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Yasunori Masubuchi, Junta Nakahara, Satomi Kikuchi, Hiromu Okano, Yasunori Takahashi, Kazumi Takashima, Mihoko Koyanagi, Robert R. Maronpot, Toshinori Yoshida, Shim-mo Hayashi, and Makoto Shibutani
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We previously reported that exposure to α-glycosyl isoquercitrin (AGIQ) from the fetal stage to adulthood facilitated fear extinction learning in rats. The present study investigated the specific AGIQ exposure period sufficient for inducing this behavioral effect. Rats were dietarily exposed to 0.5% AGIQ from the postweaning stage to adulthood (PW-AGIQ), the fetal stage to postweaning stage (DEV-AGIQ), or the fetal stage to adulthood (WP-AGIQ). Fear memory, anxiety-like behavior, and object recognition memory were assessed during adulthood. Fear extinction learning was exclusively facilitated in the WP-AGIQ rats. Synaptic plasticity-related genes showed a similar pattern of constitutive expression changes in the hippocampal dentate gyrus and prelimbic medial prefrontal cortex (mPFC) between the DEV-AGIQ and WP-AGIQ rats. However, WP-AGIQ rats revealed more genes constitutively upregulated in the infralimbic mPFC and amygdala than DEV-AGIQ rats, as well as FOS-immunoreactive(+) neurons constitutively increased in the infralimbic cortex. Ninety minutes after the last fear extinction trial, many synaptic plasticity-related genes (encoding Ephs/Ephrins, glutamate receptors/transporters, and immediate-early gene proteins and their regulator, extracellular signal-regulated kinase 2 [ERK2]) were upregulated in the dentate gyrus and amygdala in WP-AGIQ rats. Additionally, WP-AGIQ rats exhibited increased phosphorylated ERK1/2+ neurons in both the prelimbic and infralimbic cortices. These results suggest that AGIQ exposure from the fetal stage to adulthood is necessary for facilitating fear extinction learning. Furthermore, constitutive and learning-dependent upregulation of synaptic plasticity-related genes/molecules may be differentially involved in brain regions that regulate fear memory. Thus, new learning-related neural circuits for facilitating fear extinction can be established in the mPFC.(DOI: 10.1293/tox.2020-0025; J Toxicol Pathol 2020; 33: 247–263)

Keywords: α-glycosyl isoquercitrin (AGIQ), fear extinction learning, synaptic plasticity, phosphorylated ERK1/2, rat

Introduction

Alpha-glycosyl isoquercitrin (AGIQ), also known as enzymatically modified isoquercitrin, is a polyphenolic flavonol glycoside derived by the enzymatic glycosylation of rutin, which is found in several plant species such as buckwheat (Fagopyrum esculentum Moench), rue (Ruta graveolens L.), and Japanese pagoda tree (Sophora japonica L.)1. AGIQ is a mixture of quercetin glycoside, consisting of isoquercitrin and its α-glucosylated derivatives, with 1–10 or more additional linear glucose moieties1. AGIQ is highly water soluble and has antioxidant potential12. AGIQ has been reported to exert antioxidant effects3 and to have anti-inflammatory3, anti-hypertensive4, anti-allergic5, and tumor suppressive6 properties. It has been found to be safe in a 90-day toxicity study7 and in genotoxicity assays8.

