Tropisetron enhances recognition memory in rats chronically treated with risperidone or quetiapine

Abstract

While impairments of cognition in schizophrenia have the greatest impact on long-term functional outcome, the currently prescribed treatments, antipsychotic drugs (APDs), do not effectively improve cognition. Moreover, while more than 20 years have been devoted to the development of new drugs to treat cognitive deficits in schizophrenia, none have been approved to date. One area that has not been given proper attention at the preclinical or clinical stage of drug development is the chronic medication history of the test subject. Hence, very little is known about how chronic treatment with drugs that affect multiple receptors like APDs influence the response to a potential pro-cognitive agent. Therefore, the purpose of this study was to evaluate the α7 nicotinic acetylcholine receptor (α7 nAChR) partial agonist, tropisetron in rats chronically treated with APDs with distinct pharmacological profiles. Rats were treated orally with either risperidone (2.5 mg/kg/day) or quetiapine (25.0 mg/kg/day) for 30 or 90 days and then an acute injection of vehicle or tropisetron (3.0 mg/kg) was administered before training in a novel object recognition (NOR) task. After a 48 hr delay (when recollection of the familiar object was impaired in vehicle-treated animals) neither 30 nor 90 days of risperidone or quetiapine treatment improved NOR performance. In contrast, tropisetron markedly improved NOR performance in rats treated with either APD for 30 or 90 days. These animal data reinforce the argument that two commonly prescribed APDs are not pro-cognitive agents and that α7 nAChR ligands like tropisetron have potential as adjunctive treatments in schizophrenia.

Graphical abstract

1. Introduction

It has been known since the early 1900s from the pioneering work of Emil Kraepelin and Eugen Bleuler that impairments of cognitive function are among the most debilitating features of the condition now referred to as schizophrenia [1]. Cognitive deficits are evident at the onset of the psychotic (positive) symptoms and in contrast to the positive symptoms (which tend to be episodic), cognitive deficits generally persist throughout the course of the illness [2]. Moreover, compared to other symptom domains, impairments of cognition in schizophrenia have the greatest impact on key indicators of functional outcome including social skills, vocational achievements, and success in rehabilitation programs [3, 4, 5, 6].

While virtually all of the currently prescribed antipsychotic drugs (APDs) were developed to address the positive symptoms of schizophrenia (not cognitive deficits) there was some optimism beginning in the early 1990s that the compounds now known as the second-generation antipsychotics (SGAs) might improve cognition and that they were superior to the older first generation antipsychotics (FGAs) in this regard. Unfortunately, these early reports were not confirmed in subsequent randomized, double-blinded studies where acceptable dosing comparisons between SGAs and FGAs were made, and potential practice effects were considered [7, 8]. A variety of more recent studies, especially those where schizophrenia patients were followed over more extended periods of time, do not support the view that APDs (in general) significantly improve cognition in schizophrenia [9, 10, 11].

For over 20 years, drug discovery programs in both academia and industry have attempted to develop novel compounds to address the cognitive deficits of schizophrenia. Drugs designed to modulate a variety of proposed therapeutic targets have made it to clinical trials, however, none have been approved for use in schizophrenia patients to date. Both the preclinical research approaches and the clinical trial designs have been implicated in these treatment failures. On the preclinical side, the translational validity of the animal models has been criticized [12] while on the clinical side, the lack of consensus on clinical trial design to establish therapeutic efficacy of potential cognitive enhancing drugs has certainly been a barrier to successful drug development [13]. In 2004, a workshop on clinical trial design for evaluating cognitive enhancing drugs for schizophrenia was held and it included experts from the FDA, NIMH, as well as MATRICS investigators and scientists from academia and the pharmaceutical industry [13]. Some of the notable recommendations that came out of this workshop included limiting the trials to patients in the residual phase of their illness, who have a predefined level of positive, negative, and affective symptoms. Moreover, some guidelines related to concomitant drug exposure were formulated. Polypharmacy (treatment with multiple APDs) was to be avoided, as would the practice of combining a putative cognitive-enhancing agent with an APD with high affinity for the targeted receptor that could attenuate potential therapeutic effects. As an example of a situation to be avoided would be to combine agonists at the strychnine insensitive glycine receptor, such as glycine and D-cycloserine with clozapine (which is known to antagonize strychnine-sensitive glycine receptors, see [14]). Likewise, combining olanzapine or clozapine (potent enhancers of acetylcholine release, see [15]) with drugs that modulate nicotinic acetylcholine receptors would not be recommended.

