Characterization of 5-HT1A receptor and transport protein KIF13A expression in the hippocampus of stress-adaptive and -maladaptive mice
Introduction
There are now known to be seven kinds of 5-HT receptor families, 5-HT1-7, that are comprised of 14 structurally and pharmacologically distinct 5-HT receptor subtypes [1]. Among them, the 5-HT1A receptor plays a major role in pre- and postsynaptic components of serotonergic transmission and plays a crucial role in the pathophysiology of psychiatric disorders [2]. Human genetic and imaging studies have shown that differences in 5-HT1A receptor levels or regulation are associated with the pathophysiology of depression and/or anxiety, and also the response to antidepressants [3,4]. Chronic treatment with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) results in increased neurogenesis and decreased anxiety [5]. These reports indicate that 5-HT1A receptor plays an important role in mental disorders.
The ability to adapt to stress is an essential defensive function of a living body, and disturbance of this ability may contribute to some stress-related disorders. Thus, the identification of substances that have beneficial effects on the brain mechanisms that contribute to stress adaptation could help to pave the way for new therapeutic strategies for stress-related mood disorders. Dysfunction of the serotonergic system has been reported to be a vulnerability factor for major depressive disorder and other forms of affective illness [6]. Our previous studies showed that treatment with flesinoxan and 8-OH-DPAT, which were the 5-HT1A receptor full agonists, significantly suppressed the decrease in various exploratory behaviors in the hole-board test that was observed immediately after exposure to acute restraint stress [7,8]. Further, we have successfully created stress-adaptive and -maladaptive models in mice [9]; a single exposure to restraint stress for 1 h produced a decrease in the number and duration of head-dipping behaviors of mice in the hole-board test, and these acute emotional responses were recovered by exposure to repeated restraint stress for 1 h/day for 14 days, indicating the development of stress adaptation. On the other hand, such an adaptive response was not observed in mice that had been exposed to repeated restraint stress for 4 h/day for 14 days, which continued to show a decrease in head-dipping behavior in the hole-board test. Moreover, this emotional abnormality induced by stress maladaptation was alleviated by chronic treatment with flesinoxan [9]. The results of our previous studies suggest that brain 5-HT1A receptors may be involved, at least in part, in the development of adaptation to stress.
5-HT1A receptor linked to Gi/Go protein is a membrane protein that is essential for neurons to communicate through chemical signals [1]. Interestingly, KIF13A, a member of the kinesin superfamily, has been reported to be a motor protein that transports the 5-HT1A receptor from the golgi apparatus to the plasma membrane in neurons [10]. Therefore, the expression levels of 5-HT1A receptor in the plasma membrane is expected to differ between stress-adaptive and -maladaptive situations. The hippocampus is an important brain site in stress-related psychiatric disorders [11,12]. In the present study, we examined the expression of 5-HT1A receptor and KIF13A in the hippocampal cytoplasmic and membrane fractions of stress-adaptive and -maladaptive mice. Further, we carried out blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BN-SDS-PAGE) to confirm that 5-HT1A receptor and KIF13A form a complex.
Section snippets
Animals
Male ICR mice (Japan SLC Inc., