Systemic administration of a delta opioid receptor agonist, KNT-127, facilitates extinction learning of fear memory in rats.

Strategies to facilitate extinction of fear memory have attracted increasing attention for enhancing the effectiveness of exposure therapy for anxiety disorders. Previously, we demonstrated that systemic administration of a delta opioid receptor agonist, KNT-127, has clear anxiolytic-like effects in rats, without impairing memory. These observations led us to hypothesize that KNT-127 might be an appropriate therapeutic agent for anxiety disorders when combined with exposure therapy. In the present study, we demonstrate that KNT-127 (3 mg/kg) facilitates extinction learning of fear memory using the contextual fear conditioning test. As expected, a partial agonist at the glycine-binding site on the glutamatergic N-methyl-d-aspartate receptor, d-cycloserine (15 mg/kg), facilitated extinction learning of contextual fear in rats. In contrast, a benzodiazepine anxiolytic, diazepam (1 mg/kg), impaired the fear extinction learning. Interestingly, the facilitatory effect of KNT-127 on extinction learning was observed not only after a 10-min re-exposure, but also after a much shorter (2-min) re-exposure to the context, while d-cycloserine was ineffective at facilitating extinction when a short-duration exposure was given. Our findings may suggest that administration of a delta opioid receptor agonist might have therapeutic efficacy when combined with exposure therapy for treating a range of anxiety disorders.

Induction of c-Fos immunoreactivity in the amygdala of mice expressing anxiety-like behavior after local perfusion of veratrine in the prelimbic medial prefrontal cortex.

Local perfusion of the sodium channel activator veratrine in mouse prelimbic medial prefrontal cortex (PL) induced c-Fos immunoreactivity in the sub-regions of amygdala. Co-perfusion of the NMDA receptor antagonist MK-801 diminished the c-Fos expression. Significant correlations were observed between c-Fos immunoreactivity and behavioral measures in the open-field test. The PL stimulation activates a neural network projecting to the amygdala via NMDA receptor-mediated glutamatergic neurotransmission. Anxiety-like behavior induced after the PL stimulation may be partly mediated through the activation of amygdala.

Lysophosphatidic acid induces anxiety-like behavior via its receptors in mice.

Lysophosphatidic acid (LPA) is a potent bioactive lipid mediator with diverse biological properties. We previously found altered expression of the LPA-related genes in rodents after treatment with sertraline, which is widely used to treat anxiety disorders and depression. However, little is known about the behavioral effects of LPA. In the present study, we investigated the behavioral effects of intracerebroventricular injection of LPA in adult mice. LPA did not significantly affect spontaneous locomotor activity, suggesting that LPA does not induce hyperactivity, ataxia, or sedation. We next investigated the emotional effects of LPA via the hole-board test. LPA significantly increased the number of head-dips in a dose- and time-related manner. A significant induction of head-dip counts occurred 15 and 30 min after LPA administration. To clarify the involvement of LPA receptors, we examined the effect of the non-selective LPA1-4 receptor antagonist, 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl-phosphonate (BrP-LPA) co-administered with LPA. BrP-LPA dose-dependently inhibited LPA-induced head-dip counts. We next investigated anxiety-like behavior via the elevated plus-maze test. LPA significantly reduced the percentage of time spent in the open arms and BrP-LPA dose-dependently inhibited this anxiety-like behavior. In conclusion, LPA induced anxiety-like behavior in mice via LPA receptors. Our results suggest that LPA signaling plays an important role in regulating anxiety in mice.

Activation of the prelimbic medial prefrontal cortex induces anxiety-like behaviors via N-Methyl-D-aspartate receptor-mediated glutamatergic neurotransmission in mice.