Despite the recommendations cited above, there have been no improvements in the success rate of bringing new molecules to the market for treating the cognitive deficits of schizophrenia. While the subject of concomitant APD treatment has been raised (as noted above) and a few examples have been discussed where specific APDs should be avoided based on some specific pharmacological action, most of the commonly prescribed APDs have affinity for multiple receptors, transporters and other types of receptor proteins [16]. Moreover, most of receptor interactions that have been reported in the literature on specific APDs are based on acute actions in experimental models (e.g., ligand binding assays in neuronal culture or synaptosomes), whereas many of the therapeutic and adverse effects of APDs can be observed clinically only after several weeks of treatment [17, 18]. It appears likely, therefore, that these receptor interactions trigger a wide range of time-dependent cellular changes in the brain that contribute to both the pharmacology and toxicology of APDs. While there may be practical limitations in clinical trials, this suggests that the patient’s treatment history (which in many cases means multiple years of APD treatment) should be more carefully considered, not just concomitant APD treatment at the time of the clinical trial. Moreover, from the preclinical side, when potential pro-cognitive agents are evaluated in animal models, it would appear necessary to perform this evaluation in an animal that has also been treated chronically with an APD. The evaluations of potential pro-cognitive agents in animal models treated with APDs has only rarely been done and when it has, the APD has most commonly been administered acutely [19].

Therefore, the purpose of this preclinical study was to evaluate the α7 nicotinic acetylcholine receptor (α7 nAChR) partial agonist, tropisetron as a potential pro-cognitive agent in rats chronically treated with APDs, specifically, the commonly prescribed SGAs, risperidone and quetiapine. We chose to evaluate quetiapine and risperidone as the representative APDs for this study primarily for two reasons, 1) the high level of use of these particular APs across all age categories, and 2) their differential receptor pharmacology within the second-generation APD class. Thus, according to the latest information available on office-based prescribing, quetiapine and risperidone consistently ranked in the top 2–3 APDs in each age category (children, adolescents, and adults, see [20]). In terms of acute receptor pharmacology, risperidone has high affinity at D2 dopaminergic receptors while quetiapine’s affinity at D2 receptors is relatively low. In addition, risperidone is relatively devoid of affinity for histaminergic and muscarinic receptors while quetiapine binds to both receptors with relatively high affinity [16]. Our rationale for evaluating the α7 nAChR partial agonist, tropisetron as a potential pro-cognitive agent and the spontaneous novel object recognition (NOR) task as the choice of memory-related test is provided in the Discussion.

2. Materials and Methods

2.1. Chemicals and Reagents

Risperidone and 9-hydroxyrisperidone were kindly provided by the Janssen Research Foundation (Beers, Belgium). Quetiapine fumarate was purchased from Cerilliant (Round Rock, TX) and TCI America (Portland, OR), norquetiapine HCl, was purchased from Cerilliant (Round Rock, TX), midazolam was purchased from Hoffmann-La Roche (Nutley, NJ), and tropisetron was purchased from OChem Inc. (Des Plaines, IL). Isoflurane was purchased from Henry Schein Animal Health (Dublin OH). Amphetamine hemisulfate and all other chemicals including LC-MS/MS - grade solvents were purchased from Sigma–Aldrich (St. Louis, MO).