Recently, we reported that continuous exposure to 0.5% AGIQ in the diet from the fetal period through adulthood in rats facilitated fear extinction learning on the contextual fear conditioning test. Additionally, it facilitated the adult transcript upregulation of Fos, which encodes Fos proto-oncogene, AP-1 transcription factor subunit (FOS); Kif21b, which encodes kinesin family member 21B (KIF21B) in the hippocampal dentate gyrus; and Grin2d, which encodes glutamate ionotropic receptor N-methyl-D-aspartate (NMDA) -type subunit 2D (GRIN2D) in the amygdala9. AGIQ also increased the number of FOS-immunoreactive(+) hippocampal granule cells. Fos is one of the immediate-early genes (IEGs) involved in the synaptic plasticity of hippocampal granule cells10. Moreover, GRIN2D is known to function in enhancing synaptic plasticity associated with long-term memory11. These results suggest that the increases in FOS+ cells and Grin2d transcripts are associated with enhanced synaptic plasticity, which leads to the facilitation of fear extinction learning. Additionally, KIF21B was recently identified as a memory-rewriting molecule12 that is found in the hippocampal dentate gyrus, which suggests a relationship with facilitation of fear memory extinction. However, in our previous study, changes were found in the constitutive levels of gene expression and numbers of immunoreactive cells in animals that were not subjected to behavioral tests. Thus, learning-mediated neuronal cellular responses require further elucidation.

Fear memory is regulated by an interplay among the hippocampus, prelimbic cortex, infralimbic cortex, and amygdala13. The hippocampus is an important region involved in the formation and storage of context memory in the fear conditioning test13. The prelimbic cortex and infralimbic cortex are subdivisions of the medial prefrontal cortex (mPFC) and accelerate and suppress fear expression, respectively13. The amygdala is critical for fear conditioning and fear extinction and modulates fear-related learning in other structures, such as the prelimbic cortex, infralimbic cortex, and hippocampus13. Memory formation is regulated by the synaptic plasticity of related neural circuits, and the degree of the changes in synaptic plasticity can be estimated by assessing IEG responses to various stimuli, such as during the learning test14. Therefore, the induction potential of synaptic plasticity-related IEGs that play roles in neuronal signal transmission—including glutamatergic receptors/transporters in the brain—may be directly related to the facilitation of fear extinction learning. In this sense, histopathological analysis of the IEG proteins in the brain regions regulating fear memory using immunohistochemistry may provide valuable information on the mechanism involved in AGIQ-induced facilitation of fear extinction learning. Specifically, the induction pattern of the IEG proteins after the last learning test trial may help to identify the brain regions responsible for strengthening neural circuits to facilitate fear extinction learning.

Recently, some polyphenolic antioxidants have been shown to exert an ameliorating effect on post-traumatic stress disorder (PTSD), a trauma and stressor-related disorder, in animal models1516, and as a result, more attention has been given to these antioxidants. Surprisingly, a recent study has shown that dietary treatment with curcumin, a representative polyphenolic antioxidant, for 5 days impaired fear memory consolidation and reconsolidation processes in rats17. Because the sensitivity to exogenously administered antioxidants may vary among the different life stages, it is necessary to determine the optimum AGIQ exposure period for preventing or ameliorating anxiety. The present study evaluated different AGIQ exposure periods to identify the one that would be sufficient for facilitating fear extinction learning. We also examined the corresponding molecular responses in the brain regions involved in the facilitation. For these purposes, we examined the effects of AGIQ exposure in three different exposure periods: the postweaning exposure period, developmental exposure period, and entire developmental and postweaning exposure period. Behavioral tests were performed at both prepubertal and adult stages. In animals with whole period exposure to AGIQ, spontaneous recovery was also examined after the facilitation of fear extinction learning because preventing fear recovery is important for therapy related to anxiety disorders such as PTSD18. In animals subjected to spontaneous recovery, the number of immunoreactive cells for synaptic plasticity-related IEGs and their regulator, as well as constitutive changes in gene expression, in the brain regions of interest were compared among the different exposure periods. In animals that were subjected to whole period AGIQ exposure, similar immunohistochemistry and gene expression analyses were performed after the last trial of the learning test, and learning-linked responses were obtained for comparison with the changes in constitutive expression.