We investigated the possible roles of the prelimbic medial prefrontal cortex (PL) in the regulation of anxiety-like behaviors by pharmacologically activating the terminals of neuronal inputs or postsynaptic efferent neurons with a sodium channel activator veratrine. The extracellular glutamate levels were measured by in vivo microdialysis, and the behaviors were assessed with the open field (OF) test in mice simultaneously. The samples were collected every 10 min for 60 min, as basal levels of glutamate. The medium containing drugs were perfused for 30 min. The OF test was performed in the last 10 min of drug perfusion. After the drug treatments, the perfusion medium containing drugs was switched back to perfusion medium without drugs, and then samples were collected for another 90 min. The extracellular glutamate levels were significantly elevated after local perfusion of veratrine in the PL. At the same time, perfusion of veratrine in the PL produced anxiety-like behaviors in mice. Local coperfusion of a sodium channel blocker, lamotrigine, completely diminished the veratrine-induced elevated extracellular glutamate levels and the behavioral changes. Local coperfusion of an NMDA receptor antagonist, MK-801, but not a non-NMDA (AMPA/kainate) receptor antagonist, CNQX, completely diminished the behavioral changes without any effects on the veratrine-induced elevated extracellular glutamate levels. This study demonstrates that the activation of the PL with veratrine induces anxiety-like behaviors via NMDA receptor-mediated glutamatergic neurotransmission in mice.

Plasticity-related gene 1 is important for survival of neurons derived from rat neural stem cells.

Plasticity-related gene 1 (Prg1) is a membrane-associated lipid phosphate phosphatase. In this study, we first investigated the role of Prg1 in the survival of neurons derived from rat neural stem cells (NSCs) using small interfering RNA (siRNA). Prg1 knock-down decreased the cell number. Interestingly, Prg1 knock-down increased genomic DNA fragmentation, suggesting the possible induction of apoptosis. Exogenously expressed Prg1 rescued the cells from death and restored the loss of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity induced with Prg1 siRNA. However, exogenously expressed mutated-Prg1 (the 253rd amino acid, histidine253, had been changed to alanine) did not rescue the cell death or restore the MTT activity. Histidine253 of Prg1 has been reported to be important for lipid phosphate phosphatase activity. These results suggest that Prg1 is important for survival of neurons through its dephosphorylation activity.

Characterization of a WD-repeat family protein WDR3 in the brain of WDR3 hetero knockout mice.

The nuclear protein WDR3 is a member of the WD-repeat family and is a component of the 18S pre-rRNA processing complex. However, the expression and function of WDR3 in the brain remains unknown. To characterize WDR3 in the adult mouse brain, we developed Wdr3 heterozygous knockout (WDR3-HKO) mice. Notably, no homozygous Wdr3 knockout mice were born, suggesting that complete absence of WDR3 causes lethal abnormalities during embryogenesis. Brain Wdr3 mRNA expression was significantly reduced to 60% in the WDR3-HKO mice compared to wild type (WT) mice, while the expression of 18S rRNA did not decline. Using immunohistochemistry and X-gal staining, we demonstrated that WDR3 is widely expressed in the mouse brain, especially in the hippocampus, habenular nucleus, and cerebellum. We observed no differences in body weight during adulthood or developmental weight gain between the WDR3-HKO and WT mice. Interestingly, WDR3-HKO mice exhibited a slight but significant increase in spontaneous locomotor activity compared to WT littermates. In conclusion, the WDR3-HKO mice showed no significant phenotypic changes. Further studies are required to explore the behavioral characteristics of WDR3-HKO mice.

Effects of riluzole on psychiatric disorders with anxiety or fear as primary symptoms: A systematic review.

AIM: Previous behavioral pharmacology studies involving rodents suggested riluzole had potential to be an ideal psychotropic drug for psychiatric disorders with anxiety or fear as primary symptoms. Several clinical studies have recently been conducted. The purpose of this study was to gather information about the efficacy and tolerability of riluzole for patients with those symptoms. METHODS: We searched PubMed, PsycINFO, CINAHL, EMBASE, and the Cochrane database from inception until April 2021, and performed manual searches for additional relevant articles. This review included: (1) studies involving participants that were patients with generalized anxiety disorder (GAD), social anxiety disorder, panic disorder, obsessive-compulsive disorder (OCD), posttraumatic stress disorder (PTSD), acute stress disorder, or phobias; and (2) randomized controlled trials (RCTs) or intervention studies (e.g., single arm trials) examining the effects and safety of riluzole. RESULTS: Of the 795 identified articles, four RCTs, one RCT subgroup-analysis, and three open-label trials without control groups met the inclusion criteria. Most trials evaluated the efficacy of riluzole as an augmentation therapy with selective serotonin reuptake inhibitors and other antidepressants for PTSD, OCD, or GAD. However, there was insufficient evidence to confirm the effects of riluzole for patients with these psychiatric disorders. Most trials demonstrated adequate study quality. CONCLUSIONS: This review found insufficient evidence to confirm the effects of riluzole for psychiatric disorders with anxiety or fear as primary symptoms. It would be worthwhile to conduct studies that incorporate novel perspectives, such as examining the efficacy of riluzole as a concomitant medication for psychotherapy.