2.2. Test Subjects

Male albino Wistar rats (Envigo RMS, Inc., Indianapolis, IN.), approximately two months old were housed individually in a temperature controlled room (25°C), maintained on a 12-hour light/dark cycle with free access to food (Teklad Rodent Diet 8604 pellets, Harlan, Madison, WI) and water for the first week. Afterwards, drinking water was replaced with solutions that contained APDs for the remainder of the study (see drug administration section 2.3 below). All animal procedures employed during this study were reviewed and approved by the Institutional Animal Care and Use Committee and are consistent with AAALAC guidelines. Measures were taken to minimize pain or discomfort in accordance with the Guide for the Care and Use of Laboratory Animals, 11th edition, National Research Council, 2011 [21]. Significant efforts were also made to minimize the total number of animals used while maintaining statistically valid group numbers. In each of the assessments described below, plasma and brain APD analysis, open field locomotor activity assessments, 30 or 90 day effects of APDs (+/− tropisetron) on spontaneous novel object recognition, separate groups of rats were used (i.e., rats were evaluated in only one of the behavioral tests described).

2.3 Drug Administration

The oral risperidone dose was selected based on previous rodent studies in our laboratory in which time dependent behavioral and neurochemical effects were detected and the plasma levels achieved approximated those associated with antipsychotic effects in humans [22, 23, 24, 25]. In the case of quetiapine, the dose was based on published papers where notable effects on behavior were associated with chronic oral treatment [26]. Rats were thus treated with risperidone 2.5 mg/kg/day, or quetiapine 25.0 mg/kg/day orally in drinking water for 30 or 90 days. The drugs were dissolved in an acetic acid solution (1:100/0.1M acetic acid) and then subsequently diluted with distilled-deionized water for administration in drinking water. Drug dosing was based on the average daily fluid consumption and the weight of the individual test subjects.

2.4. Quantification of Antipsychotic Levels in Plasma and Brain

Plasma and Brain Collection

Plasma and brain samples were collected from groups of rats (n=6) that were treated with APDs for 30 days, but not tested in behavioral experiments. At the end of the dosing period, subjects were anesthetized with isoflurane and 3.0 mL of blood were collected via cardiac puncture into K2-EDTA blood collection tubes (Lavender Tops, Becton, Dickinson and Company, Franklin Lakes, NJ.). The blood was centrifuged for 15 min at 2500 × g at room temp and the resulting plasma was frozen until analyzed. Whole brains were subsequently removed, washed in phosphate-buffered saline, flash frozen for storage, at kept frozen at −70°C until analyzed for APD levels.

Sample Preparation and LC-MS/MS Analysis

The levels of risperidone, quetiapine and active metabolites of the APDs (9-hydroxyrisperidone and norquetiapine, respectively) in plasma and brain were quantified as we have described in detail previously [25, 27]. Briefly, liquid-liquid extraction was applied to prepare the biological samples. To each 100 μL of rat plasma or brain homogenate, 10 μL of internal standard solution (500.0 ng/mL midazolam), 100 μL of 0.5 M Na₂HPO₄ (pH 11.5) and 1.7 mL of diisopropyl ether were added. The mixture was vortexed, centrifuged, and 1.0 mL (for plasma samples) or 1.3 mL (for brain homogenate samples) of supernatant was transferred and evaporated in the vacuum concentrator. The sample was reconstituted with 150 μL of methanol: 20 mM ammonium formate (70:30, pH 3.9). After vortex and centrifugation, 100 μL of supernatant was transferred into an autosampler vial for LC-MS/MS analysis.

Analytes were separated on a Waters Atlantis dC-18 (2.1 × 30 mm, 3 μm) column using a gradient method (20%–100% mobile phase B over 5 min, mobile phase A is 5 mM ammonium formate and mobile phase B is acetonitrile). An Agilent 1100 binary pump HPLC system (Santa Clara, CA) interfaced to a Waters Micromass Quattro Micro triple quadrupole mass spectrometer was utilized for LC–MS/MS analysis. The multiple reaction monitoring (MRM) function was applied for the detection of analytes in positive ion mode, the ion transitions were 384 → 253 for quetiapine, 296 → 210 for norquetiapine, 411 → 191 for risperidone, 427 → 207 for 9-hydroxyrisperidone and 326 → 291 for midazolam.

2.5 Behavioral Experiments

Amphetamine-Induced Locomotor Activity

Rat open field activity monitors (43.2 × 43.2 cm, Med Associates St. Albans, VT) were used for amphetamine-induced locomotor experiments and ambulatory counts (horizontal photobeam breaks) were assessed. Rats treated with vehicle or APD (risperidone or quetiapine) for 30 days were habituated in the test chambers for 60 min and then injected subcutaneously with 1.5 mg/kg amphetamine. Afterwards, they were immediately returned to the activity chamber and monitored for an additional 60 min.

