The delta opioid receptor agonist KNT-127 relieves innate anxiety-like behavior in mice by suppressing transmission from the prelimbic cortex to basolateral amygdala.

AIM: Excitatory projections from the prelimbic cortex (PL) to the basolateral nucleus of the amygdala (BLA) are implicated in the regulation of anxiety-like behaviors, and we previously demonstrated that anxiolytic-like effects of the selective delta-opioid receptor (DOP) agonist KNT-127 is involved in suppressing glutamate neurotransmission in the PL. Here, we investigated the mechanisms underlying the anxiolytic-like effect of KNT-127 in mice by combining optogenetic stimulation of the PL-BLA pathway with behavioral analyses. METHODS: Four-week-old male C57BL/6J mice received bilateral administration of adeno-associated virus (AAV)2-CaMKIIa-hChR2(H134R)-enhanced yellow fluorescent protein (EYFP) into the PL to induce expression of the light-activated excitatory ionic channel ChR2. Subsequently, an optic fiber cannula connected to a wireless photo-stimulator was implanted into the BLA for optogenetic PL-BLA pathway stimulation. We evaluated innate anxiety using the elevated plus maze (EPM) and open field (OF) tests as well as learned anxiety using the contextual fear conditioning (CFC) test. RESULTS: Optogenetic activation of the PL-BLA pathway enhanced anxiety-like behaviors in the EPM and OF, while prior subcutaneous administration of KNT-127 (10 mg/kg) reduced this anxiogenic effect. In contrast, optogenetic activation of the PL-BLA pathway had no significant effect on conditioned fear. CONCLUSION: Our findings indicate that the PL-BLA circuit contributes to innate anxiety and that the anxiolytic-like effects of KNT-127 are mediated at least in part by suppression of PL-BLA transmission. The PL delta-opioid receptor may thus be an effective therapeutic target for anxiety disorders.

KNT-127, a selective delta opioid receptor agonist, shows beneficial effects in the hippocampal dentate gyrus of a chronic vicarious social defeat stress mouse model.

Delta opioid receptors (DOPs) play an important role in depression and other mood disorders. However, little is known about the underlying physiological mechanisms. The hypothalamic-pituitary-adrenal axis, adult hippocampal neurogenesis, and neuroinflammation are regarded as key pathophysiological factors in depression. In this study, we investigated the influence of DOP activation on those factors in a valid animal model of depression, chronic vicarious social defeat stress (cVSDS) mice. cVSDS mice (male C57BL/6J mice) were produced following a 10-day exposure to witness of social defeat stress, and each evaluation was performed more than 28 days after the stress period. Repeated administrations to cVSDS mice with a selective DOP agonist, KNT-127, both during (10 days) and after (28 days) the stress period respectively improved their decreased social interaction behaviors and increased serum corticosterone levels. When administered during the stress period, KNT-127 suppressed decreases in the hippocampal newborn neuron survival rate in cVSDS mice. Moreover, in both administration paradigms, KNT-127 reduced the number of Iba-1- and CD11b-positive cells in the subgranular zone and the granule cell layer of the hippocampal dentate gyrus, indicating a suppression of cVSDS-induced microglial overactivation. These results suggest that KNT-127 acts over the hypothalamic-pituitary-adrenal axis and regulates neurogenesis and neuroinflammation resulting in anti-stress effects, and the antidepressant-like effects of the DOP agonist are implicated in the suppression of the neuroinflammation. This study presents a new finding on the effects of repeated DOP activations on the pathophysiological states of depression.

Learning acquisition of consistent leader-follower relationships depends on implicit haptic interactions.

Are leaders made or born? Leader-follower roles have been well characterized in social science, but they remain somewhat obscure in sensory-motor coordination. Furthermore, it is unknown how and why leader-follower relationships are acquired, including innate versus acquired controversies. We developed a novel asymmetrical coordination task in which two participants (dyad) need to collaborate in transporting a simulated beam while maintaining its horizontal attitude. This experimental paradigm was implemented by twin robotic manipulanda, simulated beam dynamics, haptic interactions, and a projection screen. Clear leader-follower relationships were learned only when strong haptic feedback was introduced. This phenomenon occurred despite participants not being informed that they were interacting with each other and the large number of equally-valid alternative dyadic coordination strategies. We demonstrate the emergence of consistent leader-follower relationships in sensory-motor coordination, and further show that haptic interaction is essential for dyadic co-adaptation. These results provide insights into neural mechanisms responsible for the formation of leader-follower relationships in our society.