Hydrogen sulfide and polysulfides induce GABA/glutamate/D-serine release, facilitate hippocampal LTP, and regulate behavioral hyperactivity.

Hydrogen sulfide (H2S) and polysulfides (H2Sn, n ≥ 2) are signaling molecules produced by 3-mercaptopyruvate sulfurtransferase (3MST) that play various physiological roles, including the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory formation, by enhancing N-methyl-D-aspartate (NMDA) receptor activity. However, the presynaptic action of H2S/H2Sn on neurotransmitter release, regulation of LTP induction, and animal behavior are poorly understood. Here, we showed that H2S/H2S2 applied to the rat hippocampus by in vivo microdialysis induces the release of GABA, glutamate, and D-serine, a co-agonist of NMDA receptors. Animals with genetically knocked-out 3MST and the target of H2S2, transient receptor potential ankyrin 1 (TRPA1) channels, revealed that H2S/H2S2, 3MST, and TRPA1 activation play a critical role in LTP induction, and the lack of 3MST causes behavioral hypersensitivity to NMDA receptor antagonism, as in schizophrenia. H2S/H2Sn, 3MST, and TRPA1 channels have therapeutic potential for psychiatric diseases and cognitive deficits.

Dexamethasone indirectly induces Ndrg2 expression in rat astrocytes.

N-myc downstream-regulated gene 2 (Ndrg2) has been associated with cell proliferation, differentiation, and apoptosis. Ndrg2 expression in the brain is induced by glucocorticoid treatment or chronic stress in vivo. It has been postulated that glucocorticoid-induced Ndrg2 expression in astrocytes is regulated by the glucocorticoid response element half-site (GRE1/2) upstream of the Ndrg2 transcription site. Here we examined the mechanisms of dexamethasone-induced Ndrg2 expression in rat astrocytes. Ndrg2 mRNA expression in primary astrocytes was significantly increased after 24 hr of exposure to dexamethasone in a concentration-dependent manner. Dexamethasone-induced Ndrg2 mRNA and protein expression was blocked by pretreatment with RU486, a glucocorticoid receptor antagonist. Moreover, dexamethasone-induced Ndrg2 mRNA expression was reduced by pretreatment with the protein synthesis inhibitor cycloheximide. The Ndrg2 reporter assay showed that deletion of a putative GRE1/2, located upstream of Ndrg2, did not affect induction by dexamethasone. A region between -755 and -701 bp from the transcription start site was shown to regulate induction by dexamethasone using promoter constructs progressively deleted from the 5' to 3' ends. This region contained the predicted transcription factor binding sites for early B-cell factor 1 (Ebf1), nuclear factor-κB (NFκB), and paired box gene 5 (Pax5). Mutation at the NFκB- or Pax5-binding site, but not the Ebf1 binding site, abolished dexamethasone-induced promoter activation. These results indicate that Ndrg2 expression was indirectly induced by dexamethasone at the DNA level, potentially by the binding of NFκB or Pax5 to the transcription factor binding sites, and GRE1/2 was not involved in this induction.

Rhotekin modulates differentiation of cultured neural stem cells to neurons.