Spontaneous Novel Object Recognition (NOR) Task

The NOR task was adapted from Ennaceur and Delacour [28] as we have published previously [29, 30]. Briefly, test subjects were acclimated to laboratory conditions (i.e., tail marking, daily handling, and weighing) for at least 3 days prior to experimentation. During experimentation, the animals were transported to the laboratory and acclimated for 30 min prior to initiating the experimental phase; the animals remained in the laboratory for 15 min following study completion.

Habituation

The animals were acclimated, weighed, and individually placed in a dimly lit (10 lux) training/testing environment (an opaque plastic chamber, 78.7 cm × 39.4 cm × 31.7 cm with bedding on the floor) for 10 min of chamber exploration. The NOR chamber was placed on a table positioned along the short wall of the laboratory. HVAC ventilation provided masking noise to reduce any extraneous background noise, and there were no room orienting cues or wall-mounted visual cues (except for the small B/W camera positioned above the NOR chamber). At the beginning of each series of NOR experiments, fresh bedding material was placed in the chamber prior to habituation and allowed to become saturated with animal odors. Animal droppings were removed between experimental sessions, however, the same bedding remained in the chamber for remainder of each study (i.e., during training and testing), thus preventing any specific olfactory cues over the course of experimentation.

Training trial

Twenty-four hours after the habituation session, the animals were acclimated, weighed, and injected with test compound (drug or vehicle) and after the appropriate pretreatment interval, placed in the chamber with their nose facing the center of a long wall and allowed to explore two identical objects for 10 min. The animal’s behavior was observed and digitally recorded via a CCTV camera located 69 cm above the chamber; the investigator sat quietly 10–15 ft. away from the NOR chamber.

Test trial

A delay interval that reliably impairs recollection of a familiar object (48 hr) was used throughout the rest of the behavioral studies. In the NOR task, two objects, one object identical to training (familiar) and a novel object were placed in the chamber, and the animal was allowed to explore the objects for 5 min. Experimental objects to be discriminated were a plastic multicolored Duplo-Lego block configured tower (12 cm in height, 6 cm in width) paired with a ceramic conical-shaped green Christmas tree salt/pepper shaker (12 cm in height, 5 cm in diameter); all objects existed in duplicate. The objects were placed 19.3 cm from the sides of the two short walls and 19.3 cm from the sides of the long walls of the chamber; distance between the two objects was approximately 40 cm. The role of familiar and novel object as well as chamber position of object was randomly assigned across subjects and treatments, and objects were cleaned between sessions with a dilute 50% (vol/vol) ethanol solution to eliminate olfactory cues. The criteria for the observer to classify an object interaction as exploratory (investigative) behavior was direct interaction with nostrils or head positioning towards the object from a maximum distance of 2 cm. Physically climbing, rearing and digging around an object was not scored as object exploration. The primary behavioral measure was time (s) spent investigating each object. A discrimination index (d2) was calculated on each test trial and was defined as the difference in time spent exploring the novel and familiar objects divided by the total exploration time for both objects: d2 index = (novel − familiar)/(novel + familiar). This measure is considered as an index of recognition memory and takes into account individual differences in the total amount of exploration time. For data inclusion, the rat had to explore each individual object at least 4 sec and spend a minimum of 12 sec of total object exploration. Experimental groups contained 7–8 rats per treatment (or testing) condition which provided sufficient sample size to observe statistical significance. Animals were tested only once, and object exploration time was scored live under blind testing methods (i.e., the investigator was unaware of treatment assignment). As noted above, the test sessions were also digitally recorded for record-keeping purposes.

2.6 Statistical Analyses

Statistical analysis was performed using SigmaPlot 11.2 and statistical significance was assessed using an alpha level of 0.05. For one and two factor comparisons, analysis of variance (with repeated measures when indicated) was used followed by the Student Newman Keuls for post hoc analysis. All results are expressed as the mean (±SEM).

