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 potential^1, 2. 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 models^15, 16, 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 cycle^19, 20, 21. 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 powd