The delta opioid receptor agonist KNT-127 in the prelimbic medial prefrontal cortex attenuates veratrine-induced anxiety-like behaviors in mice

Accepted Manuscript Title: The delta opioid receptor agonist KNT-127 in the prelimbic medial prefrontal cortex attenuates veratrine-induced anxiety-like behaviors in mice Authors: Akiyoshi Saitoh, Satoshi Suzuki, Akinobu Soda, Masanori Ohashi, Misa Yamada, Jun-Ichiro Oka, Hiroshi Nagase, Mitsuhiko Yamada PII: DOI: Reference:

S0166-4328(17)30781-7 http://dx.doi.org/10.1016/j.bbr.2017.08.041 BBR 11060

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

12-5-2017 21-8-2017 28-8-2017

Please cite this article as: Saitoh Akiyoshi, Suzuki Satoshi, Soda Akinobu, Ohashi Masanori, Yamada Misa, Oka Jun-Ichiro, Nagase Hiroshi, Yamada Mitsuhiko.The delta opioid receptor agonist KNT-127 in the prelimbic medial prefrontal cortex attenuates veratrine-induced anxiety-like behaviors in mice.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.08.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Behavioral Brain Research

The delta opioid receptor agonist KNT-127 in the prelimbic medial prefrontal cortex attenuates veratrine-induced anxiety-like behaviors in mice Akiyoshi Saitoha*, Satoshi Suzukia,b, Akinobu Sodaa,b, Masanori Ohashia,b, Misa Yamadaa, Jun-Ichiro Okab, Hiroshi Nagasec, Mitsuhiko Yamadaa a

Department of Neuropsychopharmacology, National Institute of Mental Health, National Center of Neurology and Psychiatry, Tokyo 187-8553, Japan b Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan c International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki 305-8575, Japan

*Corresponding author: Akiyoshi Saitoh, Ph.D. Department of Neuropsychopharmacology National Institute of Mental Health National Center of Neurology and Psychiatry 4-1-1 Ogawahigashimachi, Kodaira Tokyo 187-8553, Japan Tel: +81-42-341-2711 Fax: +81-42-346-1994 E-mail: [email protected]

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Abstract: We previously reported that systemic administration of the selective delta opioid receptor (DOP) agonist KNT-127 produces potent anxiolytic-like effects in rats. Although a higher distribution pattern of DOPs was reported in the prelimbic medial prefrontal cortex (PL-PFC) of rodents, the role of DOPs in PL-PFC and in anxiolytic-like effects have not been well examined. Recently, we demonstrated that activation of PL-PFC with the sodium channel activator veratrine increases glutamatergic neurotransmission and produces anxiety-like behaviors in mice. Therefore, we investigated the effects of co-perfusion with KNT-127 in PL-PFC on veratrine-induced anxiety-like behaviors in mice. We also simultaneously measured extracellular glutamate and GABA levels. In addition, we assessed the effect of KNT-127 on the expression of c-Fos in sub-regions of the amygdala. Extracellular glutamate levels were measured in seven-week-old male C57BL/6N mice using an in vivo microdialysis-HPLC/ECD system, and behaviors were assessed simultaneously in an open field test. Basal levels of glutamate were measured by collecting samples every 10 min for 60 min. The drug-containing medium was perfused for 30 min, and the open field test was performed during the last 10 min of drug perfusion. After drug treatments, the perfusion was switched from drug-containing medium to control medium without drugs and samples were collected for another 90 min. KNT-127 co-perfusion completely diminished veratrine-induced anxiety-like behaviors and attenuated the veratrine-induced increase in extracellular glutamate levels in PL-PFC. Interestingly, KNT-127 perfusion alone in PL-PFC did not affect anxiety-like behaviors. Local perfusion of veratrine in PL-PFC induced c-Fos 2

immunoreactivity in sub-regions of amygdala. Co-perfusion of KNT-127 diminished c-Fos expression. Here we demonstrate that the DOP agonist KNT-127 in PL-PFC attenuates veratrine-induced anxiety-like behaviors in mice. These effects may be caused by the presynaptic suppression of activated glutamatergic transmission in PL-PFC, which projects to sub-regions of the amygdala. We propose that compounds like KNT-127, which inhibit glutamatergic transmission in PL-PFC, are candidates for novel anxiolytics.