3. Results

3.1. Quantification of Antipsychotic Drug (APD) Levels in Plasma and Brain

The results of the LC-MS/MS quantification of plasma levels of the APDs and their active metabolites after 30 days of treatment were as follows, plasma (n=6, mean ng/ml ± S.E.M): risperidone, 7.24±1.67; 9-OH risperidone, 27.96±4.76, combination of risperidone and 9-OH risperidone, 35.20±6.27; quetiapine, 6.24±2.13, norquetiapine, 1.67±0.60. The brain levels were as follows (n=4, mean ng/gram ± S.E.M.), risperidone, 3.84±0.93; 9-OH risperidone, 4.76±0.81, combination of risperidone and 9-OH risperidone, 8.60±1.64; quetiapine, 13.81±0.86, norquetiapine, 3.41±0.48.

3.2. Effects of chronic antipsychotic treatment on amphetamine-induced locomotor activity

Fig 1 illustrates the effects of 30 days of treatment with risperidone or quetiapine (compared to vehicle) in the amphetamine-induced locomotor activity test. As expected, amphetamine (1.5 mg/kg) produced a robust increase in locomotor activity (see arrow at the 60 min time period and the increase in activity afterwards). Post hoc analysis indicted that chronic treatment with risperidone significantly attenuated the effects of amphetamine (p<0.05 compared to vehicle + amphetamine) at four of the time points after 80 min. In the quetiapine evaluation, there was also a robust increase in activity after the amphetamine injection, an effect that was attenuated in the quetiapine-treated animals at all time points after 80 min.

Fig. 1.

Effects of risperidone 2.5 mg/kg/day (TOP) and quetiapine 25.0 mg/kg/day (Bottom) treatment in drinking water for 30 days on amphetamine (1.5 mg/kg s.c.)-induced locomotor activity in an open field arena. Amphetamine (AMP) was administered after a 60 min habituation period and the subjects were recorded for an additional 60 min period. For the risperidone evaluation, statistical analysis of ambulatory counts revealed the following, main effect of group, F(1,12) = 1.13, p=0.31, time period, F(23,276) = 55.74, p<0.001, group x time period interaction, F(23,276) = 1.95, p=0.007. In the quetiapine evaluation, statistical analysis revealed the following, main effect of group, F(1,13) = 2.93, p=0.11, time period, F(23,299) = 42.86, p<0.001, group x time period interaction, F(23,299) = 2.36, p<0.001. Data are expressed as the mean ± S.E.M. for each treatment group. * represents a significant difference (p<0.05 post hoc comparison) between the exploratory activity of antipsychotic-treated versus vehicle-treated (control) animals at the same respective time point. n = 7 per group.

3.3 Effects of chronic risperidone treatment +/− tropisetron on the performance of a novel object recognition task

The effects of 30 and 90 days of treatment with vehicle or risperidone plus an acute injection of vehicle or tropisetron 3.0 mg/kg in the NOR task interval are provided in Fig 2 and 3, respectively. A/B retention sessions after a 48 hr retention period are illustrated. The individual exploration times of the novel and familiar objects are shown in the main figures with the calculated discrimination (d2) ratios illustrated in the insets. For the 30 day risperidone exposure period, (Fig 2) only the subjects administered tropisetron (i.e., in subjects chronically treated with vehicle or risperidone) showed a significant preference for the novel object (p<0.001 versus familiar). These effects were also observed when the d2 ratios were analyzed. Similar effects were observed at the 90-day risperidone-treatment period (Fig 3). Surprisingly, there appeared to be some recollection for the familiar object in the vehicle treated animals (p<0.05 novel versus familiar) however, only the subjects administered tropisetron (i.e., in subjects chronically treated with vehicle or risperidone) showed a robust preference for the novel object (p<0.001 versus familiar). These effects were also observed when the d2 ratios were analyzed.

Fig. 2.

