter stainless steel grid rods placed on1/2' centers wired for the presentation of an electric foot shock (0.5 mA). In addition, each side of the chamberis equipped with a stimulus light, tone generator and two infrared beam source/detectors used to locate the ratwithin the chamber. Rats trained to avoid the foot shock are placed in the chambers for a 4-minute habituationperiod followed by 50 trials presented on a 15-second variable interval schedule (range = 7.5 - 22.5 seconds).Each trial consists of a 10-second warning tone and stimulus light (conditioned stimulus) followed by a 10-second shock (unconditioned stimulus), presented through the grid floor on the side where the rat is located, inthe presence of the tone and Conditioned avoidance responding. The conditioned avoidance responding model has been shown tobe highly predictive of anti-psychotic drug efficacy (Wadenburg and Hicks, 1999). The model is performedusing Shuttlebox test chambers (MED Associates, St Albans, VT) divided into two compartments by anarchway. Each chamber floor half is composed of thirteen 3/16' diameter stainless steel grid rods placed on1/2' centers wired for the presentation of an electric foot shock (0.5 mA). In addition, each side of the chamberis equipped with a stimulus light, tone generator and two infrared beam source/detectors used to locate the ratwithin the chamber. Rats trained to avoid the foot shock are placed in the chambers for a 4-minute habituationperiod followed by 50 trials presented on a 15-second variable interval schedule (range = 7.5 - 22.5 seconds).Each trial consists of a 10-second warning tone and stimulus light (conditioned stimulus) followed by a 10-second shock (unconditioned stimulus), presented through the grid floor on the side where the rat is located, inthe presence of the tone and light. If an animal crosses hrough the archway during the nitial 10 seconds of the trial, theone and light are terminated and he response is considered an jvoidance response. If an animal Bosses through the archway after a foot shock is initiated, the one, light and shock are erminated and the response is considered an escape response, f a response is made during an nter-trial interval (ITI), the animal s punished with a 0.5-secondshock (0.5 mA). A MED ssociates computer with dEDSTATE NOTATION software controls the test session and counts thenumber of trials in which the animal avoids shock, escapes Only animals displaying stable performance (approximately 90% avoidance responding on the training session prior to test day) are considered 'trained' and included on test day. Training is maintained by at least one nondrugtest session each week. On test days, drugs are typically administered 30 minutes prior to testing.Clinically effective antipsychotics reduce avoidance behavior and increase escape behavior without increasingfailures to respond that are indicative of sedation (Fig. 1).Antagonism of apomorphine-induced climbing and stereotypy (AICS). Stimulation of dopaminergicneurotransmission by administration of the dopamine D1/D2 agonist, apomorphine, produces sets ofstereotypic behaviors indicative of activation of distinct dopaminergic neuronal pathways. Stimulation byapomorphine of mesolimbic DA receptors induces climbing behavior while activation of nigrostriatal DAreceptors produces a stereotypic syndrome of sniffing-licking-gnawing behaviors termed stereotypy (Costall, etal. 1975 and 1980). Testing of anti-psychotic In order to measure these behaviors, drugs are administeredthirty minutes prior to challenge with apomorphine (1 mg/kg sc). Five minutes after the apomorphine injection,the climbing behavior exhibited on a wire cage and stereotypy are scored and recorded for each animal.Readings are repeated every 5 minutes during a 30-minute test session. Scores for each animal are totaledfor each set of behaviors and expressed as a percent of control values observed in vehicle-treated mice. Inthis model, typical anti-psychotics block both climbing and stereotypy at similar doses while atypicalantipsychotics produce a block of climbing behavior at doses that do not inhibit stereotypy. As a result, thiscommonly used preclinical screen is useful for predicting efficacy in treating the positive symptoms ofschizophrenia and provides an indication of the potential for motoric side-effects liability (Fig. 2).PCP/Amphetamine induced locomotor activity. Amphetamine induced locomotor activity represents a modelfor hyperdopaminergia and has been commonly used to predict anti-psychotic efficacy (Arnt et al. 1997). Inthis model the amphetamine administration induces dopamine release resulting in