Keywords: Microdialysis, Innate anxiety, Anxiogenic, Anxiolytic, Antidepressant, Opioid receptor,

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Introduction We previously reported that systemic administration of the selective delta opioid

receptor

(DOP)

agonist

1,2,3,4,4a,5,12,12a-octahydro-2-methyl-4aβ,1β-([1,2]benzenomethano)-2,6-diazanaphth acene-12aβ,17-diol (KNT-127) [1] produces potent anxiolytic-like effects in the open field (OF) test, elevated plus-maze test, and light-dark test in rats [2, 3]. Interestingly, radioautography revealed a higher distribution pattern of DOPs in the prelimbic medial prefrontal cortex (PL-PFC) in rat [4] and monkey brains [5]. Chung et al. suggested that DOPs are located on presynaptic excitatory terminals in the medial prefrontal cortex (mPFC) based on a conditional DOP knockout mouse line targeting the receptor gene specifically in GABAergic neurons [6, 7]. However, the role of DOPs in PL-PFC on anxiolytic-like effects is not well examined. Recently, we demonstrated that activation of PL-PFC with the sodium channel activator veratrine elicits increased glutamatergic neurotransmission and leads to anxiety-like behaviors via the N-methyl-D-aspartate (NMDA) receptor in mice [8]. Further, we found significant correlations between behavioral changes and c-Fos immunoreactivity in sub-regions of amygdala, the lateral amygdala (LA), basolateral amygdala (BLA), and central nuclei (CeA) of the amygdala [9]. Taken together, this suggests that activation of glutamatergic neurotransmission, which projects into the amygdala from PL-PFC, plays an important role in the expression of anxiety-like behaviors after veratrine perfusion in PL-PFC. Therefore, we investigated the effects of KNT-127 co-perfusion in PL-PFC on veratrine-induced anxiety-like behaviors in mice. We also simultaneously measured extracellular glutamate and GABA levels. In addition, we assessed the effect of 4

KNT-127 on the expression of c-Fos in sub-regions of the amygdala.

Materials and Methods Animals Seven-week-old male C57BL/6N mice (Japan SLC Inc., Shizuoka, Japan) were used. Mice had free access to food and water in an animal room maintained at 23 ± 1°C, with a 12 h light-dark cycle (lights were automatically switched on at 8:00 am). The study protocol was approved by the Institutional Animal Care and Use Committee of the National Center of Neurology and Psychiatry (Approval No. 2014018).

Drugs The drugs used were veratrine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and KNT-127 (synthesized by Prof. Nagase). The dose of veratrine used in this study (100 μM) was based on results from our previous experiments [8]. Veratrine hydrochloride and KNT-127 were dissolved in perfusion medium (117 mM NaCl, 4.02 mM KCl, 2.3 mM CaCl2/2H2O) and locally administered through a microdialysis probe.

Microdialysis study The microdialysis study was conducted in accordance with previously described methods [8]. Briefly, a microdialysis guide cannula was stereotactically implanted in PL-PFC (AP +1.9 mm, L +0.3 mm, and V −1.8 mm from the bregma) in mice under anesthesia. Postoperatively, to recover from surgery, animals were housed in single cages for at least 24 h before microdialysis experiments. Microdialysis probes (A-I-3-01; EICOM, Kyoto, Japan) were continuously perfused with perfusion medium 5

at a rate of 2.0 µL/min. Microdialysis sampling was initiated 120 min after the onset of probe perfusion. Dialysate samples were collected every 10 min for 40 min to simultaneously determine basal levels of glutamate and GABA. After determining basal levels, the perfusion medium was switched to a drug-containing medium by using a liquid switch (SI-60; EICOM, Kyoto, Japan). The drug-containing medium was perfused for 30 min, and the OF test was performed during the last 10 min of drug perfusion. After drug treatment, the drug-containing medium perfusion was switched back to perfusion medium without drugs, and then dialysate samples were collected for 90 min. The location of the dialysis probes was verified at the end of each experiment.

