Schizophrenia dimension-specific antipsychotic drug action and failure in amphetamine-sensitized psychotic-like rats

European Neuropsychopharmacology (2018) 28, 1382–1393

www.elsevier.com/locate/euroneuro

Schizophrenia dimension-specific antipsychotic drug action and failure in amphetamine-sensitized psychotic-like rats Taygun C. Uzuneser a, Magnus Schindehütte a, Ekrem Dere b, Stephan von Hörsten c, Johannes Kornhuber a, Teja W. Grömer a,1, Christian P. Müller a,1,∗ a

Department of Psychiatry and Psychotherapy, University Clinic, Friedrich-Alexander-University Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany b Teaching and Research Unit Life Sciences (UFR 927), Université Pierre and Marie Curie, 9 quai Saint Bernard 75005 Paris, France c Department of Experimental Therapy, Preclinical Experimental Center, Friedrich-Alexander-University Erlangen-Nuremberg, Palmsanlage 5, 91054 Erlangen, Germany Received 4 March 2018; received in revised form 7 August 2018; accepted 5 September 2018

KEYWORDS Haloperidol; Schizophrenia; Dopamine; Treatment failure

Abstract Schizophrenic patients suffer from various disruptions in their psyche, mood and cognition, most of which cannot be effectively treated with the available antipsychotic drugs. Some dimensions of the schizophrenia syndrome in man can be mimicked in animals by the amphetamine (AMPH)-sensitization-induced psychosis model. Using such a sensitization procedure, we induced a psychosis-like syndrome in rats, measured as a deficit in sensory information processing and memory deficits. We then investigated the possible restorative effects of continuous treatment with haloperidol (HAL), a typical antipsychotic drug, on distinct dimensions of the syndrome. We found that, continuous infusion of a clinically relevant dose of HAL (0.5 mg/kg/day) effectively ameliorated AMPH-sensitization-induced sensorimotor gating disruptions after seven days of treatment. However, the sensory information processing deficit reappeared after prolonged HAL treatment, suggesting a treatment failure in this dimension of the syndrome. HAL had at this dose little beneficial effects on the cognitive deficits. In contrast, a continuously administered low dose of HAL (0.05 mg/kg/day) successfully attenuated

∗ Corresponding

author. E-mail address: [email protected] (C.P. Müller). 1These authors contributed equally. https://doi.org/10.1016/j.euroneuro.2018.09.005 0924-977X/© 2018 Elsevier B.V. and ECNP. All rights reserved.

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cognitive deficits, but aggravated the sensorimotor gating deficit under both short- or longterm treatment conditions. Post mortem neurochemical analysis revealed that the psychoticlike behavior induced by our manipulations might be explained by altered monoamine levels in distinct brain regions. These findings provide evidence for dissociating and dose-dependent HAL treatment action and failure at different dimensions of schizophrenia. © 2018 Elsevier B.V. and ECNP. All rights reserved.

1.

Introduction

Schizophrenia is a neuropsychiatric disorder, causing psychotic, affective and neurocognitive impairments in patients. Even though the etiology of schizophrenia has not yet been fully understood, neurochemical abnormalities involved in the distinct symptom dimensions of the disease have been suggested (Schumann et al., 2014). A striatal dopamine (DA) hyper-function is considered as the most prominent alteration (Baumeister & Francis, 2002), causing the psychotic symptoms of the disease (Kapur et al., 2005). Conversely, brain imaging studies of schizophrenic patients suggest a hypo-function of prefrontal DA, which might contribute to the cognitive and emotional symptoms of the disease (Davis et al., 1991; Ragland et al., 2007; Simpson et al., 2010). Antipsychotic drugs mainly target the postsynaptic D2 receptors to reverse the striatal dopaminergic hyper-function (Miyamoto et al., 2005). Treatment with haloperidol (HAL), a typical antipsychotic drug, has been shown to ameliorate the psychotic symptoms in schizophrenic patients with little effects on cognitive deficits (Mortimer, 1997). However, at the same time, HAL also targets presynaptic D2 receptors. A disturbed D2 autoinhibition might contribute to treatment failure in the long-term (Amato et al., 2018). In rats, continuous HAL administration with a therapeutic dose (0.5 mg/kg/day), which resembles human D2 receptor occupancy, has been shown to reduce amphetamine (AMPH)-induced locomotor activity, a proxy model of psychotic behavior. However, this effect was lost during the ongoing treatment. A hyper-locomotor response emerged after a brief withdrawal from the drug (Samaha et al., 2007, 2008). Moreover, in vivo studies monitoring dynamic monoamine responses to aversive and appetitive stimulation showed that therapeutic HAL modulates DA and noradrenaline (NA) levels differently in short- versus longterm treatment which may be responsible for treatment failure (Amato et al., 2011a,b; 2018). However, antipsychotic treatment failure studies have been performed using healthy animals. Therapeutic action and failure of antipsychotic drugs have not been thoroughly investigated in an animal model of psychosis. Conversely, low dose HAL treatment, which mainly targets presynaptic DA D2 autoreceptors, resulting in elevated dopaminergic transmission (Dias et al., 2012), has been shown to improve cognitive symptoms in schizophrenia patients (Green et al., 2002; Keefe et al., 2004, 2006). Yet the restorative effects of low dose HAL treatment in animal models have not been thoroughly investigated. Here, we addressed the effectiveness and potential failure of antipsychotic drug treatment in an animal model of psychosis. For this purpose, we used an escalating AMPH-

sensitization regimen to induce a psychosis-like state in rats (Jones et al., 2011). AMPH-sensitized psychotic animals have been shown to respond more to an acute AMPH challenge (Featherstone et al., 2007; Kalivas & Stewart, 1991) exhibit memory impairments (Bisagno et al., 2003) and sensorimotor gating deficits (Peleg-Raibstein et al., 2006; Tenn et al., 2003). After short- and long-term continuous treatment, we investigated whether a clinically relevant or a low dose of HAL could restore the AMPH-sensitization-induced aberrations in behaviors associated with distinct disease dimensions. After the conclusion of the behavioral experiments we examined possible changes in monoamine levels in brain regions known to be associated with antipsychotic treatment effects.

2. 2.1.

Experimental procedures Animals

Male Sprague-Dawley rats (Charles River, Germany) weighing 330–380 g at the beginning of the experiment were used. Animals were housed as four animals per cage with food and water available ad libitum, in a temperature (22 ± 2 °C) and humidity (55 ± 10%) controlled room under a light-dark cycle (light on from 07:00 to 19:00). The behavioral tests were conducted during the light phase of the cycle. All experiments were carried out in accordance with the Animal Protection Law of the Federal Republic of Germany and the European Communities Council 2010 Directive (2010/63/EU). They were all approved by the local authority “Regierung von Mittelfranken”.

2.2.

