*4.4. Optical Recording*

Hippocampal slices (*n* = 8) were stained with a voltage-sensitive dye (VSD) solution (Di-2-ANEPEQ, JPW114; Invitrogen, Carlsbad, CA, USA) [39]. Briefly, 0.2 mM Di-2-ANEPEQ staining solution was applied to the OHSCs in the plexiglass mesh-attached ring. The OHSCs were maintained in a humidified chamber with a mixture of O2 and CO2 gases for 25 min. The OHSCs were rinsed with artificial cerebrospinal fluid (ACSF: composition in mM: NaCl, 124; NaHCO3, 26; glucose, 10; KCl, 3; CaCl2, 2; MgCl2, 1; HEPES, 10; pH 7.4) by dipping them through the plexiglass mesh-attached ring and kept for at least 1 h to be recovered before being used for the experiment. A stained hippocampal slice was attached to the chamber glass, which was pre-coated with 0.01% polyethylenimine for approximately 1 h and rinsed with distilled water. Stained OHSCs were stabilized in the recording chamber, which was continuously perfused with ACSF at 31 ◦C bubbled with mixed 95% O2 and 5% CO2 gases. The Schaffer collateral (SC)/commissural pathway was stimulated with a bipolar electrode (CBBPE75, FHC Inc, Bowdoin, ME, USA). Synaptic activity was observed using an optical imaging system (MiCAM02, Brain Vision Inc., Tsukuba, Japan) with a high-speed CCD camera. Optical images were recorded with 512 frames (1890 ms total) for the test stimulation and acquired at a sampling rate of 3.7 ms per frame. Trials were conducted every 20 s (0.05 Hz). Fluorescence intensity [ΔF/F: the change in the intensity of fluorescence (ΔF) in each pixel relative to the initial intensity of fluorescence (F)] was used to assess fractional changes in the amount of VSD fluorescence. Activated areas were determined by averaging images (spatial filter: 3 × 3 pixels, cubic filter: 3 × 3 pixels). Acquisition and analysis software (BV-Analyzer, Brain Vision Inc.) was used to display and analyze the optical images.

#### *4.5. Statistical Analysis*

Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Data are expressed as means ± standard errors of the mean (SEM). The data were evaluated by two-way analysis of variance (ANOVA) followed by Tukey's multiple-range post hoc comparisons. Differences were considered significant when *p*-values were less than 0.05.

#### **5. Conclusions**

In this study, we demonstrated that chronic AA treatment has a neuroprotective influence on KA-induced oxidative stress in an aging hippocampus model. Based on PI staining, chronic AA treatment also reduced age-related neurotoxicity (9 w-daily AA treatment), and this was similar to its influence on oxidative injury in younger hippocampi (3 w AA treatment). Western blot analysis demonstrated that chronic AA treatment activated SOD, a well-known detoxifying enzyme, compared to acute AA treatment in the 9 w group. Therefore, we have shown that chronic AA treatment increases antioxidant activity and decreases neuronal cell death in this model. Using optical signals to observe the functional recovery of surviving neurons, AA treatment decreased latencies of the optical signal, indicating an increase in effective neuronal transmission. These results suggest that surviving neurons, protected by chronic AA treatment, show enhanced functional recovery in this aging model.

**Author Contributions:** All authors contributed substantially to the preparation of this study. K.H.L. designed the study and drafted the manuscript. U.J.K. performed experiments and corrected and analyzed the data. M.C. assisted with the drafting of the manuscript and analyzed optical data. B.H.L. oversaw the entire project and prepared the drafting of the manuscript. K.H.L. and U.J.K. were equal contributors to this study. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (NRF-2016R1D1A3B20- 08194, NRF-2020R1A2C3008481).

**Institutional Review Board Statement:** All animal study was approved by Institutional Animal Care and Use Committee of Yonsei University Health System (permit no.: 2018-0095, approval date: 8 May 2018).

**Informed Consent Statement:** Not Applicable.

**Data Availability Statement:** Not Applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