Materials and Methods

Chemicals and animals

AGIQ (purity: >97%) was provided by San-Ei Gen F.F.I., Inc. (Osaka, Japan). Thirty-six mated female Slc:SD rats at gestational day (GD) 1 (appearance of vaginal plugs was designated as GD 0) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Rats were individually housed with their offspring in polycarbonate cages with paper bedding until day 21 post-delivery. Animals were kept in an air-conditioned animal room (temperature: 23 ± 2°C, relative humidity: 55 ± 15%) with a 12-h light/dark cycle and provided powdered basal diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) ad libitum until exposure to AGIQ began and tap water ad libitum during the experiment. Offspring were weaned at postnatal day (PND) 21 (where PND 0 was the day of delivery) and reared two animals per cage thereafter and provided powdered basal diet with or without AGIQ and tap water ad libitum.

Experimental design

Mated female rats were randomly divided into two groups of untreated controls (18 animals) and the AGIQ group (18 animals) (Fig. 1). Animals in the AGIQ group were administered 0.5% AGIQ (w/w) in their powdered basal diet from GD 6 to day 21 post-delivery. The dosage we chose has been shown to facilitate fear extinction learning with continuous exposure from fetal stages to adulthood9.

Fig. 1.
Experimental design for continuous exposure to α-glycosyl isoquercitrin (AGIQ) from fetal stages to adulthood in rats.

We measured the body weight (BW) and food and water consumption of the dams every 3–4 days from GD 6 to day 21 post-delivery. On PND 4, litters were randomly culled to preserve 6 or 7 male and 1 or 2 female offspring per dam (a total of 8 offspring per dam). The offspring were weighed every 3 or 4 days until PND 21. Dams and female offspring were euthanized by exsanguination through the abdominal aorta under CO2/O2 anesthesia on day 22 post-delivery. Male offspring were selected for behavioral tests and immunohistochemical and gene expression analyses because animal behaviors are influenced by circulating levels of steroid hormones during the estrous cycle192021. From PND 21, the remaining male offspring in the untreated controls and AGIQ group were left untreated or dietarily exposed to AGIQ, respectively. On PND 30, animals that had been subjected to prepubertal behavioral tests were subjected to brain sampling for other experimental purposes.

From PND 30 onwards, the remaining male offspring from both groups were either left untreated or exposed to AGIQ. There were four animal groups: untreated controls (Ctrl; 32 animals), the postweaning AGIQ-exposed group (PW-AGIQ; 32 animals), the developmental AGIQ-exposed group (DEV-AGIQ; 32 animals), and the whole period AGIQ-exposed group (WP-AGIQ; 32 animals) (Fig. 1). Offspring in the PW-AGIQ and WP-AGIQ groups were fed a powdered diet containing 0.5% AGIQ ad libitum. Rats in all groups were weighed once weekly thereafter, and the amounts of food and water consumed were also recorded.

On PND 76 and PND 79, animals were subjected to brain sampling after the end of the adult stage behavioral tests (adult stage test 1, PND 76; adult stage test 2, PND 79). For brain immunohistochemistry, 10 male offspring per group (1 pup per dam) were subjected to perfusion fixation through the left cardiac ventricle with ice-cold 4% (w/v) paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) at a flow rate of 35 mL/min under deep CO2/O2-induced anesthesia. For gene expression analysis, 6 male offspring per group (1 pup per dam) were euthanized by exsanguination through the abdominal aorta under CO2/O2 anesthesia and subjected to necropsy. The brains were removed and then fixed in methacarn solution at 4°C for 5 h.

All dams and offspring were checked each day to assess their general appearance (abnormal gait and behaviors). All procedures in this study were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and according to the protocol approved by the Animal Care and Use Committee of The Tokyo University of Agriculture and Technology (Approved No.: 29-82). All efforts were made to minimize animal suffering.

Behavioral tests

Animals included in the prepubertal stage test (untreated controls and the AGIQ group) and adult stage test 1 (untreated controls and the DEV-AGIQ, PW-AGIQ, and WP-AGIQ groups) were subjected to the open field test, object recognition test, and contextual fear conditioning test. Animals included in adult stage test 1 also completed the spontaneous recovery test. Animals included in adult stage test 2 (untreated controls and the WP-AGIQ group) were subjected to the contextual fear conditioning test.