Drugs and treatment procedure

D-amphetamine sulfate (AMPH; 1 ml/kg; Fagron) or 0.9% saline (SAL; 1 ml/kg) was administered intraperitoneally (i.p.). AMPH was dissolved in SAL. Our sensitization procedure was adapted from Peleg-Raibstein et al. (2006). AMPH was administered 3 times per day (at 9:00, 13:00, 17:00) for 6 consecutive days with the concentration of AMPH escalating from 1 to 8 mg/kg with an increment of 1 mg/kg after each injection. The concentration was then maintained at 8 mg/kg for the following injections. SAL-treated animals received the same number of injections as the AMPH-treated animals. Haloperidol (HAL) was dissolved in distilled water containing 0.3% ascorbic acid / 10% cyclodextrin. It was administered continuously via Alzet osmotic mini pumps (model 2ML2; 14 day delivery; DURECT Corporation). Since the half-life of HAL is approximately six times higher in humans than in rodents, compared to

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Fig. 1 Graphical depiction of the sequence of manipulations and behavioral tests. Animals were AMPH-sensitized for six consecutive days, receiving three i.p. amphetamine (or saline) injections per day with escalating doses (from day −7 till day −1). A day after the last injection, animals were implanted with mini pumps containing HAL (or vehicle, day 0). These manipulations were repeated for a second cohort of animals. The first cohort of animals (A) were tested for light-induced activity and AMPH-induced locomotion test, which were assessed twice, after short-term (3–7 days) and long-term (10–14 days) drug treatment (n = 43). The second cohort of animals (B) were tested for novel object recognition (n = 28) and pre-pulse inhibition, the latter assessed twice; after short-term (5 days) and long-term (12 days) drug treatment (n = 47). Animals were sacrificed in order to investigate post mortem monoamine levels in their brain. APD, antipsychotic drug; OF, open field; AMPH, amphetamine; HAL, haloperidol; LIA, light-induced activity; NOR, novel object recognition; PPI, pre-pulse inhibition.

the intermittent delivery strategies, the continuous delivery of HAL via subcutaneous osmotic mini-pumps mimics the pharmacokinetic properties of the clinical situation in humans (Samaha et al., 2008; Tischbirek et al., 2012). Two concentrations of HAL, 0.05 mg/kg/day (HAL-0.05) and 0.5 mg/kg/day (HAL-0.5), and a vehicle solution (VEH), containing distilled water with 0.3% ascorbic acid / 10% cyclodextrin, were prepared. HAL-0.5 corresponds to the therapeutic HAL dose used in schizophrenic patients, occupying 73% striatal D2 receptors, thus, inhibiting both pre- and post-synaptic D2 receptors. A dose of HAL-0.05 (low dose) mainly targets presynaptic D2 receptors, thus, enhancing dopaminergic neurotransmission, especially within the striatum (Amato et al., 2011a, 2018; Dias et al., 2012; Kapur et al., 2003; Möller et al., 2017). Mini pumps were filled with these solutions and implanted into the lower back of animals under isoflurane anesthesia (Isoflurane; Baxter Germany GMBH; 5% induction, 2% maintenance) one day after the offset of AMPH injections (Fig. 1). SAL-administered animals were implanted with VEH-containing pumps, whereas the AMPH-administered animals were implanted with VEH, HAL-0.05 or HAL-0.5-containing pumps. Two separate experiments were conducted using this procedure. In the first experiment (N = 43), light-induced activity (LIA), open field (OF) behavior and AMPH-induced locomotor behavior were tested (Fig. 1A). LIA was tested 5 and again 12 days after the mini pump implantation. OF and AMPH-induced locomotion was tested 7 and again 14 days after the mini pump implantation. In the second experiment, novel object recognition (NOR, N = 28) and pre-pulse inhibition (PPI) were tested (N = 47, Fig. 1B). PPI was tested twice, 5 and 12 days after the mini pump implantation. NOR was tested 8–9 days

after the treatment onset. Short-term treatment was defined as 3–7 days of treatment, and long-term treatment as 10–14 days of treatment (Amato et al., 2011a, 2018).

2.3.

Behavioral equipment and procedures

AMPH-induced locomotion was tested in a cubic gray OF arena. A 20 min baseline period was followed by a 40 min test, prior to which animals received an i.p injection of AMPH (1.5 mg/kg). Locomotor behavior was analyzed using Biobserve Viewer III (Biobserve GMBH, Germany) software. The within-session habituation and PPI of the acoustic startle response (ASR) tests were conducted using the paradigm adapted from Peleg-Raibstein et al. (2006). Soundproof boxes were used for testing. 3 pre-pulse alone trials (74, 80, 86 dB), 3 pulse alone trials (100, 110, 120 dB), and 9 pre-pulse + pulse trials were repeated 10 times in a pseudo-randomized fashion. The startle response was measured using the TSE startle response system (TSE Systems, Bad Homburg, Germany). The %PPI was calculated with the formula of: %PPI = 100 − [100 x (startle amplitude of prepulse + pulse trials / startle amplitude of pulse alone trials)] (Fendt & Fanselow, 1999). In contrast to aversively-motivated PPI, LIA is used to measure appetitively-motivated sensorimotor gating. Locomotor activity has been shown to be reliably triggered by randomized, medium-intensity light stimulation, which is sought out by animals in a free choice condition. The test was conducted in an OF arena. The 20 min baseline period was followed by a 20 min test session, which involved the presentation of 10 randomly distributed white light stimuli.

Schizophrenia dimension-specific antipsychotic drug action Each light stimulus was administered for 30 seconds with an intensity of 82 lx. The horizontal and vertical locomotor activities were detected by a TruScan system (Coulbourn Instruments, Allentown, USA; Pum et al., 2008a, 2009, 2011). NOR test was performed using a Y-maze made of gray acrylic. By using a relatively short sample - test intertrial (IT) delay period (1 h), the effects of AMPH-sensitization and HAL treatment on short-intermediate term recognition memory were examined for three minutes. A discrimination ratio was calculated to measure the performance, using the formula: (time spent exploring novel object – time spent exploring familiar object) / (time spent exploring novel object + time spent exploring familiar object) (Dere et al., 2007; Kandel, 2001; Winters et al., 2004).

2.4.

Post mortem brain monoamine analysis

One day after the last behavioral testing, animals were sacrificed. The brains were quickly harvested and frozen and 2 mm thick coronal sections were prepared. These sections were used to dissect brain areas of interest, including the prefrontal cortex (PFC), dorsal and ventral striatum (DS, VS), thalamus (TH), hypothalamus (HYP), dorsal and ventral hippocampus (DH, VH), and dorsal and ventral midbrain (DM, VM). Dissection was performed according to the rat brain atlas (Paxinos & Watson, 2014). The monoamine content of the samples was assessed using high performance liquid chromatography (Müller et al., 2017; Pum et al., 2008b).

2.5.

Data analysis

The data are shown as mean ± SEM. All statistical analyses were carried out with IBM SPSS 21 software (SPSS Inc., Chicago, Illinois). Statistical significance was set p < 0.05 for all tests. Data were analyzed by two-way ANOVAs (locomotion, rearing, startle habituation), three-way ANOVAs (PPI), or one-way ANOVAs (monoamine neurochemistry). Pre-planned analyses were calculated to compare group differences using Bonferroni-corrected least significant difference tests. Paired sample t-tests were applied when appropriate. The detailed explanation of the equipment and behavioral procedures, tissue preparation and chromatographic conditions, and data analysis are available in Supplementary Experimental Procedures.

3.