Open field (OF) test The OF test was conducted as previously described [8]. Briefly, each mouse was placed in a habituation cage (20 × 20 × 33 cm) for at least 2 h for adaptation to a new environment before the OF test. The OF apparatus consisted of a square area (50 × 50 cm) with opaque walls (height, 50 cm) placed in indirect light (50 lux). Mice were gently placed in a corner of the OF facing an opaque wall. They were allowed to freely explore the apparatus for 10 min. The total distance traveled, the percentage of the distance traveled in the center area (30 × 30 cm), and the time spent in the center area were automatically recorded using a video camera (Smart v.3.0; Panlab S.L., Barcelona, Spain). After each animal was removed, the apparatus was cleaned.

High-performance liquid chromatography (HPLC) assay The HPLC assay was conducted as previously reported [8]. Briefly, glutamate and GABA levels were analyzed via HPLC using an electrochemical detector 6

(HTEC-700, EICOM, Kyoto, Japan) after derivatization with o-phthalaldehyde (OPA; Wako Pure Chemical Industries, Ltd., Osaka, Japan). A reverse-phase column (Eicompack FA-3ODS, φ3 × 75 mm, EICOM, Kyoto, Japan) was used to separate glutamate and GABA at 40°C. The potential of the glassy carbon electrode (WE-GC, EICOM, Kyoto, Japan) was set at +0.6 V (vs. Ag/AgCl).

Brain samples Two hours after the OF test, mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and transcardially perfused with 20 ml of ice-cold heparinized-saline (10 unit/mL) followed by 20 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The brains were removed, stored in the same fixative for 24 h at 4°C, and subsequently immersed in 30% sucrose/4% paraformaldehyde in 0.1 M PB for 2 days at 4°C. The brains were then frozen and stored at −80°C.

Immunohistochemistry The brain sections (40 µm) were immunohistochemically stained using a free-floating method. The sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBST) for 30 min, treated with methanol containing 3% H2O2 for 5 min, and then blocked with PBST containing 3% normal goat serum (PBSTS) for 1 h. Sections were incubated with anti-c-Fos antibody (1:2,000; sc-52, Santa Cruz Biotechnology, Dallas, TX, USA) in PBSTS for 48 h at 4°C. After incubation with biotinylated anti-rabbit IgG antibody (1:500; Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature, sections were incubated with Elite avidin-biotin peroxidase complex according to the manufacturer’s instructions (Vector 7

Laboratories). The immunostaining reaction was developed using the ImmPACT DAB peroxidase substrate kit (Vector Laboratories). Sections were mounted on glass slides and dehydrated in ethanol solutions and xylene.

Quantification of c-Fos immunoreactive nuclei Microscopic images of brain sections were taken using an Olympus BX-60 microscope and digitized with an Olympus MP5Mc/OL CCD camera (Olympus, Tokyo, Japan). Nuclei positive for c-Fos were counted using ImageJ v1.46 software developed at the National Institutes of Health (http://rsbweb.nih.gov/ij/). In brief, the number of immunopositive nuclei (stained gray-black) was divided by the unit area (200 × 200 µm) for each sub-region of the amygdala, including the LA, BLA, and CeA. The ‘Threshold’ function of the ImageJ program, which highlights all of the target nuclei, was used. Image analysis was conducted on three sections per brain.

