## 4 Discussion
Reactive astrocytes produce excess GABA in an MAO-B-dependent manner, leading to impairment in synaptic plasticity and memory function, which is rescued by selective MAO-B inhibitors [45, 46]. Therefore, the inhibitory effect of \(\beta\)-lactolin on MAO-B may affect GABA production in astrocytes. Further studies are warranted to investigate this possibility. Lastly, we demonstrated that \(\beta\)-lactolin suppresses DA metabolism in astrocytes. The results are consistent with our previous report suggesting that \(\beta\)-lactolin improves memory function and depression-like behavior [47] by increasing DA levels in the hippocampus and frontal cortex of mice [20]. It has been reported that DA plays essential roles in cognitive control. Previous studies have demonstrated that DA levels in the prefrontal cortex are associated with cognitive performance, including working memory and attention [8, 9]. Moreover, DA modulates synaptic plasticity in the hippocampus and enhances DA binding to DA receptors, thereby improving cognitive performance in mice [48, 49]. In fact, the DA precursor levodopa improved the rate of task-based learning and task performances in elderly people [50]. Astrocytes are the main cells involved in DA metabolism in the CNS, as they express high levels of DA-metabolizing enzymes, such as MAO-B and COMT [13]. Moreover, it has been reported that DA metabolism dysfunction in astrocytes of the prefrontal cortex leads to cognitive impairment [51]. This evidence suggests that the suppressive effects of \(\beta\)-lactolin on DA metabolism in astrocytes might contribute to the memory-improving effects of \(\beta\)-lactolin. Notably, even though astrocytes express both MAO-B and COMT, the suppression of DA metabolism by \(\beta\)-lactolin was mainly considered to occur due to its inhibitory effect on MAO-B, since it did not significantly inhibit COMT activity, as demonstrated in this study. However, there are some limitations to the present study. First, we did not clarify the mechanism underlying the inhibition of ROS production by \(\beta\)-lactolin. A mechanism that might lead to reduced ROS levels is the upregulation of antioxidant activity. Although we demonstrated that MAO-B activity was suppressed by \(\beta\)-lactolin, we did not evaluate the effect of MAO-B inhibitors on ROS production in this study. Therefore, further studies are warranted to investigate the mechanisms involved in the decrease in ROS production in detail. Next, since we used only cultured astrocytes in this study, the direct effects of \(\beta\)-lactolin on astrocytes in in vivo models have not been evaluated. For instance, it is not clear to what extent the suppressive effects of \(\beta\)-lactolin on DA metabolism contribute to the memory-improving effects of \(\beta\)-lactolin via the dopaminergic system in mice. To evaluate this possibility, astrocyte-specific MAO-B knockdown using adenovirus needs to be performed in future in vivo studies. In conclusion, this study revealed novel effects of \(\beta\)-lactolin on astrocytes, including its protective effects on astrocytes under pathologic conditions, and suggested the involvement of astrocytes in the memory-improving effects of \(\beta\)-lactolin. The following are available online at [[https://www.mdpi.com/article/10.3390/app11073034/s1](https://www.mdpi.com/article/10.3390/app11073034/s1)]([https://www.mdpi.com/article/10.3390/app11073034/s1](https://www.mdpi.com/article/10.3390/app11073034/s1)), Figure S1: ROS production in astrocytes treated with \(\beta\)-lactolin, Figure S2: Effect of \(\beta\)-lactolin on COMT activity in astrocytes. Conceptualization, Y.A., and S.A.; methodology, S.A.; investigation, S.A., C.T., and R.O. ; writing--original draft preparation, S.A.; writing--review and editing, Y.A., and T.A. ; visualization, S.A.; supervision, Y.A. All authors have read and agreed to the published version of the manuscript. Kirin Holdings Company, Limited supported this study. Not applicable.