Results

3.1. Dose-dependent HAL treatment efficacy after 7 days for AMPH-induced locomotor activity During the 20 min baseline, animals habituated to the test environment. Their locomotion and rearing activities declined over time (Fig. 2A, 2C). A significant effect of treatment (Locomotion: F(3,39) = 7.189, p < 0.001; Rearing: F(3,39) = 9.406, p < 0.001) suggests differences in baseline activities between groups. In order to measure the overall baseline activities for each treatment group, we used the area under the curve (AUC) analysis. Pre-planned

1385 comparisons revealed that AMPH sensitization had no effect on baseline motor activities (p > 0.05). HAL-0.5 treatment reduced the baseline locomotion and rearing, as indicated at 5 min time points (Locomotion, min −15: p = 0.041, min −10: p = 0.038, min 0: p = 0.029; Rearing, min −15: p = 0.001, min −10: p = 0.036; 2A, 2C) and in the AUC analysis (Locomotion, p = 0.008; Rearing, p = 0.003; 2B, 2D). An AMPH challenge induced a rapid increase in locomotion (Fig. 2A) and rearing (Fig. 2C) in all treatment groups within the first 20 min interval. However, the rate of elevation was not uniform between groups (Locomotion: F(3,39) = 16.594, p < 0.001; Rearing: F(3,39) = 22.711, p < 0.001). Compared to the AMPH-naïve animals, AMPH-sensitized psychotic animals showed enhanced locomotion and rearing at single time points and at 20 min AUC level (Locomotion, min 10: p = 0.001, min15: p = 0.002, AUC: p = 0.008; Rearing, min 10: p = 0.006, AUC: p = 0.037). This potentiation was further enhanced by the low dose HAL-0.05 treatment, but only for locomotion (min 10: p = 0.027, min 15: p = 0.002, min 20: p = 0.048; AUC: p = 0.046). Therapeutic HAL-0.5 treatment reversed the locomotor activity to the level of AMPH-naïve animals and diminished the rearing activity (Locomotion: min 5: p = 0.007, min 10: p = 0.024, min 20: p = 0.045; AUC: p = 0.003; Rearing: min 5–20: p < 0.001; AUC: p < 0.001). When the longer-lasting effects of an AMPH challenge were examined, 20–40 min after injection, the ANOVA analyses revealed significant effects of treatment (Locomotion: F(3,39) = 10.269, p < 0.001; Rearing: F(3,39) = 20.533, p < 0.001). HAL-0.05 treatment persisted to induce an increase in locomotion (min 30: p = 0.035, min 35: p = 0.018; AUC: p = 0.024). HAL-0.5 treatment still effectively reduced locomotion (min 40: p = 0.006; AUC: p = 0.023) and rearing (min 25, 30, 40: p < 0.001, min 35: p = 0.014; AUC: p < 0.001). Altogether, these findings suggest that continuous low dose HAL-0.05 treatment potentiated AMPH-sensitization effects, whereas continuous therapeutic dose HAL-0.5 treatment effectively reversed these effects. This implies an effective short-term treatment.

3.2. Dose-dependent HAL treatment efficacy after 14 days for AMPH-induced locomotor activity The baseline locomotor behavior remained comparable to the behavior after short-term drug treatment. HAL-0.5 treatment still reduced baseline locomotion and rearing (Locomotion: p = 0.021; Rearing: p = 0.001). Within group comparisons of the baseline locomotor and rearing activities between treatment days 7 and 14 revealed that baseline locomotor activity remained unchanged for all treatment groups (p > 0.05; Fig. 2E, 2F). In order to analyze treatment failure, we compared the AMPH-induced locomotor and rearing activities of animals on treatment days 7 and 14. HAL-0.5 treatment was no longer effective in normalizing AMPH-induced locomotion after long-term treatment. HAL-0.5-treated animals showed increased locomotor behavior compared to treatment day 7. This suggests a loss of efficacy on treatment day 14 (0– 20 min, p = 0.002; 20–40 min, p = 0.02; Fig. 2E). For AMPHinduced rearing, animals in the other three groups showed reduced activity on treatment day 14 (p < 0.05), but the HAL-0.5-treated animals showed an elevated rearing ac-

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Fig. 2 The effects of AMPH-sensitization and continuous HAL administration on baseline and AMPH-induced locomotor behavior. Short-term (7 day) drug treatment (A)–(D) induced a significant treatment effect on the 20 min baseline activities. HAL-0.5 treatment reduced the baseline locomotion and rearing, as indicated at each 5 min time points (A), (C) and in the AUC analysis (B), (D). AMPH-sensitized psychotic animals showed elevated locomotion and rearing within the first 20 min interval after a challenge AMPH injection (1.5 mg/kg). This elevation was exacerbated by HAL-0.05 treatment (only for locomotion), and reversed by HAL-0.5 treatment. Within the second 20 min interval after the challenge AMPH injection, HAL-0.05 treatment still induced an increase in locomotion, and HAL-0.5 treatment effectively reduced locomotion and rearing. Short-term and long-term drug treatments influence the locomotor behavior differently (E), (F). HAL-0.5-treated animals showed elevated AMPH-induced locomotor behavior after long-term treatment (E). For AMPH-induced rearing, animals in the other three groups showed reduced activity on treatment day 14, but the HAL-0.5-treated animals showed an elevated rearing activity (F). Arrow represents the AMPH injection time, which is set to min 0. n = 10–12 per group. Values are shown as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 compared to AMPH/VEH in (A)–(D). ∗ p < 0.05, ∗∗ p < 0.01, compared to treatment day 7 in (E), (F). BL, baseline; A-I, AMPH-induced activity; AUC, area under curve.

tivity (0–20 min, p = 0.032; 20–40 min, p = 0.049, Fig. 2F). These findings suggest that continuous HAL treatment with a short-term effective concentration loses its antipsychotic efficacy after long-term treatment in psychotic animals. As such, treatment-failure also emerged in psychotic animals, much the same way as previously observed in normal animals (Amato et al., 2011a; Samaha et al., 2007).

3.3. Effective reversal of the habituation of the acoustic startle response by HAL-0.5 after 12 days of treatment Fig. 3A shows the habituation of animals to the pulse stimuli after 12 days of drug treatment. ANOVA analyses revealed a significant treatment effect (F(3,43) = 4.408, p = 0.009) and a near significant treatment x trial interaction (F(27,387) = 1.508, p = 0.052) for startle

habituation. Pre-planned comparisons revealed a significant increase in the startle response of psychotic animals compared to AMPH-naïve animals (p = 0.018), suggesting a lack of habituation. AMPH-naïve animals showed a clear habituation. Compared to the first block of trials, the startle response was significantly attenuated on trials 3, 8, 9, and 10 (p = 0.032, 0.024, 0.023, 0.001, respectively). AMPHsensitization disrupted the startle habituation. This was indicated by an absence of startle attenuation at all trials (p > 0.05), except trial 5 (p = 0.032), in which the startle response was rather sensitized. Treatment with HAL-0.5 restored disrupted habituation induced by AMPH-sensitization (trial 8, p = 0.014; trial 10, p = 0.039), suggesting HAL treatment efficacy. In contrast, HAL-0.05 treatment had no effect on this behavior (p > 0.05). Short-term HAL-0.5 treatment partially restored disrupted habituation induced by AMPH-sensitization, suggesting partial HAL treatment efficacy. Short-term HAL-0.05 treatment had no effect

