[1] 23w Chronic infusion of PCP via osmotic mini-pumps: A new rodent model of cognitive deficit in schizophrenia characterized by impaired attentional set-shifting (ID/ED) performance

[1] 240w The identification of animal disease-like models for cognitive symptoms in schizophrenia is of central importance to the successful development of pharmacological therapies for psychosis resulting in a functional outcome in patients. Executive function is one of the most severely affected cognitive domains in schizophrenia that remains inadequately treated by existing therapies. The rat attentional set-shifting (or intra-dimensional-extra-dimensional (ID/ED)) task has been developed to test executive function in rodents and successful translation of pre-clinical data into the clinical setting now depends on the identification of a predictive animal disease-like model. The present study investigates whether a continuous 14-day mini-pump infusion of the non-competitive NMDA receptor antagonist phencyclidine (PCP) leads to a deficit in the ID/ED task, and subsequently evaluates the effect of modafinil in this model.Lister hooded rats were implanted subcutaneously with osmotic mini-pumps containing saline or PCP (15 mg/kg/day) for 14 days followed by a 7-day drug-free recovery phase. Rats were then tested in the ID/ED task following an acute injection of either vehicle or modafinil. PCP-treated animals displayed a selective deficit at the ED shift stage resembling that observed in schizophrenic patients. This deficit was reversed by an acute injection of modafinil. The PCP-induced impairment and its reinstatement by modafinil are quantitatively and qualitatively similar to that described earlier by our group following sub-chronic intraperitoneal PCP administration, indicative that sub-chronic PCP infusion via osmotic mini-pumps may represent an attractive alternative to the systemic administration protocols generally employed to date.

[1] 151w Schizophrenia is a disease syndrome with major public health implications (Jablensky et al., 1992) that includes selective impairments across several domains of cognition. To date, there is no approved treatment for the cognitive symptoms of schizophrenia, and only very few compounds have been shown to be of any therapeutic benefit (Tamminga et al., 1998). Cognitive dysfunction in schizophrenia is well documented with executive function being particularly impaired (Nuechterlein and Dawson, 1984;Liddle et al., 1987;Elliott et al., 1995;Green, 1996;Pantelis et al., 1999;Green et al., 2000). Clinically, executive function can be assessed using the Wisconsin Card Sorting Test (WCST) or the CANTAB attentional setshifting (ID/ED) task (Elliott et al., 1995). Recently, the ID/ED test has been adapted for use in rodents (Birrell and Brown, 2000) and subsequently when combined with schizophrenia disease-like models, it was used to enable the assessment of potential pro-cognitive compounds for schizophrenia (Rodefer et al., 2005;Featherstone et al., 2007).

[2] 87w PCP has been shown to induce schizophrenic-like symptoms in healthy humans (Luby et al., 1959) (for review see (Javitt and Zukin, 1991) and rodents (Martin et al., 1979;Geyer et al., 1984;Handelmann et al., 1987;Sams-Dodd, 1996), and to exacerbate symptoms in schizophrenic patients (Luby et al., 1959;Intil et al., 1967)). Consequently, PCP has been used in rodents following various treatment paradigms to mimic symptoms of schizophrenia, including cognitive impairments (Jentsch and Roth, 1999). The use of such animal models is essential for the successful development of future medications.

[3] 80w In the present study, we assessed the effect of a continuous delivery of PCP given subcutaneously for 14 days ("two-week infusion model") using osmotic mini-pumps (15 mg/kg/day) on the ID/ED task in rats in order to examine its potential as a new schizophrenia disease-like animal model. Furthermore, the effect of an acute administration of modafinil on the performance of PCP-treated animals was evaluated, since this compound has been shown to be clinically active in schizophrenic patients (Turner et al., 2004).

[1] 162w As demonstrated in Fig. 3, an overall effect of discrimination stage [F(6,251) = 37.9; p < 0.001] and a significant discrimina- tion stage × treatment interaction [F(12, 251) = 2.0; p < 0.05] was demonstrated by the two-way ANOVA test. The subsequent post hoc test showed that an attentional set had been formed in the saline/vehicle group, since a significantly higher number of trials to reach criteria was needed for this group at the ED shift stage compared to the ID2 stage of the task (p < 0.01). Moreover, a significant increase in trials to reach criteria in the ED discrimination stage was observed in the PCP/Veh-treated group compared to Veh/Vehtreated animals (p < 0.005). This PCP-induced impairment was significantly reversed in the PCP/modafinil-treated group when compared to the PCP/Veh-treatment group (p ≤ 0.01). Additionally, rats in the PCP/modafinil-treated group needed a significantly lower number of trials to reach criteria in the EDR stage compared to the PCP/Veh-treated groups (p < 0.05).

