張 心健

Schizophrenia is a psychiatric disorder characterized by positive, negative, and cognitive symptoms. MK-801, an N-methyl-D-aspartate receptor antagonist, has been used to induce schizophrenia-like behaviors in animal models. Here, we employed IntelliCage, an automated system used for tracking behavior, to assess schizophrenia-like behaviors in MK-801-treated mice under semi-naturalistic conditions. Mice that had been treated with MK-801 for 2 weeks were analyzed for locomotion, emotional, and cognitive functions. Repeated MK-801-treated mice exhibited transient hyperactivity in a novel environment, without significant changes in overall circadian activity. Sucrose preference remained intact, suggesting preserved reward sensitivity. However, less time spent in the corner during the early phase of the competition test indicated reduced competitive behavior for limited water rewards. In the behavioral flexibility test, repeated MK-801-treated mice showed impaired reversal learning, suggesting reduced cognitive flexibility, although the acquisition of initial place discrimination was comparable to that observed in control mice. These behavioral impairments parallel core symptoms of schizophrenia, particularly in the social and cognitive domains. Our findings demonstrate the utility of IntelliCage in detecting behavioral phenotypes over prolonged periods in group-housed settings. This study provides an ecologically valid platform for assessing schizophrenia-like behaviors and may facilitate the development of translationally relevant therapeutic interventions.

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

In patients with Parkinson’s disease (PD), dopamine replacement therapy with dopamine D2/D3 receptor agonists induces impairments in decision-making, including pathological gambling. The neurobiological mechanisms underlying these adverse effects remain elusive. Here, in a mouse model of PD, we investigated the effects of the dopamine D3 receptor (D3R)-preferring agonist pramipexole (PPX) on decision-making. PD model mice were generated using a bilateral injection of the toxin 6-hydroxydopamine into the dorsolateral striatum. Subsequent treatment with PPX increased disadvantageous choices characterized by a high-risk/high-reward in the touchscreen-based Iowa Gambling Task. This effect was blocked by treatment with the selective D3R antagonist PG-01037. In model mice treated with PPX, the number of c-Fos-positive cells was increased in the external globus pallidus (GPe), indicating dysregulation of the indirect pathway in the corticothalamic-basal ganglia circuitry. In accordance, chemogenetic inhibition of the GPe restored normal c-Fos activation and rescued PPX-induced disadvantageous choices. These findings demonstrate that the hyperactivation of GPe neurons in the indirect pathway impairs decision-making in PD model mice. The results provide a candidate mechanism and therapeutic target for pathological gambling observed during D2/D3 receptor pharmacotherapy in PD patients.

Although opioids are useful narcotic analgesics in clinical settings, their misuse and addiction in the United States of America and other countries are rapidly increasing. Therefore, the development of abuse-deterrent formulations is an urgent issue. We herein investigated how to select the ratio of an opioid and the opioid receptor antagonist, naloxone in abuse-deterrent formulations for mice. The conditioned place preference (CPP) test was used to evaluate the rewarding effects of abused drugs. The opioids morphine (30 μmol/kg), oxycodone (3 μmol/kg), fentanyl (0.4 μmol/kg), and buprenorphine (0.5 μmol/kg) significantly induced place preference in mice. We also examined the optimal ratio of naloxone and opioids to inhibit the rewarding effects of the latter. Naloxone (3-5 μmol/kg) effectively inhibited place preference induced by the opioids tested. We calculated theoretical drug doses that exerted the same pharmacodynamic effects based on two parameters: μ-opioid receptor binding affinity and blood-brain barrier (BBB) permeability. Theoretical doses were very close to the drug doses at which mice showed place preference. Therefore, the CPP test is useful as a behavioral method for evaluating abuse-deterrent formulations of opioids mixed with an antagonist. The ratio of naloxone with opioids, at which mice did not show place preference, may be an effective index for developing abuse-deterrent formulations. Ratios may be calculated for other opioids based on μ-opioid receptor binding affinity and BBB permeability.

Dopamine regulates emotional behaviors, including rewarding and aversive behaviors, through the mesolimbic dopaminergic pathway, which projects dopamine neurons from the ventral tegmental area to the nucleus accumbens (NAc). Protein phosphorylation is critical for intracellular signaling pathways and physiological functions, which are regulated by neurotransmitters in the brain. Previous studies have demonstrated that dopamine stimulated the phosphorylation of intracellular substrates, such as receptors, ion channels, and transcription factors, to regulate neuronal excitability and synaptic plasticity through dopamine receptors. We also established a novel database called KANPHOS that provides information on phosphorylation signals downstream of monoamines identified by our kinase substrate screening methods, including dopamine, in addition to those reported in the literature. Recent advances in proteomics techniques have enabled us to clarify the mechanisms through which dopamine controls rewarding and aversive behaviors through signal pathways in the NAc. In this review, we discuss the intracellular phosphorylation signals regulated by dopamine in these two emotional behaviors.