In each behavior experiment, animals were transported from the animal room to the behavioral test room 1–2 h before starting the tests. After the end of each behavioral test, animals were promptly returned to their home cage and transferred to the animal room. Apparatuses were cleaned with 70% ethanol solution before and after each test. All experiments were conducted between 08:00 and 19:00, and the order of animal selection for tests among groups was counter-balanced across the test time to avoid any bias resulting from the trial times of each group.

Open field test

The open field test was performed on PND 23 and PND 24 (prepubertal stage test) and on PND 58 and PND 61 (adult stage test 1) to assess locomotor activity and anxiety-like behaviors and to habituate the rats to the arena that would be used in the object recognition test on the following day (acclimation phase). The arena comprised a square stainless-steel tray with a matte black polyvinyl plastic surface and stainless-steel walls surrounding a tray with a matte black polyvinyl plastic surface (900 mm width × 900 mm depth × 500 mm height, O’Hara & Co., Ltd., Tokyo, Japan). The illumination was set at 20 Lux at the middle of the arena. The animals were placed at the corner of the arena with their heads facing the wall and allowed to explore the arena freely for 10 min. The total distance and time spent in the center or peripheral areas of the field were recorded by a CCD camera (WAT-902B; Watec Co., Ltd., Tsuruoka, Japan) mounted above the arena and evaluated by an automatic video-tracking system (TimeOFCR1 software; O’Hara & Co., Ltd.). In the video tracking analysis, the field was divided equally into 25 square regions, and 9 central regions were defined as the center area. The percentage of time spent in the center area was calculated. The center area rate was defined as the percentage of time that an animal stayed in the center area during the observation time.

Object recognition test

The object recognition test was performed on PND 24 and PND 25 (prepubertal stage test) and on PND 59 to PND 62 (adult stage test 1) to assess object recognition memory. Experiments were conducted in the same arena that was used in the open field test. The test comprised three steps: acclimation, sample phase, and test phase. Twenty-four hours after acclimation, the animals were allowed to explore the arena for 5 min with two identical sample objects (sample phase). After a prescribed interval (1 or 4 h for the prepubertal stage test or 1 or 24 h for adult stage test 1), the animals were allowed to explore the arena for 3 min with one familiar sample object and one novel object (test phase). In the sample and test phases, the animals were placed in the middle of the wall along the inside of the field with their heads facing the wall. Objects were placed equidistant to this location to the right and left sides behind the animal in the arena. The illumination was set at 20 Lux at the middle of the arena. The sample object was a black and white striped quadrangular prism with smooth polyvinyl plastic surfaces. The novel object was a gray cone of polyvinyl plastic with a rough surface and a stainless-steel tip. These objects were heavy enough that they could not be moved by the animals. Animal behavior was recorded by a CCD camera (WAT-902B; Watec Co., Ltd.) mounted above the arena and evaluated by an automatic video-tracking system (TimeSSI software; O’Hara & Co., Ltd.). Total distance and exploration time toward each object were recorded. When a rat’s nose approached to within 3 cm of an object, the video-tracking system automatically counted it as “exploration,” and the cumulative exploration time was recorded. In the test phase, animal behavior was analyzed during the first 2 min because the novel object becomes increasingly familiar and exploration time decreases as time passes. A discrimination index for novel object recognition was determined using following formula:

Discrimination index = exploration time with novel object/(exploration time with familiar object + exploration time with novel object)

Contextual fear conditioning test

The contextual fear conditioning test was performed on PND 26 to PND 29 (prepubertal stage test), PND 65 to PND 76 (adult stage test 1), and PND 71 to PND 79 (adult stage test 2) (Fig. 2, Supplementary Fig. 1: online only).