[1] 325w The present study investigated the effect of PCP infusion delivered by osmotic mini-pumps on the ID-ED attentional set-shifting task in rats. This approach served the aim of evaluating the model's potential as a novel pre-clinical disease-like model for cognitive symptoms in schizophrenia. We demonstrated clearly that the PCP infusion regimen induced an executive function deficit similar to the one described previously in our group utilizing systemic PCP treatment (Goetghebeur and Dias, 2008). The rodent ID-ED task has been adapted from clinical paradigms assessing executive function in patients (ID-ED and the Wisconsin Card Sorting Test (WCST). In line with clinical findings in chronic schizophrenics (Joyce et al., 2002), the present mini-pump PCP dosing regimen induced a selective deficit at the ED shift discrimination stage of the ID/ED task in which a shift in attention between perceptual dimensions is required. Moreover, this selective deficit was reversed by acute treatment with modafinil, and notably, PCP/modafinil-treated rats also displayed a significant lower number of trials to reach criteria at the EDR stage compared to PCP/vehicle treated animals. This is in line with the work of Turner et al. (2004) where some improvement of performance in the EDR stage was found in modafinil-treated schizophrenic patients. Additionally, that the results of the present study support a positive role of modafinil in improving EDR performance on the osmotic mini-pump PCP-treated group is in contrast to previous findings in which the EDR improvement was not observed in the i.p. administration PCP model (Goetghebeur and Dias, 2008). Given the current limitation of lack of a saline + modafinil group for comparison, speculation on this point needs to be contained at present. Nevertheless, the differential effect on EDR performance between the two PCP regimen could suggest that either this effect is less robust than the amelioration of the PCP-induced ED deficit or provide further support that the mini-pump administration technique presents a better methodology. Such points are worthy of consideration in a follow up study.

[2] 243w Over the past years, several animal disease-like models for schizophrenia, including perinatal PCP (Wang et al., 2001;Wiley et al., 2003), intraperitoneal PCP injection (Fletcher et al., 2005;Rodefer et al., 2005Rodefer et al., , 2008;;Egerton et al., 2005;Deschenes et al., 2006;Goetghebeur and Dias, 2008) and methylazoxymethanol acetate (MAM) prenatal injection (Featherstone et al., 2007), have all been shown to induce a deficit in the rodent executive function test. However, until now, only the model applying intraperitoneal PCP dosing has been reported to be sensitive to reversal by acute administration of modafinil (Goetghebeur and Dias, 2008), i.e. replicating clinical findings from the human ID/ED task, and these findings increase the predictive validity and translational value of both these disease models. In our laboratory, we have tested two doses of PCP in the "two-week infusion model" (15 mg/kg and 20 mg/kg) with respect to their potential to induce an ED shift deficit. Clearly, a dose response relationship is required in order to find the optimal PCP dose. However, it is of interest to note that infusion of 20 mg/kg/day of PCP for 14 days did not induce a larger deficit compared to the lower dose but, rather, increased variability in the assay (personal observation by C.S. Pedersen, nonpublished data). Thus, lower doses of PCP or modified perfusion duration might increase the deficit observed under the present regimen. Furthermore, a factorial design study (in order to minimize the number of animals used) might best answer this question.

[3] 58w In comparison with the sub-chronic intraperitoneal PCP dosing model, the PCP mini-pump infusion model alleviates the burden of injection work and mis-dosing of the compound. It also reduces the stress of the animal of being injected intraperitoneal twice per day. These aspects provide increased robustness to the model and therefore decrease experimental repetition and use of laboratory animals.

[4] 136w In addition, the deficit induced in intraperitoneal treated rats is temporally restricted (Lundbeck in house, non-published data), complicating the use of longer lasting or chronic administration of a potential pro-cognitive, antipsychotic treatment. Conversely, the steady PCP infusion may induce a longer lasting deficit, which will in turn permit the required chronic studies. Future experiments will have to investigate this time window closer, which might reveal a further advantage compared to intraperitoneal dosing. Moreover, a longer lasting PCP effect would permit re-use of the animal in other relevant cognitive assays, for instance the novel object recognition test (NOR) (Ennaceur and Delacour, 1988), Morris Water Maze (Andersen and Pouzet, 2004) or fear conditioning (Schafe et al., 2001), and thus allow assessment of a broad cognitive profile of these animals, whilst reducing the number of animal used for research.

[5] 118w In conclusion, two-week's infusion of PCP via mini-pumps in rats induces schizophrenia-like cognitive deficits in the attentional set-shifting test. This deficit is reversed by modafinil, a drug that administered acutely in adjunct to an atypical antipsychotic significantly improves ED shift performance in schizophrenic patients as well (Turner et al., 2004). Using PCP mini-pump infusion instead of the well described intraperitoneal dosing bears the advantage of reducing the animal's stress levels, bypasses the risk of potential mis-dosing that could arise from multiple dosing events and consequently may reduce the number of animals needed. Next, the assessment of our two-week PCP infusion's impact on several other cognitive domains relevant to schizophrenia will be of interest to further validate the model.