Examining the usefulness of the brain network marker program using fMRI for the diagnosis and stratification of major depressive disorder: a non-randomized study protocol.

BACKGROUND: Although many studies have reported the biological basis of major depressive disorder (MDD), none have been put into practical use. Recently, we developed a generalizable brain network marker for MDD diagnoses (diagnostic marker) across multiple imaging sites using resting-state functional magnetic resonance imaging (rs-fMRI). We have planned this clinical trial to establish evidence for the practical applicability of this diagnostic marker as a medical device. In addition, we have developed generalizable brain network markers for MDD stratification (stratification markers), and the verification of these brain network markers is a secondary endpoint of this study. METHODS: This is a non-randomized, open-label study involving patients with MDD and healthy controls (HCs). We will prospectively acquire rs-fMRI data from 50 patients with MDD and 50 HCs and anterogradely verify whether our diagnostic marker can distinguish between patients with MDD and HCs. Furthermore, we will longitudinally obtain rs-fMRI and clinical data at baseline and 6 weeks later in 80 patients with MDD treated with escitalopram and verify whether it is possible to prospectively distinguish MDD subtypes that are expected to be effectively responsive to escitalopram using our stratification markers. DISCUSSION: In this study, we will confirm that sufficient accuracy of the diagnostic marker could be reproduced for data from a prospective clinical study. Using longitudinally obtained data, we will also examine whether the "brain network marker for MDD diagnosis" reflects treatment effects in patients with MDD and whether treatment effects can be predicted by "brain network markers for MDD stratification". Data collected in this study will be extremely important for the clinical application of the brain network markers for MDD diagnosis and stratification. TRIAL REGISTRATION: Japan Registry of Clinical Trials ( jRCTs062220063 ). Registered 12/10/2022.

Verification of the brain network marker of major depressive disorder: Test-retest reliability and anterograde generalization performance for newly acquired data.

BACKGROUND: Recently, we developed a generalizable brain network marker for the diagnosis of major depressive disorder (MDD) across multiple imaging sites using resting-state functional magnetic resonance imaging. Here, we applied this brain network marker to newly acquired data to verify its test-retest reliability and anterograde generalization performance for new patients. METHODS: We tested the sensitivity and specificity of our brain network marker of MDD using data acquired from 43 new patients with MDD as well as new data from 33 healthy controls (HCs) who participated in our previous study. To examine the test-retest reliability of our brain network marker, we evaluated the intraclass correlation coefficients (ICCs) between the brain network marker-based classifier's output (probability of MDD) in two sets of HC data obtained at an interval of approximately 1 year. RESULTS: Test-retest correlation between the two sets of the classifier's output (probability of MDD) from HCs exhibited moderate reliability with an ICC of 0.45 (95 % confidence interval,0.13-0.68). The classifier distinguished patients with MDD and HCs with an accuracy of 69.7 % (sensitivity, 72.1 %; specificity, 66.7 %). LIMITATIONS: The data of patients with MDD in this study were cross-sectional, and the clinical significance of the marker, such as whether it is a state or trait marker of MDD and its association with treatment responsiveness, remains unclear. CONCLUSIONS: The results of this study reaffirmed the test-retest reliability and generalization performance of our brain network marker for the diagnosis of MDD.

Repeated psychological stress, chronic vicarious social defeat stress, evokes irritable bowel syndrome-like symptoms in mice.