Data analysis Data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) was used for comparisons of more than two groups. Post-hoc individual group comparisons were made using the Bonferroni test for multiple comparisons. The student's t-test was used for comparisons of two groups. Analyses were performed using Graphpad Prism7J (Graphpad Software Inc., San Diego, CA, USA); p values <0.05 were considered statistically significant.

Results Emotional behaviors in the OF test 8

Compared with the control, veratrine (100 M) perfusion decreased the percentage of time spent in the center (one-way ANOVA: F4,42 = 4.29, p < 0.01; Bonferroni test: t = 3.42, p < 0.01; Fig. 1A), the percentage of distance moved in the center (one-way ANOVA: F4,42 = 2.36, p = 0.0687; Bonferroni test: t = 2.63, p < 0.05; Fig. 1B), and the total distance moved (one-way ANOVA: F4,42 = 5.25, p < 0.05; Bonferroni test: t = 3.02, p < 0.05; Fig. 1C). Next, we examined the effect of KNT-127 co-perfusion on emotional behaviors in the OF test after local perfusion of veratrine in PL-PFC. Compared with the control, co-perfusion with KNT-127 (3, 10, and 30 M) diminished veratrine-induced decreases in the percentage of time spent in the center (Bonferroni test: t = 2.48, t = 1.42, and t = 0.127, p > 0.05, respectively; Fig. 1A), the percentage of distance moved in the center (Bonferroni test: t = 0.908, t = 0.407, and t = 0.0646, p > 0.05, respectively; Fig. 1B), and the total distance moved (Bonferroni test t = 3.35, p < 0.05, t = 1.31, t = 0.169, p > 0.05, respectively; Fig. 1C) in a dose-dependent manner.

Effect of perfusing KNT-127 alone on emotional behaviors in the OF test We examined the effect of perfusing KNT-127 alone in PL-PFC on anxiety-like behaviors in the OF test (Fig. 1). Compared with the control group, perfusion of KNT-127 (30 M) alone did not influence the percentage of time spent in the center (t(16) = 0.400, p > 0.05; Fig. 1A), the percentage of distance moved in the center (t(16) = 0.589, p > 0.05; Fig. 1B), and the total distance moved (t(16) = 1.28, p > 0.05; Fig. 1C).

Extracellular glutamate and GABA levels 9

Basal levels of glutamate and GABA were 0.279 ± 0.0382 and 0.0178 ± 0.00167 M, respectively. We examined the effect of KNT-127 co-perfusion on the increase in extracellular glutamate and GABA levels induced by local perfusion of veratrine in PL-PFC (Fig. 2A, 2B). As shown in Fig 2A, veratrine (100 M) perfusion increased extracellular glutamate levels at 20 and 30 min after treatment compared with the control group. Co-perfusion of KNT-127 (3, 10, and 30 M) with veratrine decreased extracellular glutamate levels in a dose-dependent manner. The AUC data for extracellular glutamate levels in PL-PFC showed that veratrine (100 M) perfusion significantly increased glutamate levels after treatment compared with the control group (one-way ANOVA: F4,42 = 7.20, p < 0.01; Bonferroni test: t = 4.07, p < 0.01; Fig. 2C). Compared with the control, KNT-127 (3, 10, and 30 M) co-perfusion diminished the veratrine-induced increase in glutamate levels (Bonferroni test t = 4.54, p < 0.01, t = 2.63, p < 0.05, t = 1.22, p > 0.05, respectively; Fig. 2C) in a dose-dependent manner. As shown in Fig 2B, veratrine (100 M) perfusion increased extracellular GABA levels at 10, 20, and 30 min after treatment compared with the control group. The AUC data for extracellular GABA levels in PL-PFC showed that veratrine (100 M) perfusion significantly increased GABA levels after treatment compared with the control group (one-way ANOVA: F4,42 = 14.2, p < 0.01; Bonferroni test: t = 6.74, p < 0.01; Fig. 2D). Compared with the control group, KNT-127 (3, 10, and 30 M) co-perfusion did not diminish the veratrine-induced increase in GABA levels after

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treatment (Bonferroni test t = 5.42, p < 0.01, t = 5.83, p < 0.01, t = 3.98, p < 0.01, respectively; Fig. 2D).