Rhotekin is a downstream signal of Rho and is expressed in the central nervous system. However, the physiological role of rhotekin in the development of neural stem cells (NSCs) into neurons is unknown. In this study, we knocked down the expression of rhotekin protein with small interfering RNA (siRNA) in the NSCs and in neural differentiated cells and measured cell proliferation, differentiation, neurite length, and survival. By using immunocytochemistry and Western blot, the production of rhotekin was observed in NSCs and neuronal cells. Furthermore, rhotekin production was increased in accordance with neural differentiation. Rhotekin knock-down reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity and increased the cell death 72 hr after transfection in neurons. On the other hand, in NSCs, rhotekin knock-down increased MTT activity and the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells. In the present study, we demonstrated that rhotekin is required for maintenance and survival of neurons and positively regulates differentiation and neurite outgrowth. Moreover, we found that rhotekin is produced in NSCs and that the role of rhotekin is to regulate cell proliferation negatively. In conclusion, these results suggest that rhotekin is one of the key molecules in the differentiation of NSCs into neurons.

Indirect exposure to socially defeated conspecifics using recorded video activates the HPA axis and reduces reward sensitivity in mice.

Rodents perceive the emotional states of conspecifics using vision. In the present study, we demonstrated that exposure to the video-recorded distress of conspecifics induces stress responses in male C57BL/6J mice. A single exposure to a video-recorded scene of the social defeat stress (SDS) increased plasma corticosterone levels in these mice. This physiological change was suppressed by blocking the visual information, suggesting that vision plays a crucial role in inducing stress responses. Furthermore, after exposure to the video, there were increased numbers of c-Fos-positive neurons in the anterior cingulate cortex and other brain areas that are associated with the negative valence and empathy systems, but not in the regions related to the pain signaling. In addition, repeated exposure to SDS videos induced an apparent reduction in reward sensitivity in the sucrose preference test, but did not affect avoidance behaviour in the social interaction test or immobility behaviour in the forced swim test. Reduced reward sensitivity in mice reflects anhedonia, which is a core symptom of depression in humans. Our video SDS model therefore provides a unique opportunity to not only understand the mechanisms underlying stress-induced anhedonia, but also to screen effective candidate molecules for stress-related disorders with greater reproducibility.

The effects of emotional stress are not identical to those of physical stress in mouse model of social defeat stress.

In this study, we investigated the effects of emotional stress and physical stress using the social defeat stress (SDS) model in mice. Male C57BL/6 J mice were attacked by male non-retired ICR mice for 10 min daily for 10 days (physical stress; PS), while the other cohort of mice witnessed the defeat (emotional stress; ES). As a result, both PS and ES mice exhibited decreased social behavior in the social interaction test (SIT) and increased immobility in the forced swim test (FST). Interestingly, only ES mice exhibited decreased sucrose preference, and only PS mice exhibited decreased time spent in the open arms in the elevated plus-maze test. ES mice did not exhibit increased levels of corticosterone and epinephrine after a single stress exposure, but showed a decrease in plasma CXCL16 levels 1 month after stress exposure. Finally, a RhoA/Rho kinase inhibitor, fasudil, which has been reported to attenuate the effects of chronic stress, suppressed the increased immobility in the FST in PS mice, but not in ES mice. These results demonstrate that, although ES and PS mice shared many characteristics, the effects of emotional stress are not identical to those of physical stress in mice.

Lysophosphatidic acid levels in cerebrospinal fluid and plasma samples in patients with major depressive disorder.

Major depressive disorder (MDD) is the most common psychiatric disorders. However, a biochemical marker has yet to be established for clinical purposes. It is proposed that lysophosphatidic acid (LPA, 1-acyl-2-sn-glycerol-3-phosphoate) plays some important roles in emotional regulation of experimental animals. Therefore, in this study, we measured LPA levels using enzyme-linked immunosorbent assays of cerebrospinal fluid (CSF) and plasma samples from patients with MDD. The participants were 52 patients and 49 normal healthy controls for CSF study, and 47 patients and 44 controls for plasma study. We used the Japanese version of the GRID Hamilton Depression Rating Scale (17-item version) for the assessment of depressive symptoms. We found no associations between LPA levels (CSF or plasma) and either diagnosis or severity of MDD, or with psychotropic medication. In conclusion, our data suggest that LPA levels likely would not serve as a practical biomarker of MDD.

Levels of lysophosphatidic acid in cerebrospinal fluid and plasma of patients with schizophrenia.