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Fig. 3 The effects of AMPH-sensitization and continuous HAL administration on sensorimotor gating and startle habituation. Habituation of the startle response to an acoustic stimulus is shown after long-term (12 day) treatment (A). The startle response on the initial trial is set to 1, and responses on the subsequent trials are normalized according to the initial response (Valsamis & Schmid, 2011). The startle response of AMPH-naïve animals was attenuated on trials 3, 8, 9, and 10 compared to the first trial. AMPH-sensitized psychotic animals lacked this attenuation, which was restored by HAL-0.5 treatment on trials 8 and 10, but not restored HAL-0.05 treatment. Pre-pulse inhibition of the acoustic startle stimulus is shown for each pre-pulse-pulse pair (B), as well as overall inhibition for p100 trials (C) after long-term (12 day) treatment. AMPH-sensitization disrupted PPI both at the 80–100 dB (B), and at the 100 dB AUC level (C). Both doses of HAL effectively restored PPI at the 80–100 dB pairing. Light-induced locomotor activities are depicted after short-term (5 day) treatment (D). Light-induced locomotion was normalized to the activity on the last 5 min of baseline, which was set to 0. Light-stimulation within the first 5 min induced an elevation in locomotion in all treatment groups. Light-stimulation induced a stronger response in AMPH-sensitized animals, which was partially normalized by HAL-0.5, but not by HAL-0.05. n = 10–13 per group. Values are shown as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared to AMPH/VEH. dB, decibel; AUC, area under curve, LIA; light-induced activity P100, pulse intensity = 100 dB; P110, pulse intensity = 110 dB; P120, pulse intensity = 120 dB. Background noise = 68 dB.

on this behavior (Supplementary Fig. S1A). These findings indicate effective reversal by therapeutic HAL-0.5 treatment on AMPH-sensitization-induced disruptions in startle habituation, which is more pronounced after long-term treatment.

3.4. Reversal of the disrupted PPI after 12 days of HAL treatment Fig. 3B shows the mean PPI of animals with all possible prepulse x pulse pairings after 12 days of drug treatment. There was no significant effect of treatment (p > 0.05). However, the main effects of the pulse (F(2,172) = 11.236, p < 0.001), and the pulse x treatment interaction (F(6,172) = 2.262, p = 0.045) suggested that the amount of inhibition was strongly influenced by the pulse intensity. A pulse stimulus of 100 dB resulted in the highest level of inhibition (vs. 110 dB, p = 0.04; vs. 120 dB, p < 0.001). Similar findings suggesting a higher inhibition by weaker pulse intensities have been reported in previous studies (Yee et al., 2005; Peleg-Raibstein et al., 2006). Therefore, we restricted further analysis to the lowest pulse intensity used. This approach yielded a trend for a treatment effect (p = 0.055). Pre-planned comparisons showed that AMPHsensitization disrupted PPI both at the 80–100 dB pairings (p = 0.003, Fig. 3B), and at the 100 dB AUC level (100 dB AUC: p = 0.024, Fig. 3C). HAL treatment did not reverse the AMPH-sensitization induced overall inhibition (p > 0.05). But for the 80–100 dB pairings, both doses of HAL effectively restored PPI (HAL-0.05: p = 0.027, HAL-0.5: p = 0.019). A

stimulus-specific reversal of PPI was also assessed after short-term treatment. Both doses of HAL restored the PPI deficits for 80–100 dB pairings after 5 days of treatment with the therapeutic dose being most effective (Supplementary Fig. S1B, S1C). Altogether, these findings suggest that psychotic-like animals show a stimulus-specific deficit in their aversively-motivated sensorimotor gating. This was improved by both doses of continuous HAL treatment after both, short- and long-term treatment.

3.5. Reversal of the light induced hyperactivity after 5 days of HAL-0.5 treatment The locomotor-stimulant effect of light is short-lasting (Amato et al., 2015). It is best observed within the first 10 min after the onset of light-stimulation (Fig. 3D). Lightstimulation within the first 5 min induced an elevation in locomotion in all treatment groups (F(1,39) = 63.598, p < 0.001). A main effect of treatment was observed 5 min (F(3,39) = 3.691, p = 0.02) and 10 min (F(3,39) = 5.329, p = 0.004) after the light stimulation. Pre-planned comparisons suggested that AMPH-sensitized psychotic-like animals respond stronger to the locomotion-enhancing effect of light-stimulation after 5 min (p = 0.04) and 10 min (p = 0.007). HAL-0.5 treatment reversed the exaggerated response to the light stimulation. It showed a normalization of locomotion after 5 min (p = 0.075) and 10 min (p = 0.03). HAL-0.05 treatment had no significant effect (p > 0.05) after 5 days of treatment. However, HAL-0.05 treatment preserved the locomotion-enhancing effect of

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T.C. Uzuneser et al. motor deficits or increased novel-object induced neophobia (Supplementary Fig. S2A). Between-group comparisons revealed a reduction of the object exploration time in the AMPH-sensitized animals (p = 0.015), which confirms previous reports (Peleg-Raibstein et al., 2009). This reduction was not restored by either HAL treatment (p > 0.05, Fig. 4B). These findings suggest that AMPH-sensitization disrupts short-intermediate term recognition memory performance, which was effectively reversed by only continuous low dose HAL treatment.

Fig. 4 The effects of AMPH-sensitization and continuous HAL administration on the short term recognition memory after 9 days of treatment. Recognition memory was tested using 1 h inter-trial delay. A discrimination ratio was calculated for the overall performance of each group (A). A significantly reduced preference for the novel object was observed in the AMPHsensitized animals, which was reversed by HAL-0.05, but not by HAL-0.5 treatment. A significant reduction of the object exploration time was observed in the AMPH-sensitized animals, which was not restored by treatment with either dose of HAL (B). Discrimination ratio: (time spent exploring novel object – time spent exploring familiar object) / (time spent exploring novel object + time spent exploring familiar object); equal preference = 0. N = 6–8 per group. Values are shown as mean ± SEM. ∗ p < 0.05 compared to AMPH/VEH. Disc. ratio, Discrimination ratio.

light-stimulation, which disappeared in psychotic-like animals after 12 days of treatment (Supplementary Fig. S1D). These findings suggest that short-term continuous treatment with a clinically relevant dose of HAL can reverse appetitively-motivated sensorimotor gating deficits induced by AMPH-sensitization. A low dose HAL treatment induces a sustained deficit after long-term treatment.