increasedlocomotor activity.Antipsychotics exert their effects by competing for postsynaptic dopamine D2 receptors in the mesolimbicpathway reducing the induced locomotor response. In similar fashion, NMDA antagonists, e.g. phencyclidine(PCP), which stimulate locomotor activity, have also been employed to assess the impact of glutamatergichypofunction in schizophrenia (Coyle et al, 2003; Lewis et at 2003). Atypical antipsychotics have been shownto block hyperactivity induced with NMDA antagonists in rodents (Gleason and Shannon, 1997). In this modelthe capacity of anti-psychotic drugs to block spontaneous, amphetamine - induced and PGP - inducedlocomotor activity are compared. Drugs are administered to the mice concurrently with d-amphetamine (3mg/kg, sc), phencyclidine (3 mg/kg, sc) or vehicle. The animals are then immediately placed in locomotoractivity monitoring chambers for 70-minute tests during which time locomotor activity data is recorded underroom light and white noise using Accuscan infrared beam activity monitors with enclosed Plexiglas chambers(8 in. x 8 in.). Accuscan Versamax and Versadat software (Columbus, OH) are used to convert the infraredbeam breaks into horizontal activity counts in 5-minute bins (Fig. 3).Prepulse inhibition (PPI) of the acoustic startle response. PPI is a measure of sensorimotor gating that iscommonly used to characterize antipsychotic compounds. PPI is reduced in schizophrenia patients and inrodents treated with dopamine agonists or NMDA receptor antagonists (Geyer et al. 2001). The PPI responsein normal individuals is displayed when subjects are exposed to a startling auditory pulse a diminished startleresponse to a loud noise if it is preceded by a non-startling auditory tone (prepulse). Schizophrenic patientsshow a response whether stimulus is prepulse. Schizophrenic patients show aresponse whether stimulus is prepulse. Animals are subjected paradigm similar startle regardless of the startling preceded by a Animals are to the PPI using testing chambers (SR-LAB system, San Diego Instruments,San Diego, CA) that consist of a Plexiglas cylinder fitted with a piezoelectric accelerometer that detectsmovement of animal within the cylinder. A loudspeaker mounted above the cylinder provides backgroundwhite noise, acoustic noise bursts and acoustic prepulses. Presentation of acoustic pulse and prepulse stimuli are controlled by the SR-LAB software and interface system, which also digitizes, rectifies and records the responses from the accelerometer. Mean startle amplitude is determined by averaging 100 1-ms readings taken from the beginning of the pulse stimulus onset. Test sessions consist of 60 total trials with a 15 sec inter-trial interval. Following a 5 min acclimation to a 64 dB background noise, four trial types (120 dB pulse, or a 69, 74, or 79 dB prepulse paired with a 120 dBpulse) are presented in a pseudorandom order. Drug is typically administered 30 min prior to test withdisrupting agents, d-amphetamine (4 mg/kg, sc) and MK-801 (0.15 mg/kg, ip), administered afterwards.Prepulse inhibition is defined as 100-[(startle amplitude on prepulse trials/startle amplitude on pulse alonetrials) x 100] (Fig. 4). This model measures the ability of anti-psychotic compounds to reverse deficits inauditory sensorimotor gating in rats induced by the indirect DA agonist amphetamine and the NMDAantagonist MK-801.Catalepsy. Assessment of drug-induced catalepsy is widely used as a preclinical screen for potentialextrapyramidal motor side effect liability of anti-psychotic drug candidates (Hoffman and Donovan, 1995). Highlevels of blockade of dopamine D2 receptor, typically exceeding 80 %, results in catalepsy, which is defined asa lack of movement and maintenance of awkward posture. While typical anti-psychotic compounds producecatalepsy at doses comparable to their efficacious dose, atypical antipsychotic drugs exhibit a distinctseparation between cataleptogenic and efficacious doses in models predictive of antipsychotic activity. Inorder to measure the cataleptogenic potential of anti-psychotic compounds, animals are monitored forcataleptic behavior at 30-minute intervals for 2 hours post dosing with drug. The cataleptic behaviormeasurement consists of draping the forelegs of the treated animals a thin horizontal rod 1 3/4' high. Theamount of time (in seconds) for which the animal maintains this awkward position is recorded (60 secondmaximum). Maintenance of this position is considered catalepsy. Mean seconds spent in the catalepsyposition for each dose at each time point is calculated. (Fig. 5).
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