It is suggested that lysophosphatidic acid (LPA) plays a key role in the pathophysiology of schizophrenia. In this study, we measured LPA levels by enzyme-linked immunosorbent assay in cerebrospinal fluid (CSF) and plasma samples. The participants were 49 patients with schizophrenia and 49 normal healthy controls for CSF study, and 42 patients and 44 controls for plasma study. We found that LPA levels in the patients were not significantly different from those of controls in CSF (controls: 0.189 ±â€¯0.077 µM, patients: 0.175 ±â€¯0.067 µM; P = 0.318) and plasma samples (controls: 0.131 ±â€¯0.067 µM, patients: 0.120 ±â€¯0.075 µM; P = 0.465). On the other hand, CSF levels in medicated patients (0.162 ±â€¯0.061 µM) were significantly lower than those observed in unmedicated patients (0.224 ±â€¯0.067 µM, P = 0.038), suggesting that our findings could be masked by the influence of medication with antipsychotics. Interestingly, we detected significant negative correlation between PANSS scores and plasma LPA levels, especially in males and in unmedicated patients. Our result suggests that LPA levels in CSF and plasma samples would not serve as a diagnostic biomarker, but plasma levels could be used for symptomatic assessment of schizophrenia.

Prg1 is regulated by the basic helix-loop-helix transcription factor Math2.

Math2 (NEX-1/NeuroD6) is a member of the basic helix-loop-helix transcription factor family and is involved in neuronal differentiation and maturation. In this study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. In the promoter region of rat Prg1, we identified four E-boxes [designated -E1 to -E4 (CANNTG)] recognized by the basic helix-loop-helix transcription factor. Using chromatin immunoprecipitation assays, we found that Math2 directly bound to at least one of these E-boxes. The Prg1 reporter assay showed that -E1 was critical for the regulation of Math2-mediated Prg1 expression. Investigation of the functional roles of Math2 and Prg1 in PC12 cells revealed that 72 h after transfection with either Math2 or Prg1, neurite length and number were significantly induced. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and that the Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.

Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway.

Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimer's disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1DeltaR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1DeltaR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.

Antidepressant-like effects of the delta-opioid receptor agonist SNC80 ([(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide) in an olfactory bulbectomized rat model.

The responses of olfactory bulbectomized (OBX) rats to antidepressant treatment are similar to those of depressed patients since chronic administration of an antidepressant reverses OBX-induced behavioral and physiological changes. Previously, using several animal models, it was demonstrated that single treatment with delta-opioid receptor agonists produced an antidepressant-like effect. This study examined the antidepressant effects resulting from subchronic exposure for 8 days to the delta-opioid receptor agonist SNC80 in an OBX rat model of depression. The olfactory bulbs were removed by suction. The emotionality of rats was measured by scoring their responses to given stimuli, i.e., attack, startle, struggle, and fight responses. The OBX rats chronically treated with vehicle for 7 days at 14 days following surgery showed a significant increase in emotionality score and a decrease in the time spent and entries in the open arm of a plus-maze. In the case of OBX rats, these changes were dose- and time-dependently reversed by chronic SNC80 treatment (1-10 mg/kg, s.c.) for 7 days, as same as desipramine (10 mg/kg, i.p.). Moreover, the concentration of 5-HT and its metabolite 5-HIAA in the frontal cortex, hippocampus, and amygdala were decreased in OBX rats, and these changes were also normalized by SNC80 treatment, rather than desipramine treatment. In addition, SNC80 also significantly reversed the loss of TH-positive cells produced by OBX in the dorsal raphe. In conclusion, we demonstrated that subchronic SNC80 treatment could completely reverse OBX-induced behavioral abnormalities and defects in serotonergic function.

Administration of riluzole to the basolateral amygdala facilitates fear extinction in rats.