3.6. Reversal of disrupted recognition memory after a 1 h retention delay by HAL-0.05 During the sample trial, both objects were equally preferred (data not shown). Fig. 4A shows the overall discrimination ratios of the test phase after a 1 h retention delay. Pre-planned comparisons revealed a significantly reduced preference for the novel object in the AMPH-sensitized animals (p = 0.017), and a reversal of this deficit by HAL-0.05 (p = 0.045), but not by HAL-0.5 treatment (p > 0.05). The comparison of the novel object preference with equivocal preference (chance level performance; no recognition) provided further support for these findings. Unlike AMPHnaïve animals (p = 0.002), AMPH-sensitized psychotic-like animals did not have a significant preference towards the novel object (p > 0.05). HAL-0.05-treated animals showed a preference towards the novel object (p = 0.021), suggesting a reversal effect. HAL-0.5-treated animals did not show such a preference (p > 0.05). When a zero retention delay was used, animals in all treatment groups showed a preference towards the novel object (p < 0.05). This suggests that memory performance was not influenced by treatmentinduced side effects, such as motivational changes, gross

3.7. Effects of AMPH-sensitization and continuous HAL treatment on post mortem monoamine levels in the brain Schizophrenia is commonly associated with altered monoamine homeostasis in certain brain regions. We examined nine brain regions (dorsal and ventral striatum (DS, VS), prefrontal cortex (PFC), dorsal and ventral hippocampus (DH, VH), thalamus (TH), hypothalamus (HYP), and dorsal and ventral midbrain (DM, VM)) and two monoamines (DA and NA) of the post mortem tissue samples in order to assess psychosis-related monoaminergic disturbances, and their possible reversal by continuous HAL treatment (Fig. 5). AMPH-sensitized animals showed significantly reduced DA levels in the DS (p = 0.027, Fig. 5A), and significantly elevated DA levels in the DH (p = 0.016, Fig. 5D). Reduced DA levels in the DS were only partially restored by treatment with HAL-0.5. However, increased DA levels in the DH were successfully reversed by treatment with both doses of HAL (p < 0.05). Therapeutic HAL-0.5 treatment significantly increased DA levels in the HYP (p = 0.031, Fig. 5G) and VM (p = 0.031, Fig. 5I). HAL-0.05 treatment also increased DA levels in the VM (p = 0.006, Fig. 5I). AMPH-sensitized animals exhibited significantly reduced NA levels in the VH (p = 0.002, Fig. 5O), which were only partially restored by treatment with either dose of HAL. Despite AMPH-sensitization did not influence NA levels in the PFC, HAL-0.05 treatment significantly increased it (p = 0.035, Fig. 5M). Therapeutic HAL-0.5 treatment increased NA levels in the DH (p = 0.042, Fig. 5N), the TH (p = 0.021, Fig. 5P), and in the VM (p = 0.021, Fig. 5S). Altogether, these findings suggest that monoamine levels are region-specifically influenced by AMPH-sensitization and dose-dependent HAL treatment (Fig. 5J and 5T). AMPH, especially when administered chronically in a high dose, and HAL can produce adverse behavioral responses, such as dystonia, catalepsy, irregular limb movement or body posture, freezing, or stereotypies. Therefore, during drug administration, we thoroughly monitored the animals. Neither AMPH nor HAL produced any of these behaviors or significant weight loss in the animals. Therefore, extrapyramidal side effects are ruled out as an explanation for the altered behavioral responses in the animals.

4.

Discussion

In this study, we investigated the treatment efficacy and failure of antipsychotic drug treatment in psychotic-like rats

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Fig. 5 The effects of AMPH-sensitization and continuous HAL administration on post mortem monoamine levels. Post mortem tissue levels of dopamine (DA; A-I) are shown for nine brain regions. AMPH-sensitized animals exhibited significantly reduced DA levels in DS, which was only partially restored by HAL treatment with either dose (A). AMPH-sensitized animals exhibited elevated DA levels in DH (D), which was significantly restored by HAL treatment with either dose. DA levels in VM were increased by HAL treatment with either dose (I). HAL-0.5 treatment significantly increased DA levels in the HYP (G). Post mortem tissue levels of noradrenaline (NA; K-S) are shown for nine brain regions. AMPH-sensitized animals exhibited significantly reduced NA levels in VH, which was only partially restored by HAL treatment with either dose (O). HAL-0.05 treatment significantly increased NA levels in the PFC (M). HAL-0.5 treatment increased NA levels in the DH (N), TH (P), and VM (S). The monoamine levels are depicted altogether for DA (J) and NA (T), normalized to the levels of SAL/VEH group for each tissue sample examined. N = 7–9 per group. Values are shown as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01 compared to AMPH/VEH. hipp, hippocampus.

1390 along two major symptom dimensions of schizophrenia. We found that therapeutic HAL treatment (HAL-0.5) was effective in the remission of psychotic-like symptoms. This effect was abolished after long-term treatment, suggesting a treatment failure. HAL-0.5 treatment was also effective in the reversal of disruptions in sensorimotor gating and startle habituation, with a higher efficacy after long-term treatment. However, HAL-0.5 treatment had little beneficial effects on the cognitive deficits. In contrast, low dose HAL-0.05 treatment restored these cognitive deficits more effectively than therapeutic HAL-0.5 treatment. However, HAL-0.05 treatment caused an exacerbation of psychoticlike behavior, both after short- and long-term treatment. Furthermore, it was less beneficial for the restoration of the deficits in sensorimotor gating and startle habituation. Here we report contrasting effects of dose-dependent continuous HAL treatment on treatment efficacy, failure, and treatment-induced aggravation in a schizophrenia dimension-specific manner in rats with a psychotic-like state. Imaging studies show that AMPH administration induces an elevated DA release in schizophrenia patients (Laruelle & Abi-Dargham, 1999). Similarly, AMPH-sensitized animals displayed an elevated motor response to an AMPH challenge compared to AMPH-naïve animals, suggesting psychotic-like behavior. However, this elevation was limited to the initial 20 min after the AMPH challenge. This can be explained by D2 receptors regulating two temporally distinct signaling cascades. An acute AMPH administration has been shown to initially activate the canonical G-protein-dependent signaling pathway, which peaks 10–15 min after its administration. In addition to this, a non-canonical ßarrestin-2-Akt-GSK3-dependent signaling cascade is induced. This activation is progressive and longer-lasting, and regulates DA-related behavior (Beaulieu et al., 2007; Beaulieu & Gainetdinov, 2011). Schizophrenia has been associated with the over-activation of either pathway (Boyd & Mailman, 2012; Emamian et al., 2004; Park et al., 2016). Both of which have been shown to be regulated by antipsychotic drugs (Sutton et al., 2007; Sutton & Rushlow, 2011). According to our findings, continuous HAL0.5 treatment inhibited both pathways after short-term treatment, suggesting an effective treatment response. Using the atypical antipsychotic drug aripiprazole and the identical AMPH-sensitization procedure, we have previously shown that continuous therapeutic aripiprazole treatment (1.5 mg/kg/day), similar to continuous therapeutic HAL treatment, attenuated the motor response of rats to an AMPH challenge after short term treatment (Möller et al., 2017). A similar attenuation of locomotor responses was also observed in psychotic-like mice after short-term therapeutic olanzapine (10 mg/kg/day) treatment (Groos et al., submitted). Collectively, these findings suggest comparable antipsychotic efficacy of typical and atypical antipsychotic drugs after short-term treatment in rodents. After long-term treatment, the efficacy of HAL on the inhibition of the hyperlocomotor response to an AMPHchallenge decreased, thus, suggesting treatment failure (Samaha et al., 2007). The loss of antipsychotic efficacy during the ongoing treatment can explain the highly prevalent psychotic relapse rate observed in schizophrenic patients,

