Potentiation of M1 Muscarinic Receptor Reverses Plasticity Deficits and Negative and Cognitive Symptoms in a Schizophrenia Mouse Model
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Neuropsychopharmacology (2016) 41, 598–610 © 2016 American College of Neuropsychopharmacology. All rights reserved 0893-133X/16 www.neuropsychopharmacology.org Potentiation of M1 Muscarinic Receptor Reverses Plasticity Deficits and Negative and Cognitive Symptoms in a Schizophrenia Mouse Model 1,2 1,2 1 1 1 1 1 A Ghoshal , JM Rook , JW Dickerson , GN Roop , RD Morrison , N Jalan-Sakrikar , A Lamsal , 1 1 1 1 1 1 1 1 MJ Noetzel , MS Poslusney , MR Wood , BJ Melancon , SR Stauffer , Z Xiang , JS Daniels ,CMNiswender, 1 1 ,1 CK Jones , CW Lindsley and PJ Conn* 1 Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN, USA Schizophrenia patients exhibit deficits in signaling of the M1 subtype of muscarinic acetylcholine receptor (mAChR) in the prefrontal cortex (PFC) and also display impaired cortical long-term depression (LTD). We report that selective activation of the M1 mAChR subtype induces LTD in PFC and that this response is completely lost after repeated administration of phencyclidine (PCP), a mouse model of schizophrenia. Furthermore, discovery of a novel, systemically active M positive allosteric modulator (PAM), VU0453595, allowed us to 1 evaluate the impact of selective potentiation of M1 on induction of LTD and behavioral deficits in PCP-treated mice. Interestingly, VU0453595 fully restored impaired LTD as well as deficits in cognitive function and social interaction in these mice. These results provide critical new insights into synaptic changes that may contribute to behavioral deficits in this mouse model and support a role for selective M1 PAMs as a novel approach for the treatment of schizophrenia. Neuropsychopharmacology (2016) 41, 598–610; doi:10.1038/npp.2015.189; published online 15 July 2015 INTRODUCTION patients diagnosed with schizophrenia (Hasan et al, 2013; Strube et al, 2014). Furthermore, a common preclinical Schizophrenia is a chronic debilitating psychiatric disorder model of schizophrenia involving transient blockade of that is characterized by positive (hallucinations, delusions), N-methyl-D-aspartate receptors (NMDARs) with repeated negative (affective flattening, social withdrawal), and cogni- administration of NMDAR antagonists during a critical tive symptoms (working memory and attentional deficits) period of adolescent development induces profound and (Lewis and Lieberman, 2000). A strong body of evidence has lasting deficits in LTD at synapses from the hippocampus implicated a dysfunction of prefrontal cortex (PFC) as a key to PFC (Ghoshal and Conn, 2015; Thomases et al, 2014). component of the pathophysiology underlying symptomol- This has been postulated to contribute to the increased ogy of schizophrenia. These studies have primarily demon- activity of PFC neurons observed following phencyclidine strated task-related hypofrontality in patients as well as (PCP) treatment in rodents (Katayama et al, 2007; Suzuki animal models (for review, see Manoach, 2003). However, a et al, 2002; Thomases et al, 2014; Wang et al, 2007) recent and rapidly emerging body of clinical and preclinical as well as the subsequent behavioral changes associated studies also suggest that excessive activation of the PFC with negative and cognitive symptoms in the disorder by subcortical excitatory projections may contribute to the (Neill et al, 2010, 2014). negative symptoms and cognitive deficits observed in Muscarinic acetylcholine receptors (mAChRs) play schizophrenia (Jodo, 2013; Manoach, 2003; Woodward important roles in regulating synaptic plasticity in the et al, 2013). It is possible that this excessive activation of PFC and activation of mAChRs induces LTD at the the PFC may be due to an imbalance in the two common hippocampo-PFC synapse (Lopes-Aguiar et al, 2013; Parent forms of plasticity in PFC, namely, long-term potentiation et al, 2010; Wang and Yuan, 2009). Interestingly, multiple (LTP) and long-term depression (LTD). Indeed, recent studies suggest that cholinergic signaling is disrupted in clinical literature suggests that there are deficits in LTD in schizophrenia patients and in animal models of schizophrenia (Berman et al, 2007; Dean et al, 2002; Scarr *Correspondence: Dr PJ Conn, Department of Pharmacology, Vanderbilt and Dean, 2009b; Zavitsanou et al, 2004). If these changes Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, 1205 Light Hall, Nashville, TN 37232-0679, USA, Tel: +1 615 936 in cholinergic signaling lead to impairments in muscarinic 2478, Fax: +1 615 343 3088, E-mail: [email protected] LTD (mLTD), this could contribute to the underlying 2These authors contributed equally to this work pathophysiology in PFC