Increasing evidence has demonstrated that emotional states and intestinal conditions are inter-connected in so-called "brain-gut interactions." Indeed, many psychiatric disorders are accompanied by gastrointestinal symptoms, such as the irritable bowel syndrome (IBS). However, the functional connection remains elusive, partly because there are few useful experimental animal models. Here, we focused on a highly validated animal model of stress-induced psychiatric disorders, such as depression, known as the chronic vicarious social defeat stress (cVSDS) model mice, which we prepared using exposure to repeated psychological stress, thereafter examining their intestinal conditions. In the charcoal meal test and the capsaicin-induced hyperalgesia test, cVSDS model mice showed a significantly higher intestinal transit ratio and increased visceral pain-related behaviors, respectively. These changes persisted over one month after the stress session. On the other hand, the pathological evaluations of the histological and inflammatory scores of naive and cVSDS model mice did not differ. Furthermore, keishikashakuyakuto-a kampo medicine clinically used for the treatment of IBS-normalized the intestinal motility change in cVSDS model mice. Our results indicate that cVSDS model mice present IBS-like symptoms such as chronic intestinal peristaltic changes and abdominal hyperalgesia without organic lesion. We therefore propose the cVSDS paradigm as a novel animal model of IBS with wide validity, elucidating the correlation between depressive states and intestinal abnormalities.

High-frequency ultrasound exposure improves depressive-like behavior in an olfactory bulbectomized rat model of depression.

OBJECTIVES: According to previous studies, ultrasound exposure appears to be a noninvasive method for modulating brain activity related to cognition and consciousness; however, its effects on emotional states remain unclear. Therefore, an animal model is required in which the effects and effect mechanisms of ultrasound exposure can be investigated. Thus, we used olfactory bulbectomized rats as an animal model of depression and investigated their emotional state following ultrasound exposure. METHODS: In male Wistar/ST olfactory bulbectomized rats, hyperemotionality was evaluated according to hyperemotionality scoring and the scores before and after 24-h ultrasound exposure were compared. Elevated plus maze (EPM) tests were also conducted after 24-h ultrasound exposure, and blood samples were collected in which plasma corticosterone concentrations were measured. RESULTS: Following exposure to high-frequency (~50 kHz) ultrasound vocalizations (USVs) associated with the pleasant emotions of rats, the hyperemotionality scores of olfactory bulbectomized rats were significantly reduced. Additionally, the latency of the first entry into the open arm of the EPM was significantly decreased in USV-exposed olfactory bulbectomized rats, as were their plasma corticosterone levels. Furthermore, artificial ultrasound (50 kHz) at a similar frequency to that of USV also significantly decreased the hyperemotionality score of olfactory bulbectomized rats. CONCLUSIONS: Ultrasound exposure improved depressive-like behavior in olfactory bulbectomized rats and reduced their plasma corticosterone levels. Thus, we recommend the use of olfactory bulbectomized rats as an animal model for investigating the effects and effect mechanisms of ultrasound exposure.

Disulfiram Produces Potent Anxiolytic-Like Effects Without Benzodiazepine Anxiolytics-Related Adverse Effects in Mice.

Disulfiram is an FDA approved drug for the treatment of alcoholism. The drug acts by inhibiting aldehyde dehydrogenase, an enzyme essential to alcohol metabolism. However, a recent study has demonstrated that disulfiram also potently inhibits the cytoplasmic protein FROUNT, a common regulator of chemokine receptor CCR2 and CCR5 signaling. Several studies have reported that chemokine receptors are associated with the regulation of emotional behaviors in rodents, such as anxiety. Therefore, this study was performed to clarify the effect of disulfiram on emotional behavior in rodents. The anxiolytic-like effects of disulfiram were investigated using an elevated plus-maze (EPM) test, a typical screening model for anxiolytics. Disulfiram (40 or 80 mg/kg) significantly increased the amount of time spent in the open arms of the maze and the number of open arm entries without affecting the total open arms entries. Similar results were obtained in mice treated with a selective FROUNT inhibitor, disulfiram-41 (10 mg/kg). These disulfiram-associated behavioral changes were similar to those observed following treatment with the benzodiazepine anxiolytic diazepam (1.5 mg/kg). Moreover, disulfiram (40 mg/kg) significantly and completely attenuated increased extracellular glutamate levels in the prelimbic-prefrontal cortex (PL-PFC) during stress exposure on the elevated open-platform. However, no effect in the EPM test was seen following administration of the selective aldehyde dehydrogenase inhibitor cyanamide (40 mg/kg). In contrast to diazepam, disulfiram caused no sedation effects in the open-field, coordination disorder on a rotarod, or amnesia in a Y-maze. This is the first report suggesting that disulfiram produces anxiolytic-like effects in rodents. We found that the presynaptic inhibitory effects on glutaminergic neurons in the PL-PFC may be involved in its underlying mechanism. Disulfiram could therefore be an effective and novel anxiolytic drug that does not produce benzodiazepine-related adverse effects, such as amnesia, coordination disorder, or sedation, as found with diazepam. We propose that the inhibitory activity of disulfiram against FROUNT function provides an effective therapeutic option in anxiety.