T.C. Uzuneser et al. even after long-acting depot application (Schooler, 2003). HAL-0.05 treatment exacerbated the hyper-locomotor response to AMPH, including both canonical and noncanonical pathways. These findings provide evidence that low dose HAL may enhances dopaminergic neurotransmission (Dias et al., 2012) and aggravate the psychotic-like symptoms in animals. We observed disruptions in appetitive and aversive sensorimotor gating in our AMPH-sensitized animals. Such disruptions have been shown to occur in schizophrenic patients (Braff et al., 1992; Geyer et al., 1990) and were classified as a part of the positive symptoms of the disease (Featherstone et al., 2007). LIA has not been investigated in patients, yet acute administration of cocaine, another indirect agonist of DA, has been shown to elevate the locomotion-inducing effects of light in rats (Pum et al., 2011). We found a similar effect after short-term abstinence from AMPH-sensitization (Möller et al., 2017). Despite the mixed findings reported about the effects of AMPHsensitization on PPI (Murphy et al., 2001; Peleg-Raibstein et al., 2006; Tenn et al., 2003), the sensitization protocol used by Peleg-Raibstein et al. (2006) caused reduced PPI, which was more pronounced using weaker pulse intensities. Using an identical sensitization regimen and a PPI protocol, we induced a sensory overload in animals, disrupting the gating and filtering mechanisms. Therapeutic HAL-0.5 treatment restored the elevated motor response to lightstimulation after short-term treatment, and deficits in the startle habituation and PPI after both short-term and longterm treatment. This suggests an effective treatment action in psychotic-like animals. We have previously shown beneficial effects of continuous aripiprazole treatment in psychotic-like rats (Möller et al., 2017), and of continuous olanzapine treatment in psychotic-like mice (Groos et al., submitted) in LIA after short term-treatment. These findings provide evidence for comparable treatment efficacy of typical and atypical antipsychotic drugs on sensorimotor gating disruptions caused by a psychosis-like disease state. Chronic long term amphetamine abusers have been shown to display impaired performance on attentional setshifting and pattern recognition memory (Ornstein et al., 2000), executive functioning and memory consolidation (Schouw et al., 2013). Such disruptions are also observed in schizophrenia as parts of cognitive symptoms. Low dose HAL administration has previously been suggested to improve neurocognitive disruptions in schizophrenia patients (Green et al., 2002; Keefe et al., 2004, 2006). An improvement in attention by low dose HAL treatment in AMPHsensitized rats (Martinez & Sarter, 2008), and an improvement of object recognition memory by a single low dose HAL injection in heterozygous DA transporter knockout mice (França et al., 2016) have been reported. Our findings expand these reports. Hippocampus and adjacent cortical areas are involved in memory function. Among those adjacent areas, perirhinal cortex is mostly associated with object memory, and its connection with the DH is critical for the consolidation of recognition memory (Antunes & Biala 2012). Our post mortem neurochemistry findings revealed a significant dopaminergic aberration in the DH region in AMPH-sensitized psychotic rats, and its reversal by HAL

Schizophrenia dimension-specific antipsychotic drug action treatment. This may provide a possible explanation for the altered memory function. It should be noted that the perirhinal cortex DA levels are elevated after a single AMPH injection in freely moving rats (Pum et al., 2007), suggesting that AMPH-sensitization might disrupt object recognition memory through this mechanism as well. We have not found an effect of either AMPH-sensitization or HAL treatment on the post mortem DA levels in the VS, a center of converging input from various brain regions involved in the modulation of PPI of the ASR (Koch, 1999). However, AMPH-sensitization significantly increased post mortem DA levels in the DH. Local administration of AMPH or quinpirole, a DA D2/D3 receptor agonist, applied to the CA1 area of the DH, has been shown to disrupt PPI (Ellenbroek et al., 2002). Furthermore, recordings from the CA3 of hippocampus have suggested that this region might mediate the gating of auditory-evoked responses in rats, which were disrupted by AMPH and reversed by HAL (Bickford-Wimer et al., 1990). Our HAL-treatment, at either dose, reversed the DA levels in the DH to the level of control animals. It also reversed the deficits in PPI after long-term treatment, further implicating dorsal hippocampal DA in the regulation of PPI. Treatment with both doses of HAL caused elevated DA levels in the VM, a mesencephalic region that includes dopaminergic and noradrenergic neurons. Altered DA levels in post mortem tissue should be caused by altered DA synthesis within these regions. Increased tyrosine hydroxylase levels, key enzyme for DA synthesis, have been reported in the post mortem VM tissue of schizophrenic patients (Howes et al., 2013). However, these patients were receiving antipsychotic treatment, which might also cause this elevation. There are some limitations in our study. Our post mortem samples were harvested from animals after 14 days of treatment. Since therapeutic HAL treatment was effective in reversing psychotic-like behavior on treatment day 7, but lost this effect on treatment day 14, our post mortem samples from this treatment group do not necessarily reflect the beneficiary effects of therapeutic HAL in psychosis. Therefore, changes in the DA and NA levels in the post mortem tissue samples might predominantly reflect treatment failure of psychosis for therapeutic HAL-treated animals. Furthermore, AMPH-sensitization-induced psychosis, even though a useful model with face and predictive validity, is not an etiology-driven model, ignoring genetic factors associated with schizophrenia. Thus, developing methods with optimized construct validity can provide a better understanding about the efficacy of antipsychotic drugs in preclinical research.

Role of funding source This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grant (MU 2789/7-1, GR 4549/1-1); the DFG had no further role in study design; in the collection, analysis and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication.

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Contributors Authors CPM and TG designed the study and managed the literature search. Authors TCU and MS were involved in the data acquisition. Author TCU undertook the statistical analysis. Authors TCU and CPM wrote the first draft of the manuscript. Author ED, SvH and JK revised the manuscript. All authors contributed to and have approved the final manuscript.

Conflict of interest None declared.

Acknowledgments We thank Mr. Benedikt Quinger for his technical assistance in completing this work.

Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.euroneuro. 2018.09.005.

References Amato, D., Natesan, S., Yavich, L., Kapur, S., Müller, C.P., 2011a. Dynamic regulation of dopamine and serotonin responses to salient stimuli during chronic haloperidol treatment. Int. J. Neuropsychopharmacol. 14, 1327–1339. Amato, D., Natesan, S., Kapur, S., Müller, C.P., 2011b. Haloperidol modulates noradrenergic responses to aversive stimulation depending on treatment duration. Behav. Brain. Res. 221, 311–313. Amato, D., Pum, M.E., Groos, D., Lauber, A.C., Huston, J.P., Carey, R.J., de Souza Silva, M.A., Müller, C.P., 2015. Neuropharmacology of light-induced activation. Neuropharmacology 95, 243–251. Amato, D., Canneva, F., Cumming, P., Maschauer, S., Groos, D., Wrosch, J.K., Grömer, T.W., Chiofalo, L., Dahlmanns, M., Zheng, F., Kornhuber, J., Prante, O., Alzheimer, C., von Hörsten, S., Müller, C.P., 2018. A dopaminergic mechanism of antipsychotic drug efficacy, failure, and failure reversal: the role of the dopamine transporter. Mol. Psychiatry doi:10.1038/ s41380- 018- 0114- 5, In press. Antunes, M., Biala, G., 2012. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cognit. Process. 13, 93–110. Baumeister, A.A., Francis, J.L., 2002. Historical development of the dopamine hypothesis of schizophrenia. J. Hist. Neurosci. 11, 265–277. Beaulieu, J.M., Gainetdinov, R.R., Caron, M.G., 2007. The Akt-GSK-3 signaling cascade in the actions of dopamine. Trends. Pharmacol. Sci. 28, 166–172. Beaulieu, J.M., Gainetdinov, R.R., 2011. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol. Rev. 63, 182–217. Bickford-Wimer, P.C., Nagamoto, H., Johnson, R., Adler, L.E., Egan, M., Rose, G.M., Freedman, R., 1990. Auditory sensory gating in hippocampal neurons: a model system in the rat. Biol. Psychiatry 27 (2), 183–192.

