Ameliorative effects of yokukansan on learning and memory deficits in olfactory bulbectomized mice

Abstract

Aim of the study

Yokukansan (YKS) is a Japanese traditional herbal medicine and has been used for the treatment of the behavioral and psychological symptoms of dementia (BPSD). The present study aimed to clarify the effects of YKS on learning and memory impairments, and its mechanisms of action in olfactory bulbectomized (OBX) mice, one of the animal models of Alzheimer's disease (AD).

Materials and methods

OBX or sham-operated ddY mice were treated with YKS or donepezil (DPZ), a reference drug, and their cognitive performances were tested by the modified Y-maze test, novel object recognition test, and fear conditioning test to elucidate the spatial working memory, non-spatial short-term memory, and long-term memory, respectively. After completing the behavioral experiments, the expression level of cholinergic marker proteins and the activity of acetylcholinesterase (AChE) in the brain were analyzed by western blotting and Ellman's method, respectively.

Results

OBX caused spatial working memory and non-spatial working memory impairments that were reversed by YKS and also by DPZ; however, YKS failed to affect the long-term memory deficits. Amelioration of the spatial working memory by YKS was reversible by scopolamine, a muscarinic receptor antagonist. YKS treatment reversed OBX-induced down-regulation of choline acetyltransferase and muscarinic muscarinic M₁ receptor expression without affecting muscarinic M₃ receptor expression or AChE activity.

Conclusion

These results demonstrate that YKS improves short-term memory deficit caused by OBX and that the effect is at least partly mediated by muscarinic receptor stimulation and the normalization of central cholinergic systems. The present findings also suggest that YKS has a therapeutic effect not only on BPSD, but also on memory impairment of AD.

Introduction

Alzheimer's disease (AD) is a progressive and neurodegenerative disorder that is characterized not only by memory dysfunction but also by the behavioral and psychological symptoms of dementia (BPSD). An increasing population of AD patients is a serious social and economic problem in super-aging societies such as Japan; however, only a few drugs are clinically available for this disease. Therefore, new drug discovery and the establishment of new therapeutic methods are pressing needs.

Yokukansan (YKS) is one of the Kampo prescriptions, which consists of 7 different medicinal herbs: Atractylodis Lanceae Rhizoma (rhizome of Atractylodes lancea De Candolle, Compositae), Poria (sclerotium of Poria cocos Wolf, Polyporaceae), Cnidii Rhizoma (rhizome of Cnidium officinale Makino, Umbelliferae), Uncariae Uncis Cum Ramulus (hook of Uncaria rhynchophylla Miquel, Rubiaceae), Angelicae Radix (root of Angelica acutiloba Kitagawa, Umbelliferae), Bupleuri Radix (root of Bupleurum facatum LINNĖ, Umbelliferae), and Glycyrrhizae Radix (root and stolon of Glycyrrhiza uralensis Fisher, Leguminosae). This formula has been used for the treatment of neurosis, insomnia, night cry, and peevishness in infants. Above all, it has been demonstrated that YKS ameliorates BPSD such as hallucination, delusion, depression, excitation, anxiety, and aggression in clinical study of AD (Iwasaki et al., 2005, Monji et al., 2009, Okahara et al., 2010). While accumulated evidence indicates the beneficial effect of YKS on the BPSD, little information is available on the effects of YKS on memory impairment, a core symptom of AD. For example, yokukansan-ka-chimpi-hange, a prescription that is derived from YKS and additionally includes Aurantii Nobilis Pericarpium (Citrus unshiu Markovich) and Pinelliae tuber (tuber of Pinellia ternate Breitenbach), reportedly improves scopolamine-induced amnesia in the eight-arm radial maze test (Egashira et al., 2001). Moreover, when administered as 0.5–1.0% powdered diet for 10 months, YKS improved spatial cognitive deficit of Tg2576 mice (Tabuchi et al., 2009) and intellicage performances in Aβ (i.c.v.)-injected AD animal model (Sekiguchi et al., 2010). Accumulated scientific evidence, however, is still needed not only to clarify the mechanism(s) of the action of YKS but also to promote more possible usages of YKS in cognitive deficit therapy.

To gain a better understanding of the pharmacological mechanism underlying the action of YKS on the core symptoms of AD, a clinically relevant animal model and evaluation methods are required. In this study, we focused on an animal model of olfaction deficits to elucidate the anti-dementia effect of YKS for a couple of reasons. First, olfactory bulbectomy (OBX) in rodents has been used as one of the AD models since the impairment of olfactory perceptual acuity is present not only at the early stage of AD (Serby, 1987, Bahar-Fuchs et al., 2010) and in mild cognitive disorder (MCI) patients (Devanand et al., 2000, Djordjevic et al., 2008), but also in a transgenic AD model of mice with over-expression of a mutant form of the human amyloid-β-precursor protein (Wesson et al., 2010). Second, OBX induces not only a loss of olfactory cue, but also various behavioral and biochemical alterations such as increases in locomotor activity (Breuer et al., 2009) and aggressiveness (Hsuchou et al., 2002). Moreover, it also causes learning and memory impairments in rodents by inducing neurodegeneration of septo-hippocampal cholinergic innervation (Hozumi et al., 2003) and elevation of Aβ level in the brain (Aleksandrova et al., 2004), indicating that OBX provides a beneficial animal model of AD that is independent from transgenic animal models.

In the present study, we took advantage of the OBX model to elucidate the anti-dementia effects of YKS. For this aim, we employed three different behavioral paradigms, namely, a modified Y-maze test (Dellu et al., 2000), a novel object recognition test (Zhao et al., 2007), and the contextual and auditory versions of the fear memory test (Gulick and Gould, 2007) to elucidate short-term spatial working memory, short-term non-spatial memory, and long-term memory, respectively, in OBX animals. Moreover, to understand the mechanism underlying the action of YKS, we investigated whether YKS administration affects the central cholinergic systems in OBX animals.