Development of a classifier for gambling disorder based on functional connections between brain regions.

AIM: Recently, a machine-learning (ML) technique has been used to create generalizable classifiers for psychiatric disorders based on information of functional connections (FCs) between brain regions at resting state. These classifiers predict diagnostic labels by a weighted linear sum (WLS) of the correlation values of a small number of selected FCs. We aimed to develop a generalizable classifier for gambling disorder (GD) from the information of FCs using the ML technique and examine relationships between WLS and clinical data. METHODS: As a training dataset for ML, data from 71 GD patients and 90 healthy controls (HCs) were obtained from two magnetic resonance imaging sites. We used an ML algorithm consisting of a cascade of an L1-regularized sparse canonical correlation analysis and a sparse logistic regression to create the classifier. The generalizability of the classifier was verified using an external dataset. This external dataset consisted of six GD patients and 14 HCs, and was collected at a different site from the sites of the training dataset. Correlations between WLS and South Oaks Gambling Screen (SOGS) and duration of illness were examined. RESULTS: The classifier distinguished between the GD patients and HCs with high accuracy in leave-one-out cross-validation (area under curve (AUC = 0.89)). This performance was confirmed in the external dataset (AUC = 0.81). There was no correlation between WLS, and SOGS and duration of illness in the GD patients. CONCLUSION: We developed a generalizable classifier for GD based on information of functional connections between brain regions at resting state.

Cold-Restraint Stress-Induced Ultrasonic Vocalization as a Novel Tool to Measure Anxiety in Mice.

Ultrasonic vocalization (USVs) is a promising tool to measure behavioral anxiety in rodents as USV recording is noninvasive, behaviorally relevant, ethological, and reproducible. Studies reporting the effects of stress-induced USVs in adult mice remain limited and debated. We investigated the conditions under which mice emit aversive USVs and evaluated the effects of psychiatric drugs on stress-induced USVs. Male C57BL/6J mice were used. USVs during entire stress sessions were recorded according to their frequency. To investigate the effect of psychiatric drugs on USVs, the number of USVs under cold-restraint stress conditions before and after drug administration was compared. Immediately after stress exposure, blood samples were collected and plasma corticosterone levels were measured. The combination of cold and restraint stress conditions significantly increased the USV numbers and plasma corticosterone levels compared with each stress alone. A benzodiazepine anxiolytic (midazolam) and δ-opioid receptor agonist putative anxiolytic (KNT-127) significantly reduced the stress-induced USV number and plasma corticosterone levels; however, a monoaminergic antidepressant (duloxetine) and N-methyl-D-aspartic acid receptor antagonist antidepressant (ketamine) did not reduce the USV numbers. No changes were noted in the USV numbers after repeated exposure to cold-restraint stress on days 1 and 3. The suppressive effect of midazolam on day 3 was comparable to that on day 1. Stress-induced USV may be used as a quantitative measure of anxiety to systematically assess the effects of anxiolytics. Therefore, cold-restraint stress-induced USVs may be used as a novel tool to measure rodent anxiety and as a useful anxiolytic-screening system.

Chronic vicarious social defeat stress attenuates new-born neuronal cell survival in mouse hippocampus.

Increasing evidence has shown that adult hippocampal neurogenesis is closely related to the pathophysiological condition of depressive disorders. Recently, chronic social defeat stress paradigms have been regarded as important animal models of depression, accompanied with neural plastic changes in the hippocampus. However, little is known about influences of non-physical stress on neurogenesis. In the present study, we focused on the chronic vicarious social defeat stress paradigm and examined the effect of psychological stress on mouse hippocampal neurogenesis. Immediately after the chronic psychological stress, the cell survival rate in the dentate gyrus of the hippocampus was significantly diminished without modifying the cell proliferation rate. The decreased ratio in cell survival persisted for 4 weeks after the stress-loading period, while the differentiation and maturity of new-born neurons were identical to control groups. Furthermore, treatment with the chronic antidepressant fluoxetine reversed the social behavioral deficits and promoted new-born neurons survival. These results demonstrate that emotional stress in the vicarious social defeat stress paradigm influences neuronal cell survival in the hippocampus, which reinforces its validity as an animal model of depression.