1392 Bisagno, V., Ferguson, D., Luine, V.N., 2003. Chronic D -amphetamine induces sexually dimorphic effects on locomotion, recognition memory, and brain monoamines. Pharmacol. Biochem. Behav. 74, 859–867. Boyd, K.N., Mailman, R.B., 2012. Dopamine receptor signaling and current and future antipsychotic drugs. Handb. Exp. Pharmacol. 212, 53–86. Braff, D.L., Grillon, C., Geyer, M.A., 1992. Gating and habituation of the startle reflex in schizophrenic patients. Arch. Gen. Psychiatry 49, 206–215. Davis, K.L., Kahn, R.S., Ko, G., Davidson, M., 1991. Dopamine in schizophrenia: a review and reconceptualization. Am. J. Psychiatry 148, 1474–1486. Dere, E., Huston, J.P., de Souza Silva, M.A., 2007. The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents. Neurosci. Biobehav. Rev. 31, 673–704. Dias, F.R., de Matos, L.W., Sampaio, M., Carey, R.J., Carrera, M.P., 2012. Opposite effects of low versus high dose haloperidol treatments on spontaneous and apomorphine induced motor behavior: evidence that at a very low dose haloperidol acts as an indirect dopamine agonist. Behav. Brain Res. 229 (1), 153–159. Ellenbroek, B.A., Lubbers, L.J., Cools, A.R., 2002. The role of hippocampal dopamine receptors in prepulse inhibition. Eur. J. Neurosci. 15, 1237–1243. Emamian, E.S., Hall, D., Birnbaum, M.J., Karayiorgou, M., Gogos, J.A., 2004. Convergent evidence for impaired AKT1-GSK3beta signaling in schizophrenia. Nat. Genet. 36, 131–137. Featherstone, R.E., Kapur, S., Fletcher, P.J., 2007. The amphetamine-induced sensitized state as a model of schizophrenia. Prog. Neuropsychopharmacol. Biol. Psychiatry 31, 1556–1571. Fendt, M., Fanselow, M.S., 1999. The neuroanatomical and neurochemical basis of conditioned fear. Neurosci. Biobehav. Rev. 23, 743–760. França, A.S., Muratori, L., Nascimento, G.C., Pereira, C.M., Ribeiro, S., Lobão-Soares, B., 2016. Object recognition impairment and rescue by a dopamine D2 antagonist in hyperdopaminergic mice. Behav Brain Res. 308, 211–216. Geyer, M., Swerdlow, N., Mansbach, R., Braff, D., 1990. Startle response models of sensorimotor gating and habituation deficits in schizophrenia. Brain. Res. Bull. 25, 485–498. Green, M.F., Marder, S.R., Glynn, S.M., McGurk, S.R., Wirshing, W.C., Wirshing, D.A., Liberman, R.P., Mintz, J., 2002. The neurocognitive effects of low-dose haloperidol: a two-year comparison with risperidone. Biol, Psychiatry 51, 972–978. Groos, D., Zheng, F., Kornhuber, J., Müller, C.P., Alzheimer, C., Chronic antipsychotic treatment reverses GIRK current suppression in dopaminergic neurons, loss of accumbal LTD, and behavioral sensitization in a mouse model of amphetamine psychosis, submitted. Howes, O.D., Williams, M., Ibrahim, K., Leung, G., Egerton, A., McGuire, P.K., Turkheimer, F., 2013. Midbrain dopamine function in schizophrenia and depression: a post-mortem and positron emission tomographic imaging study. Brain 136, 3242–3251. Jones, C.A., Watson, D.J., Fone, K.C., 2011. Animal models of schizophrenia. Br. J. Pharmacol. 164, 1162–1194. Kalivas, P.W., Stewart, J., 1991. Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain. Res. Rev. 16, 223–244. Kandel, E.R., 2001. The molecular biology of memory storage: a dialogue between genes and synapses. Science 294, 1030–1038. Kapur, S., Mizrahi, R., Li, M., 2005. From dopamine to salience to psychosis—linking biology, pharmacology and phenomenology of psychosis. Schizophr. Res. 79 (1), 59–68. Kapur, S., Vanderspek, S.C., Brownlee, B.A., Nobrega, J.N., 2003. Antipsychotic dosing in preclinical models is often unrepresen-

T.C. Uzuneser et al. tative of the clinical condition: a suggested solution based on in vivo occupancy. J. Pharmacol. Exp. Ther. 305, 625–631. Keefe, R.S., Seidman, L.J., Christensen, B.K., Hamer, R.M., Sharma, T., Sitskoorn, M.M., Lewine, R.R., Yurgelun-Todd, D.A., Gur, R.C., Tohen, M., Tollefson, G.D., Sanger, T.M., Lieberman, J.A., 2004. Comparative effect of atypical and conventional antipsychotic drugs on neurocognition in first-episode psychosis: a randomized, double-blind trial of olanzapine versus low doses of haloperidol. Am. J. Psychiatry 161, 985–995. Keefe, R.S., Seidman, L.J., Christensen, B.K., Hamer, R.M., Sharma, T., Sitskoorn, M.M., Rock, S.L., Woolson, S., Tohen, M., Tollefson, G.D., Sanger, T.M., Lieberman, J.A.HGDH Research Group, 2006. Long-term neurocognitive effects of olanzapine or low-dose haloperidol in first-episode psychosis. Biol. Psychiatry 59, 97–105. Koch, M., 1999. The neurobiology of startle. Prog. Neurobiol 59, 107–128. Laruelle, M., Abi-Dargham, A., 1999. Dopamine as the wind of psychotic fire: new evidence from brain imaging studies. J. Psychopharmacol. 13, 358–371. Martinez, V., Sarter, M., 2008. Detection of the moderately beneficial cognitive effects of low-dose treatment with haloperidol or clozapine in an animal model of the attentional impairments of schizophrenia. Neuropsychopharmacology 33, 2635–2647. Miyamoto, S., Duncan, G.E., Marx, C.E., Lieberman, J.A., 2005. Treatments for schizophrenia: a critical review of pharmacology and mechanisms of action of antipsychotic drugs. Mol. Psychiatry 10, 79–104. Möller, D., Banerjee, A., Uzuneser, T.C., Skultety, M., Huth, T., Hübner, H., Alzheimer, C., Friedland, K., Müller, C.P., Bouvier, M., Gmeiner, P., 2017. Discovery of G protein-biased dopaminergics with a pyrazolo[1,5-a]pyridine substructure. J. Med. Chem. 60, 2908–2929. Mortimer, A.M., 1997. Cognitive function in schizophrenia–do neuroleptics make a difference? Pharmacol. Biochem. Behav. 56 (4), 789–795. Müller, C.P., Kalinichenko, L.S., Tiesel, J., Witt, M., Stöckl, T., Sprenger, E., et al., 2017. Paradoxical antidepressant effects of alcohol are related to acid sphingomyelinase and its control of sphingolipid homeostasis. Acta. Neuropathol. 133 (3), 463–483. Murphy, C.A., Fend, M., Russig, H., Feldon, J., 2001. Latent inhibition, but not prepulse inhibition, is reduced during withdrawal from an escalating dosage schedule of amphetamine. Behav. Neurosci. 115, 1247–1256. Ornstein, T.J., Iddon, J.L., Baldacchino, A.M., Sahakian, B.J., London, M., Everitt, B.J., Robbins, T.W., 2000. Profiles of cognitive dysfunction in chronic amphetamine and heroin abusers. Neuropsychopharmacology 23, 113–126. Park, S.M., Chen, M., Schmerberg, C.M., Dulman, R.S., Rodriguiz, R.M., Caron, M.G., Jin, J., Wetsel, W.C., 2016. Effects of beta-arrestin-biased dopamine D2 receptor ligands on schizophrenia-like behavior in hypoglutamatergic mice. Neuropsychopharmacology 41, 704–715. Paxinos, G., Watson, C., 2014. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press. Peleg-Raibstein, D., Sydekum, E., Feldon, J., 2006. Differential effects on prepulse inhibition of withdrawal from two different repeated administration schedules of amphetamine. Int. J. Neuropsychopharmacol. 9, 737–749. Peleg-Raibstein, D., Yee, B.K., Feldon, J., Hauser, J., 2009. The amphetamine sensitization model of schizophrenia: relevance beyond psychotic symptoms? Psychopharmacology (Berl) 206, 603–621. Pum, M., Carey, R.J., Huston, J.P., Müller, C.P., 2007. Dissociating effects of cocaine and D-amphetamine on dopamine and serotonin in the perirhinal, entorhinal, and prefrontal cortex of freely moving rats. Psychopharmacology (Berl) 193, 375–390.