A general understanding exists that inhibition of glutamatergic neurotransmission in the basolateral amygdala (BLA) impairs fear extinction in rodents. Surprisingly, we recently found that systemic administration of riluzole, which has been shown to inhibit the glutamatergic system, facilitates extinction learning in rats with a preconditioned contextual fear response. However, the mechanisms underlying this paradoxical effect of riluzole remain unclear. In this study, adult male Wistar rats were bilaterally cannulated in the BLA to examine the effects of intra-BLA administration of riluzole. We also compared the effects of riluzole with those of d-cycloserine, a partial agonist at the glycine-binding region of the N-methyl-d-aspartate (NMDA) receptor. In this study, intra-BLA administration of either riluzole or d-cycloserine facilitated extinction learning of contextual fear in conditioned rats. In addition, both riluzole and d-cycloserine enhanced the acquisition of recognition memory in the same model. However, intra-BLA injections of riluzole, but not d-cycloserine, had a potent anxiolytic-like effect when investigated using an elevated plus-maze test. Our findings suggest that riluzole-induced facilitation of extinction learning in rats with a preconditioned contextual fear reflects an indirect effect, resulting from the intra-BLA administration of the drug, and might not be directly related to inhibition of glutamatergic signaling. Further research is needed to clarify the mechanisms underlying the paradoxical effect of riluzole on fear extinction learning observed in this study.

Post-reexposure administration of riluzole attenuates the reconsolidation of conditioned fear memory in rats.

Recently, we demonstrated that riluzole, which has been shown to block the glutamatergic system, facilitates fear extinction in rats. Here, we undertook experiments on contextual fear conditioning to clarify the actions of riluzole on the reconsolidation of fear memory in rats. We used the fast-acting benzodiazepine midazolam as a reconsolidation-inhibiting control drug. We demonstrated that riluzole (3 mg/kg) and midazolam (1 mg/kg) impaired the reconsolidation of contextual fear memory. Results from spontaneous recovery experiments also suggested that riluzole attenuated reconsolidation. Indeed, conditioned fear did not recover spontaneously 4 weeks after a short (3 min) reexposure and riluzole administration, whereas it recovered after a long (10 min) reexposure. Using western blotting, we demonstrated that a short reexposure increased the phosphorylation of cyclic adenosine monophosphate response element binding protein significantly in the dorsal part of hippocampus, but not in the medial prefrontal cortex. Interestingly, this phosphorylation was attenuated by riluzole with short reexposure. In addition, bilateral microinjection of riluzole (2 µM/0.2 µl/side) directly into the dorsal hippocampus clearly attenuated the reconsolidation. These findings suggested that the attenuating effect of riluzole on the reconsolidation of fear memory involves, at least in part, the dorsal part of the hippocampus. In conclusion, we demonstrated that riluzole attenuates the reconsolidation of fear memory in rats.

Administration of a delta opioid receptor agonist KNT-127 to the basolateral amygdala has robust anxiolytic-like effects in rats.

RATIONALE: We previously reported that systemic administration of a selective delta opioid receptor (DOP) agonist, KNT-127, produced potent anxiolytic-like effects in rats. Interestingly, DOPs are highly distributed in the basolateral region of the amygdala (BLA). OBJECTIVES: In this study, we investigated the effect of intra-BLA administration of KNT-127 on anxiety-like behaviors in rats. METHODS AND RESULTS: In the elevated plus maze test, bilateral injection of KNT-127 into the BLA significantly and dose-dependently increased time spent in the open arms. The magnitude of KNT-127 (0.08 µg/0.2 µl)-induced anxiolytic-like effects was similar to muscimol (0.1 µg/0.2 µl), which is a selective agonist for the gamma amino butyric acid type A receptors. Further, anxiolytic-like effects of KNT-127 were abolished by pretreatment with naltrindole, a selective DOP antagonist, suggesting that KNT-127-induced anxiolytic-like effects are mediated by DOPs. These anxiolytic-like effects were confirmed using another innate anxiety model, the open field test. Interestingly, intra-BLA administration of KNT-127 also induced anxiolytic-like effects in the contextual fear conditioning test. Moreover, these effects were also abolished by naltrindole pretreatment. Finally, we demonstrated that intra-BLA administration of KNT-127 facilitates extinction learning of contextual fear in conditioned rats. CONCLUSIONS: Altogether, our findings clearly demonstrate that intra-BLA administration of KNT-127 in rats has robust anxiolytic-like effects not only in innate anxiety-like behavioral tests but also in the contextual fear conditioning test.