Acetylcholine is a neuromodulator critical for learning and memory. The cholinesterase inhibitor donepezil increases brain acetylcholine levels and improves Alzheimer's disease (AD)-associated learning disabilities. Acetylcholine activates striatal/nucleus accumbens dopamine receptor D2-expressing medium spiny neurons (D2R-MSNs), which regulate aversive learning through muscarinic receptor M1 (M1R). However, how acetylcholine stimulates learning beyond M1Rs remains unresolved. Here, we found that acetylcholine stimulated protein kinase C (PKC) in mouse striatal/nucleus accumbens. Our original kinase-oriented phosphoproteomic analysis revealed 116 PKC substrate candidates, including Rac1 activator β-PIX. Acetylcholine induced β-PIX phosphorylation and activation, thereby stimulating Rac1 effector p21-activated kinase (PAK). Aversive stimulus activated the M1R-PKC-PAK pathway in mouse D2R-MSNs. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system regulated dendritic spine structural plasticity and aversive learning. Donepezil induced PAK activation in both accumbal D2R-MSNs and in the CA1 region of the hippocampus and enhanced D2R-MSN-mediated aversive learning. These findings demonstrate that acetylcholine stimulates M1R-PKC-β-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning and imply the cascade's therapeutic potential for AD as aversive learning is used to preliminarily screen AD drugs.

Glutamate induces Ca²⁺ influx in neurons through NMDA receptors (NMDARs) and activates Ca²⁺-dependent protein kinases, including CaMKII, which play critical roles in synaptic plasticity and learning. However, how these kinases regulate synaptic plasticity and learning remains largely unknown. Here, we performed phosphoproteomics and identified 160 proteins including ArhGEF2 whose phosphorylation were promoted by NMDA. CaMKII phosphorylated ArhGEF2 and stimulated its RhoGEF activity. Aversive stimuli induced CaMKII-mediated ArhGEF2 phosphorylation and Rho-kinase/ROCK activation in the nucleus accumbens (NAc). Inhibition of Rho-kinase in the NAc attenuated aversive learning. We also screened Rho-kinase substrates and identified 221 proteins including Shank3 which links actin filaments with NMDARs and AMPA receptors via Dlgap3. The Rho-kinase-mediated phosphorylation of Shank3 increased its interaction with Dlgap3. Manipulation of Shank3 in the NAc regulated dendritic spine formation and aversive learning in a phosphorylation-dependent manner. These results demonstrate that NMDA activates the CaMKII-ArhGEF2-Rho-kinase pathway to induce Shank3 phosphorylation for aversive learning.

The nucleus accumbens (NAc) plays a crucial role in various mental activities, including positive and negative reinforcement. We previously hypothesized that a balance between dopamine (DA) and adenosine signals regulates the PKA-Rap1 pathway in medium spiny neurons expressing DA D1 receptors (D1R-MSNs) or D2 receptors (D2R-MSNs) and demonstrated that the PKA-Rap1 pathway in D1R-MSNs is responsible for positive reinforcement. Here, we show the role of the PKA-Rap1 pathway in accumbal D2R-MSNs in negative reinforcement. Mice were exposed to electric foot shock as an aversive stimulus. We monitored the phosphorylation level of Rap1gap S563, which leads to the activation of Rap1. Electric foot shocks increased the phosphorylation level of GluN1 S897 and Rap1gap S563 in the NAc. The aversive stimulus-evoked phosphorylation of Rap1gap S563 was detected in accumbal D2R-MSNs and inhibited by pretreatment with adenosine A2a receptor (A2aR) antagonist. A2aR antagonist-treated mice showed impaired aversive memory in passive avoidance tests. AAV-mediated inhibition of PKA, Rap1, or MEK1 in accumbal D2R-MSNs impaired aversive memory in passive avoidance tests, whereas activation of this pathway potentiated aversive memory. Optogenetic inactivation of mesolimbic DAergic neurons induced place aversion in real-time place aversion tests. Aversive response was attenuated by inhibition of PKA-Rap1 signaling in accumbal D2R-MSNs. These results suggested that accumbal D2R-MSNs regulate aversive behaviors through the A2aR-PKA-Rap1-MEK pathway. Our findings provide a novel molecular mechanism for regulating negative reinforcement.