学位論文内容の要旨（ Summary of dissertation）
博士の専攻分野の名称
博士（医 学）氏 名 安 燕（Degree conferred: Doctor of Philosophy）（Name of recipient: An Yan
学 位 論 文 題 名（Title of dissertation））

Biological basis of the anxiolytic-like effect of mirtazapine in the rat conditioned fear stress model(ラット恐怖条件付けモデルにおける mirtazapine の抗不安効果の生物学的基盤に関する研究)【Background and Objectives】 Mirtazapine, a NaSSA, which blocks the adrenergic α2 auto- andhetero-receptors, has been proven to be effective in the treatment of various anxiety disorders inaddition to depressive disorders. However, the mechanism whereby mirtazapine exerts its anxiolytic
effect has not been fully clarified. Previous studies have shown that the systemic administration ofmirtazapine has the anxiolytic-like effect in the contextual fear conditioning model and increases
extracellular serotonin levels in the hippocampus. These effects might be related because a numberof studies have demonstrated that the facilitation of 5-HT neurotransmission decreases theexpression of contextual conditioned freezing. To further explore the anxiolytic mechanism of
mirtazapine, in the present study, we investigated the brain area(s) in which mirtazapine exerts its
anxiolytic-like effect in the contextual fear conditioning test by using the microinjection ofmirtazapine into the median raphe nucleus (MRN) , hippocampus and amygdala. These brain areas
are reportedly related to the contextual fear conditioning and the MRN, like the dorsal raphe nucleus,
is a main source of serotonergic innervation to the forebrain structures involved in anxiety regulation,such as the hippocampus. Furthermore, I investigated augmentation strategies for the anxiolytic-likeeffect of mirtazapine. Recently, evidence from animal and clinical studies has shown that lithium has
an effect on monoamine systems to potentiate the antidepressant action of various antidepressants.
However, only a few studies have investigated the effect of lithium with mirtazapine on anxiety
clinically but not preclinically. Previous studies have shown that both lithium and mirtazapine
influence serotonergic systems and anxiety. These suggest the possibility that the combination oflithium and mirtazapine may have better efficacies for anxiety disorders through the effect onserotonin.
【Methods】First, I examined the effect of the two intervals between conditioning and exposure toconditioned fear on acute systemic mirtazapine treatment. One day after footshock, the rats received
a single injection of mirtazapine 0, 1, 3 and 10 mg/kg at 30 min before testing. One and 7 days afterfootshock, the rats received a single injection of mirtazapine 10 mg/kg at 30 min before testing. Next,
I explored target brain sites of the anxiolytic-like effect of mirtazapine. One day after footshock,mirtazapine (3 μg/site) was directly injected into three brain structures, the MRN, hippocampus andamygdala at 10 min before testing. In chapter 2, I explored augmentation strategies for theanxiolytic-like effect of mirtazapine. In the exepriment of subchronic lithium co-treatment with acutesystemic mirtazapine∶immediately after footshock, the rats received standard laboratory rat chowcontaining 0%, 0.05% or 0.2% of Li2CO3 for 7 days. On the eighth day, the rats received an
intraperitoneal injection of mirtazapine 10 mg/kg at 30 min before testing. In the exepriment of
subchronic lithium combined with local mirtazapine treatment∶immediately after footshock, rats
received standard laboratory rat chow containing 0 or 0.2% of Li2CO3 for 7 days. On the eighth day,

mirtazapine was injected into the hippocampus, amygdala and MRN at 10 min before testing.
【Results】When the interval between conditioning and testing was 1 day, acute mirtazapine showeda dose-dependent reduction in freezing time. Moreover, intra-MRN injection of mirtazapine reduced
freezing behavior significantly, while mirtazapine injections into the hippocampus or amygdala didnot. When the interval was 7 days, acute systemic mirtazapine 10 mg/kg treatment did not reduce theexpression of conditioned freezing significantly. The combination of subchronic 0.2% Li2CO3 but
not 0.05% Li2CO3 with acute mirtazapine (10 mg/kg) reduced freezing significantly. Subchronic
treatment with 0.2% Li2CO3 enhanced the effect of mirtazapine (3 μg/site) on freezing behavior
significantly when mirtazapine was infused into the MRN but not hippocampus or amygdala.
【Discussion】 Acute systemic administration of mirtazapine dose-dependently reduced freezing
one day after fear conditioning, whereas the anxiolytic-like effect of mirtazapine diminished when
the interval between fear conditioning and testing was prolonged for seven days. These results are
consistent with our previous reports, which showed that systemic mirtazapine reduced anxiety-likebehavior, freezing, in the contextual fear conditioning test and that the inhibitory effect of an acutechallenge of SSRIs on conditioned freezing diminished by prolonging the interval between
conditioning and testing. Memory consolidation processes after the fear acquisition may be involvedin this effect. The intra-MRN infusion of mirtazapine showed the anxiolytic effect in the contextual
fear conditioning test. The intra-MRN infusion of mirtazapine is supposed to increase extracellular
5-HT levels in the nerve terminal areas such as the hippocampus, because mirtazapine an
α2-adrenergic antagonist but has very weak α1-adrenergic antagonistic action. Previous reports
revealed that the local administration of mirtazapine or the α2-antatgonist idazoxan into the MRN
increased extracellular 5-HT levels in both the MRN and the entorhinal cortex. Their results supportmy hypothesis that local mirtazapine stimulates MRN activity by blocking α2-adrenoceptors in thenerve terminals of noradrenergic neurons and increases the extracellular 5-HT levels in the nerve
terminal areas of serotonergic neurons, leading to the anxiolytic effect in the contextual fear
conditioning test. In the future, the effect of microinjection of a selective α2-adrenergic antagonist
into the MRN on anxiety-like behaviors should be examined. Subchronic 0.2% Li2CO3treatment
enhanced the anxiolytic-like effects of acute systemic and local mirtazapine administration in thecontextual fear conditioning test. Earlier in vivo microdialysis studies have reported that systemic
administration of mirtazapine increases extracellular serotonin concentrations in the hippocampus
and that subchronic lithium treatment increases extracellular serotonin levels in the medial prefrontal
cortex and hippocampus. Taken together, these data suggest that the lithium augmentation of theanxiolytic-like effect of mirtazapine may be mediated by the enhancement of 5-HT
neurotransmission in the hippocampus. The mechanism, by which lithium facilitates central 5-HTneurotransmission, is not yet completely understood at present. Previous biochemical studies haveshown that lithium is linked to several factors affecting the extracellular 5-HT levels, such assynthesis, storage, release, reuptake and metabolism. The result of lithium-induced enhancement of
the anxiolytic-like effect of the intra-MRN mirtazapine administration supports the hypothesis that
this enhancement is mediated by the effect on serotonin.
In conclusion, this study showed that the anxiolytic-like effect of mirtazapine was mediated by
its action on the MRN, and that subchronic 0.2% Li2CO3 treatment enhanced the anxiolytic-like
effect of mirtazapine probably through its effect on 5-HT.