Effects of risperidone 2.5 mg/kg/day in drinking water for 30 days plus vehicle or tropisetron on the performance of a spontaneous novel object recognition task in rats. In these experiments, tropisetron (or vehicle) was administered by subcutaneous injection 30 min before the training (A/A) trial at the end of the antipsychotic treatment period. The mean (± S.E.M.) exploration times of the familiar and novel objects after 48 hr delays (A/B retention sessions) are illustrated in the main figures. The insets illustrate the mean (± S.E.M.) discrimination (d2) ratios. d2 ratio = (novel − familiar)/(novel + familiar). For the exploration times, the following statistical results were obtained, main effect of group F(3,28)=1.17, p=0.34, object type, F(1,28)= 226.64, p<0.001), group by object type interaction, F(3,28)= 64.74, p<0.001). For the D2 ratios, the following statistical results were obtained, F(3,28)= 44.58, p<0.001. Post hoc results were as follows: +++p<0.001 novel vs familiar object; *** p<0.001 vs VEH-VEH. n=8 for each group. Abbreviations, VEH (vehicle), RISP (risperidone), TROP (tropisetron).

Fig. 3.

Effects of risperidone 2.5 mg/kg/day in drinking water for 90 days plus vehicle or tropisetron on the performance of a spontaneous novel object recognition task in rats. In these experiments, tropisetron (or vehicle) was administered by subcutaneous injection 30 min before the training (A/A) trial at the end of the antipsychotic treatment period. The mean (± S.E.M.) exploration times of the familiar and novel objects after 48 hr delays (A/B retention sessions) are illustrated in the main figures. The insets illustrate the mean (± S.E.M.) discrimination (d2) ratios. d2 ratio = (novel − familiar)/(novel + familiar). For the exploration times, the following statistical results were obtained main effect of group F(3,27)=0.54, p=0.66, object type F(1,27)= 81.19, p<0.001, group by object type interaction, F(3,27)= 9.44, p<0.001. For the D2 ratios, the following statistical results were obtained, F(3,27)=15.22, p<0.001. Post hoc results were as follows: +p<0.05, +++p<0.001 novel vs familiar object; *** p<0.001 vs VEH-VEH. n=7–8 for each group. Abbreviations, VEH (vehicle), RISP (risperidone), TROP (tropisetron).

3.4 Effects of chronic quetiapine treatment +/− tropisetron on the performance of a novel object recognition task

The effects of 30 and 90 days of treatment with vehicle or quetiapine plus an acute injection of vehicle or tropisetron 3.0 mg/kg in the NOR task interval are provided in Fig 4 and 5, respectively. For the 30 day quetiapine exposure period, only the subjects administered tropisetron (i.e., in subjects chronically treated with vehicle or quetiapine) demonstrated a significant preference for the novel object (p<0.001 versus familiar). These effects were also observed when the d2 ratios were analyzed. Similar effects were observed at the 90-day quetiapine-treatment period (Fig 5). Again, only the subjects administered tropisetron (i.e., in subjects chronically treated with vehicle or quetiapine) showed a robust preference for the novel object (p<0.001 versus familiar). These effects were also observed when the d2 ratios were analyzed.

Fig. 4.

Effects of quetiapine 25.0 mg/kg/day in drinking water for 30 days plus vehicle or tropisetron on the performance of a spontaneous novel object recognition task in rats. In these experiments, tropisetron (or vehicle) was administered by subcutaneous injection 30 min before the training (A/A) trial at the end of the antipsychotic treatment period. The mean (± S.E.M.) exploration times of the familiar and novel objects after 48 hr delays (A/B retention sessions) are illustrated in the main figures. The insets illustrate the mean (± S.E.M.) discrimination (d2) ratios. d2 ratio = (novel − familiar)/(novel + familiar). For exploration times, the following statistical results were obtained, main effect of group, F(3,28)=1.12, p=0.36, object type F(1,28)= 184.54, p<0.001), group x by object type interaction (F(3,28)= 60.02, p<0.001. For the D2 ratios, the following statistical results were obtained, F(3,28)=49.84, p<0.001. Post hoc results were as follows: +++p<0.001 novel vs familiar object; *** p<0.001 vs VEH-VEH. n=8 for each group. Abbreviations, VEH (vehicle), QUET (quetiapine), TROP (tropisetron).

Fig. 5.

