Ninjinyoeito improves social behavior disorder in neuropeptide Y deficient zebrafish

Abstract

Sociability is an essential component of the linkage structure in human and other vertebrate communication. Low sociability is defined as a poor social approach, including social withdrawal and apathy, and is implicated in a variety of psychiatric disorders. Ninjinyoeito (NYT), a traditional Japanese herbal medicine, has been used in the medical field. This study aimed to determine the effect of NYT on low sociality in NPY-KO zebrafish. NPY-KO zebrafish were fed a 3% NYT-supplemented diet for 4 days and subjected to behavioral tests. In the mirror test, NPY-KO zebrafish fed a control diet showed avoidance behavior toward their mirror counterparts. In contrast, the treatment of NPY-KO zebrafish with NYT significantly increased their interaction with their counterparts in the mirror. In addition, a 3-chambers test was conducted to confirm the effect of NYT on the low sociality of NPY-KO zebrafish. NPY-KO zebrafish fed the control diet showed less interaction with fish chambers, while NYT treatment increased the interaction. Phosphorylation of ERK, a marker of neuronal activity, was significantly reduced in the whole brain of NYT-fed NPY-KO zebrafish, compared to the control diet. NYT treatment significantly suppressed hypothalamic-pituitary-adrenal-related genes (gr, pomc, and crh) and sympathetic-adrenal-medullary-related genes (th1, th2, and cck) in NPY-KO zebrafish. NYT administration significantly reduced mRNA levels of gad1b compared to the control diet, suggesting the involvement of GABAergic neurons in NYT-induced improvement of low sociability. Furthermore, the expression of CREB was suppressed when NPY-KO zebrafish were fed NYT. Next, we attempted to identify the effective herb responsible for the NYT-induced improvement of low sociability. NPY-KO zebrafish were fed an experimental diet containing the target herb for 4 days, and its effect on sociability was evaluated using the 3-chambers test. Results showed that Cinnamon Bark and Polygala Root treatments significantly increased time spent in the fish tank area compared to the control diet, while the other 10 herbs did not. We confirmed that these two herbs suppressed the activity of HPA-, SAM-, and GABAergic neurons, as well as NYT-treated zebrafish, accompanied by downregulation of CREB signaling. This study suggests the potential use of NYT as a drug for sociability disorders.

Social zebrafish:Danio rerioas an emerging model in social neuroendocrinology

“…Importantly, this area is a central hub of oxytocin neurons, and a homolog to the mammalian supraoptic nucleus of the hypothalamus, where CRH type‐2 receptors are coexpressed on oxytocin neurons 39,66 . In a separate study, it has been demonstrated that the expression of neuropeptide Y, which is involved in cell growth and homeostasis, can also negatively impact social interaction tendencies in zebrafish 112 . The deficiencies were recovered by the herbal medicine Ninjinyoeito, but this recovery did not induce changes in oxytocin levels, but it similarly affected CRH and dopamine precursor genes, suggesting that the activity of neuropeptides other than oxytocin also implicates the two systems.…”

“…39,66 In a separate study, it has been demonstrated that the expression of neuropeptide Y, which is involved in cell growth and homeostasis, can also negatively impact social interaction tendencies in zebrafish. 112 The deficiencies were recovered by the herbal medicine Ninjinyoeito, but this recovery did not induce changes in oxytocin levels, but it similarly affected CRH and dopamine precursor genes, suggesting that the activity of neuropeptides other than oxytocin also implicates the two systems. Nonetheless, the question for the anxiolytic oxytocin controls on dopaminergic signaling as a candidate system for the regulation of social motivation remains an open question.…”

Review of frequently used Kampo prescriptions: Part 4, Ninjin'yoeito

“…Previous studies indicated that NYT ameliorated dementia or depression/anxiety [94][95][96] by increasing the levels of neurotrophic factors [94,97], attenuating the inhibition of neurogenesis [95,98], suppressing microglial overactivity [99], increasing cerebral blood flow [100], and enhancing cerebral glucose metabolism [92]. Furthermore, NYT improved diminished motivation [86,87,101,102] and suppressed the hypothalamic-pituitary-adrenal (HPA) axis by downregulating the sympathetic-adrenal-medullary (SAM) axis and GABA neurons to improve sociability [103].…”

Comparative study of the effects of the three kinds of Kampo-hozai: Ninjinyoeito, Hochuekkito, and Juzentaihoto on anxious and low sociability behavior using NPY-knockout zebrafish

“…They have established protocols for assessing sociability using the three-Chambers test ( Ariyasiri et al, 2019 ). Thus, they are suitable for drug discovery research and used in Kampo medicine research ( Kawabe et al, 2021 ; Kawabe et al, 2022 ). Recently, neuropeptide Y-knockout (NPY-KO) zebrafish were established as models for anxiety-like and low social behavior ( Shiozaki et al, 2020 ).…”

“…The zebrafish NPY amino acid sequence and the NPY function are very similar to the human NPY ( Singh et al, 2017 ; Shiozaki et al, 2020 ). The NPY-KO zebrafish showed low sociability in the three-Chambers test and exhibited anxiety-like behaviors such as freezing under acute stress ( Kawabe et al, 2022 ).…”