Hierarchical motor adaptations negotiate failures during force field learning.

Humans have the amazing ability to learn the dynamics of the body and environment to develop motor skills. Traditional motor studies using arm reaching paradigms have viewed this ability as the process of 'internal model adaptation'. However, the behaviors have not been fully explored in the case when reaches fail to attain the intended target. Here we examined human reaching under two force fields types; one that induces failures (i.e., target errors), and the other that does not. Our results show the presence of a distinct failure-driven adaptation process that enables quick task success after failures, and before completion of internal model adaptation, but that can result in persistent changes to the undisturbed trajectory. These behaviors can be explained by considering a hierarchical interaction between internal model adaptation and the failure-driven adaptation of reach direction. Our findings suggest that movement failure is negotiated using hierarchical motor adaptations by humans.

Discovery of δ opioid receptor full agonists lacking a basic nitrogen atom and their antidepressant-like effects.

We have recently reported that the elaboration of the N-substituent in the δ opioid receptor (DOR) antagonist naltrindole (NTI) enabled the regulation of the DOR activities from full inverse agonists to weak partial agonists. The investigations of amide-type NTI derivatives revealed that N-phenylacetyl and N-dihydrocinnamoyl derivatives 3a and 3b were DOR full agonists. The same transformations were applied to a DOR agonist KNT-127 to provide the more potent DOR agonists 6a and 6b. Among the tested compounds, the most efficacious compound 6a showed dose-dependent antidepressant-like effects in the mouse forced swim test. The antidepressant-like effects by 6a seemed to be more potent than those of KNT-127, which is a more potent DOR agonist in in vitro assays. The amide-type compound like 6a may more fully penetrate into the central nervous system.

Control strategy of hand movement depends on target redundancy.

Reaching toward a point target has been intensively studied in human motor control. However, little is known about reaching toward a redundant target, such as grasping a bar, in which the grasping point is irrelevant to the achievement of a task. We examined whether humans could solve the target-redundancy and control problems in a serial fashion or control their body without solving the target-redundancy problem. We equalized the target ranges between two reaching tasks: a point-to-point reaching task without target-redundancy and a point-to-bar reaching task with target-redundancy. In the both tasks, we measured hand viscoelasticity at movement end as parameters that reflect the adopted control strategy. As a result, the hand viscoelasticity in the point-to-bar reaching task was smaller than that in the point-to-point reaching task, even under the same kinematics. These results indicate that the hand viscoelasticity was modulated depending on the target-redundancy. Moreover, it is suggested that a human reaches toward a redundant target by effectively utilizing information of target redundancy rather than explicitly solving the target-redundancy problem.

Effort, success, and nonuse determine arm choice.

How do humans choose one arm or the other to reach single targets in front of the body? Current theories of reward-driven decisionmaking predict that choice results from a comparison of "action values," which are the expected rewards for possible actions in a given state. In addition, current theories of motor control predict that in planning arm movements, humans minimize an expected motor cost that balances motor effort and endpoint accuracy. Here, we test the hypotheses that arm choice is determined by comparison of action values comprising expected effort and expected task success for each arm, as well as a handedness bias. Right-handed subjects, in either a large or small target condition, were first instructed to use each hand in turn to shoot through an array of targets and then to choose either hand to shoot through the same targets. Effort was estimated via inverse kinematics and dynamics. A mixed-effects logistic-regression analysis showed that, as predicted, both expected effort and expected success predicted choice, as did arm use in the preceding trial. Finally, individual parameter estimation showed that the handedness bias correlated with mean difference between right- and left-arm success, leading to overall lower use of the left arm. We discuss our results in light of arm nonuse in individuals' poststroke.

The influence of an uncertain force environment on reshaping trial-to-trial motor variability.