Effect of perfusing KNT-127 alone on extracellular glutamate and GABA levels We examined the effect of perfusing KNT-127 alone in PL-PFC on extracellular glutamate and GABA levels (Fig. 3). The basal levels of glutamate and GABA were 0.178 ± 0.0362 and 0.0183 ± 0.00315 M, respectively. The perfusion of KNT-127 (30 M) alone increased extracellular glutamate levels compared with the control group (Fig. 3A). The maximum rate of increase in glutamate levels was 164% ± 44.4% at 20 min after KNT-127 perfusion alone (Fig. 3A). The AUC data for extracellular glutamate levels in PL-PFC showed that KNT-127 (30 M) perfusion alone significantly increased glutamate levels after local perfusion (t (16) = 2.43, p < 0.05; Fig. 3C). The AUC data of the control group was 2810 ± 102 (Fig. 3C). The AUC data of the KNT-127 group was 4130 ± 599 (Fig. 3C). The perfusion of KNT-127 (30 M) alone decreased extracellular GABA levels compared with the control group (Fig. 3B). The maximum rate of decrease in GABA levels was 89.8% ± 3.40% at 10 min after perfusion of KNT-127 alone (Fig. 3B). The AUC data for extracellular GABA levels in PL-PFC showed that KNT-127 (30 M) perfusion alone significantly decreased GABA levels after local perfusion (t (16) = 2.67, p < 0.05; Fig. 3D). The AUC data of the control group was 3110 ± 70.5 (Fig. 3D). The AUC data of the KNT-127 group was 2810 ± 86.3 (Fig. 3D).

Effect of local co-perfusion of KNT-127 on c-Fos expression in the amygdala

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The expression pattern of c-Fos protein in the mouse amygdala after local perfusion of drugs in PL-PFC is shown in Fig. 4. Compared with the control, local perfusion of veratrine in PL-PFC induced c-Fos immunoreactivity in the amygdala (Fig. 4A). We quantified the number of c-Fos immunoreactive nuclei in sub-regions of the amygdala, including the LA (Fig. 5B), BLA (Fig. 4C), and CeA (Fig. 4D) after veratrine perfusion in PL-PFC. The number of c-Fos-immunoreactive nuclei significantly increased after veratrine perfusion compared with the control in LA (one-way ANOVA: F3,27 = 3.74, p < 0.05; Bonferroni test: t = 3.30, p < 0.05), BLA (one-way ANOVA: F3,27 = 6.64, p < 0.05; Bonferroni test: t = 3.41, p < 0.05), and CeA (one-way ANOVA: F3,27 = 3.21, p < 0.05; Bonferroni test: t = 2.79, p < 0.05). Interestingly,

local

co-perfusion

of KNT-127 (30

µM) diminished

veratrine-induced c-Fos expression (Fig. 4A). Compared with the control, co-perfusion with KNT-127 (30 µM) diminished veratrine-induced increases in the number of c-Fos-immunoreactive nuclei in LA, BLA, and CeA (Bonferroni test: t = 1.83, t = 2.25, and t = 0.574, p > 0.05, respectively; Fig. 4B, 4C, 4D). KNT-127 alone had no significant effect on c-Fos immunoreactivity in any sub-region of the amygdala (Bonferroni test: t = 1.63, t = 0.652, and t = 0.0878, p > 0.05, respectively; Fig. 4B, 4C, 4D).