[1] 122w Adult male Lister Hooded rats (180-300 g at the time of testing) from Charles River, Germany, were housed 2 rats per cage under controlled conditions (12 h of light starting at 06:00; 20 ± 2 • C; 30-70% humidity) in Macroloncages (type III) with standard sawdust bedding and environmental enrichment (plastic houses and wooden chew blocks). The rats were given access to food and water ad libitum until the day before the first training session, where the food was removed. During training and test, each rat received approximately 11 g of food per day, given after the training and/or test sessions. All animal procedures were carried out in compliance with the EC Directive 86/609/EEC and with Danish law regulating experiments on animals.

[2] 63w Phencyclidine (PCP; synthesized in-house) was dissolved in 0.9% saline using a 1.15 salt-base ratio. Modafinil (Sequoia Research Products, UK) was suspended in 0.5% methylcellulose (Unikem, DK). On the day of testing, 0.5% methylcellulose or modafinil (64 mg/kg) was dosed orally (p.o.) with a 30 min pretreatment time, and again every hour during the test due to the relatively short half-life of this compound.

[3] 114w The test apparatus used was a 44 cm × 64 cm × 30 cm black nontransparent Perspex test box, divided into 3 equal-sized areas (a starting area separated from a choice area subdivided in two) (Fig. 1). In the choice area, two 11 cm diameter terracotta pots were placed recessed into the box floor (with a 2 cm lip remaining above the floor) and separated by a divider. Odor (oils from The Body Shop ® , UK and DK) and media cues (not previously presented to the rats) were added to the pots. The test-box was placed on a down flow table, in order to minimize the spread of allergens and mixing the odor.

[4] 84w 36 Lister hooded male rats were implanted with osmotic minipumps (Alzet ® model 2 ml 2 , 14 days, Durect Corporation, Canada) filled with either saline or PCP (15 mg/kg/day) subcutaneously on the back of the animal parallel to the spine (Balla et al., 2001). On day 14, pumps were surgically removed. During both procedures, animals were anesthetized with Servorane ® (Abbott Scandinavia AB, Sweden)/N2/oxygen vaporous mixture, and treated post-surgically with Baitril ® (Bayer HealthCare, Germany) and Rimadyl ® (Nomeco, Denmark) for 5 days.

[5] 401w The attentional set-shifting procedure was adopted from Goetghebeur and Dias (2008). Following removal of the osmotic mini-pumps, animals were given a 7-day washout period, during which rats were subjected to (from day 3) a habituation period and trained to dig for a food reward (half a Honey Loop cereal, Nestlé ® ) in pots filled with sawdust. On day 7 (end of washout period), rats were trained within a single session for digging in pots and discriminating them according to either the media they contained or the odors they were scented with. Eight days after pump removal rats were presented with a series of discriminations: a simple discrimination (SD), a compound discrimination (CD), two intra-dimensional shifts (ID1, ID2), an intra-dimensional shift reversal (ID2R), an extra-dimensional shift (ED) and an extradimensional shift reversal (EDR) (Fig. 2). Rats only progressed from one discrimination problem to the next (always presented in the same order) after reaching a criterion performance level of six consecutive correct responses. Please note that the procedure used contains multiple ID discriminations (i.e. ID1 and ID2), and fewer reversal-learning stages, in contrast to earlier studies (Birrell and Brown, 2000;Barense et al., 2002). Such a regime appears more robust and/or successful in inducing the formation of an attentional set and, possibly, helps to minimize potential reduction or cessation of responding that can occur during the reversal discrimination stages. The first trial of each discrimination stage was a discovery trial in which rats were allowed to dig in both pots, and to self-correct if digging in the wrong pot. Rats were only given one discovery trial, since using multiple trials would provide further and unwanted learning opportunities to the animal about the rule to adopt, and this may impact on their performance. Hence, we decided to only allow one discovery trial per discrimination stage to enable detection of both media and both odors, not giving any further information on which rule to follow. The discovery trial was included in the trials to criterion if the rat dug first in the correct pot, but not if the rat self corrected an incorrect initial dig. If a rat showed no interest in the pots (no digging or sniffing in either of the pots) for more than 15 min, it was returned to their home cage to rest for approximately 30 min before resuming the test. All rats were tested within 14 days after pump removal.

[6] 49w The number of trials to reach the performance criterion at each discrimination stage was recorded. Two-way analysis of variance (ANOVA) repeated measure was performed on log-transformed data and then followed by Student Newman-Keuls test for post hoc comparison between drug groups. Statistical significance was set at p < 0.05.