Schizophrenia dimension-specific antipsychotic drug action Pum, M.E., Huston, J.P., de Souza Silva, M.A., Müller, C.P., 2008a. Visual sensory-motor gating by serotonin-activation in the medial prefrontal and occipital, but not the rhinal, cortices in rats. Neuroscience 153, 361–372. Pum, M.E., Carey, R.J., Huston, J.P., Müller, C.P., 2008b. Role of medial prefrontal, entorhinal, and occipital 5-HT in cocaine-induced place preference and hyperlocomotion: evidence for multiple dissociations. Psychopharmacology 201, 391–403. Pum, M.E., Huston, J.P., Müller, C.P., de Souza Silva, M.A., 2009. Light-induced activity in the activity box is not aversively motivated and does not show between-trial habituation. Physiol. Behav. 96, 434–439. Pum, M.E., Rubio, A.R., Carey, R.J., de Souza Silva, M.A., Müller, C.P., 2011. The effects of cocaine on light-induced activity. Brain. Res. Bull. 84, 229–234. Ragland, J.D., Yoon, J., Minzenberg, M.J., Carter, C.S., 2007. Neuroimaging of cognitive disability in schizophrenia: search for a pathophysiological mechanism. Int. Rev. Psychiatry 19, 417–427. Samaha, A.N., Seeman, P., Stewart, J., Rajabi, H., Kapur, S., 2007. “Breakthrough” dopamine supersensitivity during ongoing antipsychotic treatment leads to treatment failure over time. J. Neurosci. 27, 2979–2986. Samaha, A.N., Reckless, G.E., Seeman, P., Diwan, M., Nobrega, J.N., Kapur, S., 2008. Less is More: Antipsychotic efficacy is greater with transient rather than continuous drug delivery. Biol. Psychiatry 64, 145–152. Schooler, N.R., 2003. Relapse and rehospitalization: comparing oral and depot antipsychotics. J. Clin. Psychiatry 64, 14–17. Schouw, M.L.J., Caan, M.W.A., Geurts, H.M., Schmand, B., Booij, J., Nederveen, A.J., Reneman, L., 2013. Monoaminergic dysfunction in recreational users of dexamphetamine. Eur. Neuropsychopharmacol. 23, 1491–1502. Schumann, G., Binder, E.B., Holte, A., de Kloet, E.R., Oedegaard, K.J., Robbins, T.W., Walker-Tilley, T.R., Brown, V.J., Buitelaar, J., Ciccocioppo, R., Cools, R., Escera, C., Fleischhacker, W., Flor, H., Frith, C.D., Heinz, A., Johnsen, E., Kirschbaum, C., Klingberg, T., Lesch, K.P., Lewis, S., Maier, W., Mann, K., Martinot, J.L., Meyer-Lindenberg, A., Müller, C. P., Müller, W.E., Nutt, D.J., Persico, A., Perugi, G., Pessiglione, M., Preuss, U.W., Roiser, J., Rossini, P.M., Rybakowski, J.K., Sandi, C., Stephan, K.E., Tricklebank, M., Undurraga, J., Vieta, E., van der Wee, N., Wykes, T., Haro, J.M., Wittchen, H.U., 2014. Stratified medicine for mental disorders. Eur. Neuropsychopharmacol. 24 (1), 5–50.

1393 Simpson, E.H., Kellendonk, C., Kandel, E., 2010. A possible role for the striatum in the pathogenesis of the cognitive symptoms of schizophrenia. Neuron 65, 585–596. Sutton, L.P., Honardoust, D., Mouyal, J., Rajakumar, N., Rushlow, W.J., 2007. Activation of the canonical Wnt pathway by the antipsychotics haloperidol and clozapine involves dishevelled-3. J. Neurochem. 102, 153–169. Sutton, L.P., Rushlow, W.J., 2011. The dopamine D2 receptor regulates Akt and GSK-3 via Dvl-3. Int. J. Neuropsychopharmacology 22, 1–15. Tenn, C.C., Fletcher, P.J., Kapur, S., 2003. Amphetamine-sensitized animals show a sensorimotor gating and neurochemical abnormality similar to that of schizophrenia. Schizophr. Res. 64, 103–114. Tischbirek, C.H., Wenzel, E.M., Zheng, F., Huth, T., Amato, D., Trapp, S., Denker, A., Welzel, O., Lueke, K., Svetlitchny, A., Rauh, M., Deusser, J., Schwab, A., Rizzoli, S.O., Müller, C.P., Alzheimer, C., Kornhuber, J., Groemer, T.W., 2012. Use-dependent inhibition of synaptic transmission by the secretion of intravesicularly accumulated antipsychotic drugs. Neuron 74 (5), 830–844. Valsamis, B., Schmid, S., 2011. Habituation and prepulse inhibition of acoustic startle in rodents. J. Vis. Exp. 55, e3446. Winters, B.D., Forwood, S.E., Cowell, R.A., Saksida, L.M., Bussey, T.J., 2004. Double dissociation between the effects of peri-postrhinal cortex and hippocampal lesions on tests of object recognition and spatial memory, heterogeneity of function within the temporal lobe. J. Neurosci. 24, 5901–5908. Yee, B.K., Chang, T., Pietropaolo, S., Feldon, J., 2005. The expression of prepulse inhibition of the acoustic startle reflex as a function of three pulse stimulus intensities, three prepulse stimulus intensities, and three levels of startle responsiveness in C57BL6/J mice. Behav. Brain Res. 163, 265–276.