Discussion In the present study, perfusion of veratrine in PL-PFC of mice significantly decreased the percentage of time and distance spent in the center during the OF test, suggesting that activation of PL-PFC produces anxiety-like behaviors in mice. These results are consistent with our previous reports [8-11]. Interestingly, we found that 12

co-perfusion of KNT-127 completely abolished veratrine-induced decreases in the percentage of time and distance spent in the center during the OF test. These results suggest that KNT-127 in PL-PFC attenuates veratrine-induced anxiety-like behaviors in mice. We previously reported that systemic administration of KNT-127 produces its anxiolytic-like effect via DOPs in animal models of innate anxiety [2]. These results suggest that the anxiolytic-like effects induced by the systemic administration of KNT-127 might be mediated, at least in part, by the attenuation of PL-PFC activation. This study indicated that the total distance in the OF significantly decreased after perfusion of veratrine alone in PL-PFC. These results are also consistent with our previous reports [8-11]. Further, co-perfusion of KNT-127 completely abolished veratrine-induced decreases in the total distance in the OF. PL-PFC has connections with motor structures, which enables direct transmission of PL-PFC function to behavior. For example, a previous report indicated that inactivation of PL-PFC using cytotoxic lesioning increased the total distance moved by rats in the OF test [12]. The decreased activity in total distance in the OF may be considered an anxiety-like behavior in which a behavioral motor system is inhibited [13]. In the present study, co-perfusion of KNT-127 decreased the veratrine-induced increase in extracellular glutamate levels in PL-PFC. Several studies suggested that activation of DOPs causes presynaptic inhibition of glutamatergic excitatory neurons after electrophysiological stimulation. For example, Ostermeier et al. (2000) reported that in rat cortical slices, the endogenous DOP ligand [Met5] enkephalin and the selective peptidic DOP agonist D-Ala2-D-Leu5-enkephaline reduced the amplitude of excitatory postsynaptic currents (EPSCs) evoked by high-frequency electrical stimulation [14]. Further, Atwood et al. (2014) reported that [D-Pen2,5]-Enkephalin 13

(DPDPE) induced an increase in the ratio of synaptic responses to paired pulses in EPSCs, which was reliably associated with the release probability of presynaptic neurotransmission in rat dorsal striatum slices, suggesting that DOP agonists presynaptically reduced the glutamate release probability [15]. Interestingly, in the present study, the veratrine-induced increase in extracellular GABA levels in PL-PFC did not change significantly after the co-perfusion of KNT-127. The present result is consistent with previous reports suggesting that DOPs are mainly located on presynaptic excitatory terminals in the mouse mPFC [6, 7]. Thus, we propose that KNT-127 in PL-PFC could cause presynaptic inhibition of glutamatergic excitatory neurons. Interestingly, perfusion of KNT-127 alone in PL-PFC caused a slight but significant increase in glutamate levels. The present result is inconsistent with previous reports suggesting that DOP stimulation causes presynaptic inhibition of glutamatergic excitatory neurons. Further studies are needed to understand the complicated effects of KNT-127 in the mouse PL-PFC. Although perfusion of KNT-127 alone in PL-PFC caused a significant increase in glutamate levels, KNT-127 had no effect on the percentages of time and distance in the center and the total distance in the OF test at any dose. Therefore, these findings do not alter our conclusion that co-perfusion of KNT-127 in PL-PFC attenuates veratrine-induced anxiety-like behaviors. In contrast, perfusion of KNT-127 alone in PL-PFC caused a small but significant decrease in GABA levels. Recently, Chung et al. (2015) suggested that DOPs are mainly located on presynaptic excitatory terminals in the mPFC because, in a conditional DOP knockout mouse line targeting the receptor gene specifically in GABAergic neurons, the expression of DOPs remained intact in the frontal cortex compared with control mice [7]. Following KNT-127 perfusion, the slight inhibition of 14