Effects of quetiapine 25.0 mg/kg/day in drinking water for 90 days plus vehicle or tropisetron on the performance of a spontaneous novel object recognition task in rats. In these experiments, tropisetron (or vehicle) was administered by subcutaneous injection 30 min before the training (A/A) trial at the end of the antipsychotic treatment period. The mean (± S.E.M.) exploration times of the familiar and novel objects after 48 hr delays (A/B retention sessions) are illustrated in the main figures. The insets illustrate the mean (± S.E.M.) discrimination (d2) ratios. d2 ratio = (novel − familiar)/(novel + familiar). For exploration times, the following statistical results were obtained main effect of group F(3,25)=0.47, p=0.71, object type F(1,25)= 76.51, p<0.001, group by object type interaction, F(3,25)= 6.38, p=0.002. For the D2 ratios, the following statistical results were obtained, F(3,25)=12.10, p<0.001. Post hoc results were as follows: +p<0.05, +++p<0.001 novel vs familiar object; *** p<0.001 vs VEH-VEH. n=7–8 for each group. Abbreviations, VEH (vehicle), QUET (quetiapine), TROP (tropisetron).

4. Discussion

The results of this study can be summarized as follows: 1) the therapeutic relevance of the oral dosing approach used in this study (chronic APD treatment in drinking water) was validated in both the LC-MS/MS plasma and brain analyses and the open field locomotor activity experiments (see further discussion below), 2) neither the SGA risperidone nor quetiapine administered for 30 or 90 days improved NOR performance; 3) the nAChR partial agonist, tropisetron markedly improved NOR performance in rats that had been treated with vehicle or either APD for 30 or 90 days.

As was evident in the LC-MS/MS studies, 30 days of administration of risperidone or quetiapine resulted in easily detectible (ng/ml and ng/gram, respectively) levels of both of the APDs and their active metabolites in plasma and brain in the test subjects. The combination of risperidone and 9-hyroxyrisperidone is considered the most clinically relevant measurement for this SGA and the therapeutic range in the plasma of adult schizophrenia patients for the combination has been estimated to be 20.0–60.0 ng/ml [31, 32]. The mean level for the combination in our study in rat plasma was approximately 35 ng/ml, which is clearly within this human therapeutic range. In the case of quetiapine, the therapeutic range in human plasma has not been clearly established and a review of several clinical studies discussed ranges detected from less than 10 ng/ml to well over 500 ng/ml [33]. The mean plasma level of quetiapine in our study was approximately 6.2 ng/ml, which is somewhat below the lowest levels detected in the plasma of human patients. Never the less, the ability of both risperidone and quetiapine to attenuate the elevated locomotor activity associated with amphetamine administration reinforced the argument that our dosing approach resulted in concentrations in the brain and D2 occupancy levels that are therapeutically relevant. Moreover, there were no significant differences in locomotor activity between vehicle and APD treated groups during the habituation period before the amphetamine was injected, which argues against gross motor effects of the APDs confounding the amphetamine-related responses or the NOR-related results.