Motor memory is updated to generate ideal movements in a novel environment. When the environment changes every trial randomly, how does the brain incorporate this uncertainty into motor memory? To investigate how the brain adapts to an uncertain environment, we considered a reach adaptation protocol where individuals practiced moving in a force field where a noise was injected. After they had adapted, we measured the trial-to-trial variability in the temporal profiles of the produced hand force. We found that the motor variability was significantly magnified by the adaptation to the random force field. Temporal profiles of the motor variance were significantly dissociable between two different types of random force fields experienced. A model-based analysis suggests that the variability is generated by noise in the gains of the internal model. It further suggests that the trial-to-trial motor variability magnified by the adaptation in a random force field is generated by the uncertainty of the internal model formed in the brain as a result of the adaptation.

Imposed visual feedback delay of an action changes mass perception based on the sensory prediction error.

While performing an action, the timing of when the sensory feedback is given can be used to establish the causal link between the action and its consequence. It has been shown that delaying the visual feedback while carrying an object makes people feel the mass of the object to be greater, suggesting that the feedback timing can also impact the perceived quality of an external object. In this study, we investigated the origin of the feedback timing information that influences the mass perception of the external object. Participants made a straight reaching movement while holding a manipulandum. The movement of the manipulandum was presented as a cursor movement on a monitor. In Experiment 1, various delays were imposed between the actual trajectory and the cursor movement. The participants' perceived mass of the manipulandum significantly increased as the delay increased to 400 ms, but this gain did not reach significance when the delay was 800 ms. This suggests the existence of a temporal tuning mechanism for incorporating the visual feedback into the perception of mass. In Experiment 2, we examined whether the increased mass perception during the visual delay was due to the prediction error of the visual consequence of an action or to the actual delay of the feedback itself. After the participants adapted to the feedback delay, the perceived mass of the object became lighter than before, indicating that updating the temporal prediction model for the visual consequence diminishes the overestimation of the object's mass. We propose that the misattribution of the visual delay into mass perception is induced by the sensorimotor prediction error, possibly when the amount of delay (error) is within the range that can reasonably include the consequence of an action.

Vangl2, the planar cell polarity protein, is complexed with postsynaptic density protein PSD-95 [corrected].

Vangl is a component of the non-canonical Wnt/planar cell polarity pathway, which is implicated in various cell polarity functions. However, little is known about its synaptic localization in neurons. Here, we show that Vangl1 and Vangl2 are expressed in adult rat neurons, where they are tightly associated with the postsynaptic density (PSD) fraction. Vangl2 forms a complex with PSD-95 through direct binding. Furthermore, the C-terminal PDZ-binding motif of Vangl2 is required for localization to dendritic spines. These results suggest that Vangl2 is a new component of the PSD that forms a complex with PSD-95 in the adult brain.

Physical and functional interaction of the active zone protein CAST/ERC2 and the ß-subunit of the voltage-dependent Ca(2+) channel.

In the nerve terminals, the active zone protein CAST/ERC2 forms a protein complex with the other active zone proteins ELKS, Bassoon, Piccolo, RIM1 and Munc13-1, and is thought to play an organizational and functional role in neurotransmitter release. However, it remains obscure how CAST/ERC2 regulates the Ca(2+)-dependent release of neurotransmitters. Here, we show an interaction of CAST with voltage-dependent Ca(2+) channels (VDCCs), which are essential for regulating neurotransmitter release triggered by depolarization-induced Ca(2+) influx at the active zone. Using a biochemical assay, we showed that CAST was coimmunoprecipitated with the VDCC ß(4)-subunit from the mouse brain. A pull-down assay revealed that the VDCC ß(4)-subunit interacted directly with at least the N- and C-terminal regions of CAST. The II-III linker of VDCC α(1)-subunit also interacted with C-terminal regions of CAST; however, the interaction was much weaker than that of ß(4)-subunit. Furthermore, coexpression of CAST and VDCCs in baby hamster kidney cells caused a shift in the voltage dependence of activation towards the hyperpolarizing direction. Taken together, these results suggest that CAST forms a protein complex with VDCCs, which may regulate neurotransmitter release partly through modifying the opening of VDCCs at the presynaptic active zones.

Prickle2 is localized in the postsynaptic density and interacts with PSD-95 and NMDA receptors in the brain.

The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.