GABA release may reflect the characteristic anatomical distribution of DOP in PL-PFC. Contrarily, Tanahashi et al. (2012) reported that intraperitoneal administration of KNT-127 produced significant increases in the release of GABA in mPFC [16]. This report is inconsistent with our present findings that perfusion of KNT-127 alone in PL-PFC decreases the release of GABA. In the present study, we examined the effects on GABA release after local perfusion in PL-PFC, whereas Tanahashi et al. examined the effects of systemic administration of KNT-127 [16]. In addition, Tanahashi et al. (2012) inserted cannulas in mPFC without distinguishing between PL- and the infralimbic (IL)-PFC, which is a heterogeneous cortical structure of PFC [16]. We previously reported that these two regions have different functions in neural transmission, although IL-PFC is adjacent to PL-PFC [10]. These differences could affect how KNT-127 alters the release of GABA. The effect of a selective DOP antagonist naltrindole to KNT-127 treatment on veratrine-induced glutamate and/or GABA release was examined in our pilot study by co-perfusion of naltrindole and KNT-127 (data not shown). Surprisingly, we found that naltrindole perfusion alone diminishes the veratrine-induced increase in extracellular glutamate levels. Thus, our results still have some limitations; KNT-127 may have a pathway other than delta activity to induce anxiolytic-like effect. In the present study, PL-PFC stimulation by veratrine perfusion increased c-Fos expression in sub-regions of the amygdala, including the LA, BLA, and CeA, suggesting that PL-PFC stimulation activates a neural network which projects to sub-regions of amygdala. These results are consistent with our previous reports [9]. It was reported that there is a direct projection from PL-PFC to the amygdala. For example, early studies in rats demonstrated that significant PL-PFC fibers distribute 15

selectively to intercalated nuclei of the amygdala, LA, BLA, and capsular CeA [17, 18], whereas McDonald et al. (1996) demonstrated that PL-PFC targets the anterior amygdaloid area, BLA, and CeA [19]. Vertes (2004) also demonstrated that PL-PFC fibers primary project to BLA and CeA and light projects to LA [20]. These findings support the present results that indicate that local perfusion of veratrine in PL-PFC induces c-Fos immunoreactivity in LA, BLA, and CeA. We previously reported that the NMDA receptor antagonist MK-801 completely diminished the increase in c-Fos expression in sub-regions of the amygdala [9]. Interestingly, co-perfusion of KNT-127 in PL-PFC diminished the veratrine-induced increase in c-Fos expression in sub-regions of the amygdala. These inhibitory effects after KNT-127 co-perfusion were accompanied by the suppression of veratrine-induced elevation in extracellular glutamate levels. These findings also support our hypothesis that KNT-127 in PL-PFC presynaptically attenuates the release of glutamate. The present results indicated that KNT-127 exhibited weak inhibition in BLA compared to other sub-regions of amygdala. A previous study suggested that the selective excitation of the BLA-CeA pathway in mice expressed the excitatory channelrhodopsin 2 in BLA neurons decreases the time spent open arm on an elevated plus-maze [21], suggesting the promotion of anxiogenic-like behavior. In contrast, the activation of the BLA-lateral central nucleus of amygdala pathway in mice causes the anxiolytic-like behavioral phenotype of exploration in the open arm on the elevated plus-maze [21]. These results suggest that there are distinct neuronal pathways in BLA, which could cause either promotion or inhibition of anxiety-like behaviors [22]. These findings may explain the weak inhibition of c-Fos expression in BLA after KNT-127 co-perfusion. Further studies are necessary to elucidate the complicated modulation of 16

BLA after local perfusion of DOP agonist in PL-PFC.

Conclusion Here we demonstrated that the DOP agonist KNT-127 in PL-PFC attenuates veratrine-induced anxiety-like behaviors in mice. These effects may be caused by the presynaptic suppression of activated glutamatergic transmission in PL-PFC, which projects to sub-regions in the amygdala. We propose that compounds like KNT-127, which inhibit glutamatergic transmission in PL-PFC, are possible candidates for novel anxiolytics.

Funding and Disclosure This research was financially supported by research grants from an Intramural Research Grant (27-1) for Neurological and Psychiatric Disorders, Japan Society for the Promotion of Science (JSPS) KAKENHI, Grant Number 26461730, 17K10286.