Fig. 2.
Experimental design of the contextual fear conditioning test and freezing rate (%) during the fear conditioning, fear acquisition, fear extinction, and spontaneous recovery tests in the untreated controls and each α-glycosyl isoquercitrin (AGIQ)-exposed group at adult stage tests 1 and 2. (A) Adult stage test 1. (B) Adult stage test 2. Values are expressed as the mean + SD or mean ± SD. n = 13–16/group for adult stage test 1 (conditioning and acquisition: untreated controls (Ctrl), 16; postweaning AGIQ-exposed group (PW-AGIQ), 16; developmental AGIQ-exposed group (DEV-AGIQ), 16; whole period AGIQ-exposed group (WP-AGIQ), 16; fear extinction 1–3 and spontaneous recovery: Ctrl, 16; PW-AGIQ, 14; DEV-AGIQ, 16; WP-AGIQ, 13). n = 14–16/group for adult stage test 2 (conditioning and acquisition: Ctrl, 16; WP-AGIQ, 16; fear extinction 1–3: Ctrl, 16; WP-AGIQ, 14). *P < 0.05, significantly different from the untreated controls by Dunnett’s test or Aspin-Welch’s t-test with Bonferroni correction (adult stage test 1) or significantly different from the untreated controls by Student’s t-test or Aspin–Welch’s t-test (adult stage test 2).

Conditioning and testing took place in a rodent observation cage (30 × 37 × 25 cm; CL-3001; O’Hara & Co., Ltd.) that was placed in a sound-attenuating chamber (CL-4211; O’Hara & Co., Ltd.). The side walls and door of the observation cage were constructed of Plexiglas. The floor comprised 21 steel rods through which a scrambled footshock from a shock generator (SGA-2020; O’Hara & Co., Ltd.) could be delivered. During experiments, the chamber was ventilated, kept at a background white noise level of 50 dB, and illuminated at 200 Lux by white light-emitting diode bulbs. Animal behavior was video recorded by a CCD camera (WAT-902B; Watec Co., Ltd.) and analyzed using an automatic video-tracking system (TimeFZ2 software; O’Hara & Co., Ltd.). Body freezing time was measured, and the freezing rate was defined as the percentage of time that the animal was immobile during the test.

Contextual fear conditioning: 138 s after transferring the animals to the cage, they received two 2-s footshocks (0.5 mA intensity) 100 s apart. The animals were removed from the cage 60 s after the second footshock and returned to their home cages. Thus, fear conditioning took 5 min.

Fear acquisition, fear extinction, and spontaneous recovery: Animals were placed back in the same context as used for conditioning for 5 min without footshock. Ninety minutes after completion of adult stage tests 1 and 2 (the spontaneous recovery test or the fear extinction test), the animals were euthanized for immunohistochemistry and real-time reverse transcription PCR analysis of the brain.

Immunohistochemistry

Because IEG peak protein expression in response to acute stimuli occurs within approximately 90–120 min22, animals were euthanized for perfusion fixation 90 min after the last trial of the contextual fear conditioning test. Perfusion-fixed brains were additionally fixed with the same PFA buffer solution overnight. Coronal slices were prepared at +3.0 mm and −3.0 mm from the bregma after adult stage tests 1 and 2 (n = 10/group). For immunohistochemistry of the hippocampal dentate gyrus and amygdala, a 3 mm-thick slice posterior to the –3.0 mm coronal plane from the bregma was prepared. For immunohistochemistry of the prelimbic and infralimbic cortices, a 3 mm-thick slice anterior to the +2.0 mm coronal plane from the bregma was prepared.Brain slices were further fixed with the same PFA buffer solution overnight at 4°C and routinely processed for paraffin embedding and sliced into 3-μm-thick sections.

Brain sections were subjected to immunohistochemical analysis using primary antibodies against the following: activity-regulated cytoskeleton-associated protein (ARC), FOS, early growth response 1 (EGR1), and cyclooxygenase 2 (COX2), which are IEGs involved in synaptic plasticity1023, and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2),