function. Importantly, mAChR Received 17 January 2015; revised 9 June 2015; accepted 11 June 2015; antagonists exacerbate cognitive deficits and negative symp- accepted article preview online 25 June 2015 toms in schizophrenia patients (Veselinovic et al, 2014) Role of M1 PAM in PCP-treated mice A Ghoshal et al 599 and xanomeline, a mAChR agonist, reduces all symptom Extracellular Field Potential Recordings clusters in schizophrenia patients (Shekhar et al, 2008) and Extracellular field potential recordings were performed with corresponding animal models (Barak and Weiner, 2011; 8–9-week-old male C57BL6/J mice (Jackson Laboratories) Stanhope et al, 2001). These data are consistent with mice using coronal slices (400 μm) containing prelimbic the hypothesis that changes in mAChR signaling could PFC. Paired-pulse field excitatory postsynaptic potentials contribute to the symptoms observed in schizophrenia patients. Of the five mAChR subtypes (M –M ), M is (fEPSPs) were recorded from the layer V of the prelimbic 1 5 1 prefrontal cortex and evoked by paired electrical stimulus the predominant mAChR subtype expressed in the PFC pulses (100 μs duration, every 20 s; interpulse interval of (Levey et al, 1991) and likely participates in induction of 50 ms) of the superficial layers II–III, delivered through a mLTD (Caruana et al, 2011) and other physiological concentric bipolar stimulating electrode. For studies invol- responses that are important for PFC-dependent cognitive ving optical stimulation, blue light (470 nm) was delivered functions (Digby et al, 2012; Shirey et al, 2009). We now using a High Power LED (Thorlabs, Newton, NJ). Detailed report discovery and optimization of VU0453595 as a novel, methods are described in Supplementary Methods. highly selective positive allosteric modulator (PAM) for M1 that provides excellent pharmacokinetic properties and brain exposure in mice after systemic administration. We Whole-Cell Patch-Clamp Recordings used VU0453595, along with M1-knockout (KO) mice and mice selectively expressing channelrhodopsin in cholinergic Whole-cell patch-clamp recordings were performed using μ – neurons, to show that mLTD in the PFC is mediated coronal slices (300 m) prepared from 8 9-week-old male C57BL6/J mice (Jackson Laboratories) and a K-gluconate- exclusively by the M1 mAChR subtype. Interestingly, daily administration of the NMDAR antagonist PCP to juvenile based intracellular solution as described previously (Shirey mice led to a complete and lasting loss of M -mediated et al, 2009). Spontaneous EPSCs were recorded at a holding 1 − LTD in PFC slices. Incubation of PFC slices with the potential of 70 mV that is close to the reversal potential of chloride ions; therefore, GABAA receptor-mediated inhibi- M1-selective PAM restored the deficits in mLTD and systemic administration of VU0453595 reversed deficits tory currents were undetectable under these conditions. The in social interaction and cognitive function observed in this interevent intervals of sEPSCs from 2 min episodes during rodent model of schizophrenia. These data provide new baseline and drug application were used to generate cumu- lative probability plots. The interevent intervals from each insights into the functional impact of changes in M1 signaling in a model of schizophrenia and raise the exciting experiment were then expressed as frequency and the mean values from the 2 min episodes were grouped and compared. possibility that highly selective M1 PAMs may provide a novel approach for reducing cognitive deficits and negative Inward current data analysis was performed using Clampfit symptoms associated with changes in cortical plasticity software where the peak amplitude of the inward current was in schizophrenic patients. measured (v10.2, Molecular Devices, Sunnyvale, CA). Detailed methods are described in Supplementary Methods. MATERIALS AND METHODS Social Interaction Assay Animals Mice were allowed to habituate to a plexiglass arena consisting of white walls and a clear lid (36 × 26 × 18 cm) for 60 min. Mice All animal studies were approved by the Vanderbilt were then injected with vehicle (20% β-cyclodextrin (BCD)), 1, University Medical Center Institutional Animal Care and 3, or 10 mg/kg of VU0453595 (intraperitoneal (i.p.); 10 ml/kg) Use Committee and were conducted in accordance with the and placed back into the testing arena. Following a 30-min National Institutes of Health Guide for the Care and Use of pretreatment, an age/weight-matched noncagemate (intruder) Laboratory Animals. Male C57BL6/J mice (Jackson Labora- mouse was placed in the testing arena with the test mouse and tories, Bar Harbor, ME) were used in electrophysiology and their interaction behavior was recorded for 5 min. The amount behavioral studies (8–9 weeks old). The 8–9-week-old male of time spent in