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Figure captions Fig. 1. Emotional behaviors in the OF test after local perfusion of the selective DOP agonist KNT-127 alone and with the sodium channel activator veratrine in PL-PFC. Panel A shows the percentage of time spent in the center area after local perfusion of veratrine, veratrine + KNT-127 or KNT-127 alone in PL-PFC. Panel B shows the percentage of the distance traveled in the center area after local perfusion of veratrine, veratrine + KNT-127 or KNT-127 alone in PL-PFC. Panel C shows the total distance after local perfusion of veratrine, veratrine + KNT-127 or KNT-127 alone in PL-PFC. Panel D illustrates video-tracked movements in the OF after local perfusion of veratrine, veratrine + KNT-127 or KNT-127 alone in PL-PFC. Each column represents the mean and SEM (n = 8–10 mice/group). *p < 0.05, ** p < 0.01 (one-way ANOVA followed by the Bonferroni test). ns (no significant effect) vs. control groups (one-way ANOVA or student’s t-test).

Fig. 2. Percentage change in extracellular glutamate (A) and GABA (B) levels in PL-PFC after local perfusion of the selective DOP agonist KNT-127 with the sodium channel activator veratrine. After stabilization of extracellular glutamate levels, the perfusion medium was switched to drug-containing medium with veratrine or veratrine 20

+ KNT-127 for 30 min (0–30 min, dotted line). The OF test was performed simultaneously with veratrine perfusion for 20–30 min (closed column). Glutamate and GABA levels are expressed as a percentage of basal levels, and they were calculated from four consecutive samples before the perfusion of veratrine was initiated. Each column represents the mean and SEM (n = 9–10 mice/group). The area under curve (AUC) for extracellular glutamate (C) and GABA (D) levels in PL-PFC after local perfusion of the selective DOP agonist KNT-127 with the sodium channel activator veratrine. The AUC was calculated from glutamate and GABA release curves and plotted using their extracellular levels during drug perfusion in PL-PFC over a 30 min period (0–30 min). Each column represents the mean and SEM (n = 9–10 mice/group). *p < 0.05, **p < 0.01, ns (no significant effect) vs. control groups (one-way ANOVA followed by the Bonferroni test).

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Fig. 3. Percentage changes in extracellular glutamate (A) and GABA (B) levels in PL-PFC after local perfusion of the selective DOP agonist KNT-127 alone. After stabilization of extracellular glutamate levels, the perfusion medium was switched to drug-containing medium with KNT-127 for 30 min (0–30 min, dotted line). The OF test was performed simultaneously with veratrine perfusion for 20–30 min (closed column). Glutamate and GABA levels are expressed as a percentage of basal levels and calculated from four consecutive samples before the perfusion of veratrine. Each column represents the mean and SEM (n = 8–10 mice/group). The area under curve (AUC) for extracellular glutamate (C) and GABA (D) levels in PL-PFC after local perfusion of the selective DOP agonist KNT-127. The AUC calculated from the glutamate and GABA release curves was plotted using their extracellular levels during drug perfusion in PL-PFC over a period of 30 min (0–30 min). Each column represents the mean and the SEM (n = 8–10 mice/group). *p < 0.05 vs. control groups (student's t-test).

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Fig. 4. Immunoreactivity of c-Fos in the amygdala after local perfusion of drugs in PL-PFC. Representative photomicrographs of c-Fos expression in the amygdala after local perfusion of the selective DOP agonist KNT-127 with the sodium channel activator veratrine (scale bar = 200 µm) (A). Immunoreactive nuclei of c-Fos in the LA (B), BLA (C), and CeA (D) were quantified after local perfusion of drugs into PL-PFC. Each column represents the mean and SEM (n = 6–10 mice/group). *p < 0.05, ns (no significant effect) vs. control group (one-way ANOVA followed by the Bonferroni test).

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