In the NOR experiments the observation that neither risperidone nor quetiapine improved performance was not particularly surprising, given our extensive previous evaluations of APDs (including both FGAs and SGAs) administered chronically in rodents. We have often observed that these agents (when administered chronically) tend to impair as opposed to improve the performance of attention and memory-related tasks in rodents [see 22, 23, 25]. These observations (while in normal rats) also support the aforementioned clinical view that APDs (in general) do not significantly improve cognition in psychiatric conditions such as schizophrenia [9, 10, 11]. The observation that the α7 nicotinic acetylcholine receptor (nAChR) partial agonist, tropisetron improved NOR performance in rats that had been treated with either risperidone or quetiapine for 30 or 90 days was somewhat surprising especially in the case of risperidone. Notably, we previously observed that risperidone treatment can lead to time-dependent decreases in the expression of α7 nAChRs in the brains of rats [23, 34] an effect that might be expected to limit the pro-cognitive effects of a ligand at α7 nAChRs. We chose to evaluate tropisetron as the pro-cognitive agent for these studies for several reasons (including its effects at nAChRs, see below). Tropisetron is a potent 5-hydroxytryptamine type 3 (5-HT₃) receptor antagonist widely used outside the United States to treat patients with chemotherapy-induced nausea and vomiting [35, 36]. However, in addition to its prominent effects at 5-HT₃ receptors, tropisetron is also a high affinity partial agonist at α7 nicotinic acetylcholine receptors (α7 nAChRs) [37, 38] which have been considered viable therapeutic targets in schizophrenia for several years. This premise is supported by postmortem evidence of α7 nAChR deficits in the frontal cortex and hippocampus schizophrenic patients [39] and by linkage analysis implicating chromosome 15q14 (the region that includes the α₇ neuronal nicotinic acetylcholine receptor gene). Polymorphisms in the core promoter of the α7 nAChR gene (CHRNA7; GeneBank accession no. Z23141) have been associated with reduced inhibition of the P50 evoked response to repeated auditory stimuli (i.e., indicative of sensory gating abnormalities) in schizophrenia [40]. α7 nAChR deficits may also contribute to abnormalities of smooth pursuit eye movements, sustained attention, and other domains of cognition often observed in schizophrenia [41].

In previous studies, tropisetron was shown to improve learning and memory [42] and sensory gating deficits [43, 44] in animal models of schizophrenia. These tropisetron-related improvements appeared to be mediated by α7 nAChRs (as opposed to 5-HT₃ receptors) since they were attenuated by the α7 nAChR antagonist methyllycaconitine (MLA) [45]. In some initial clinical studies, tropisetron was also shown to improve auditory sensory gating (P50) deficits, cognitive impairments and negative symptoms in patients with schizophrenia [46, 47, 48]. We also recently observed robust cognitive enhancing effects of tropisetron in both young and aged rats in an NOR task as well as in aged male and female monkeys in a delayed match to sample, working/short-term memory task [30]. In sum, the fact that tropisetron can exert pro-cognitive effects in cases where α7 nAChRs are likely decreased (e.g., after chronic APD treatment in rodents as we have observed and in schizophrenia patients, who have commonly been exposed to APDs chronically) is particularly encouraging from an adjunctive treatment standpoint.

We chose the rodent spontaneous novel object recognition (NOR) task [28], for evaluations of the effects of the APDs and tropisetron in this study for several important reasons. The NOR task is often utilized as a test of “recognition memory” and it has been proposed that it maps well onto the NIMH sponsored “MATRICS” visual learning and memory’ domain in schizophrenia [49]. Recognition memory is believed to consist of a recollective (episodic) and a familiarity component [50] and it is demonstrated in the NOR task when subjects explore a novel object more than a familiar one. This form of memory is often impaired in patients with schizophrenia [51] and they commonly show deficits in 2-D object recognition tasks that are similar to the NOR [52, 53, 54, 55]. There is also is considerable (albeit debated) evidence that the hippocampus (an important structure in the neuropathology of schizophrenia and other neuropsychiatric disorders) is actively involved in object recognition memory in both rodents [56, 57, 58] and humans [59, 60]. Collectively, the studies cited above support the translational validity of the NOR task and accordingly, it has been used extensively in schizophrenia-related preclinical studies for several years [61].

In conclusion, the results of this study indicate that neither 30 nor 90 days of risperidone or quetiapine treatment improved performance of a task designed to evaluate recognition memory in rodents. In contrast, tropisetron markedly improved NOR performance in rats treated with either APD for 30 or 90 days. These animal data reinforce the argument that two of the commonly prescribed APDs are not pro-cognitive agents and that α7 nAChR ligands like tropisetron have potential as adjunctive medications in schizophrenia since the pro-cognitive effect was maintained in the presence of chronic APD treatment. Given that tropisetron has a long history of safe usage in human patients, studies such as this one support its evaluation in neuropsychiatric disorders as a repurposed agent to improve cognitive function.

Abbreviations

APD

antipsychotic drugs

NOR

novel object recognition

nAChR

nicotinic acetylcholine receptor

SGA

second-generation antipsychotics

FGA

first-generation antipsychotics

MLA

methyllycaconitine

MRM

multiple reaction monitoring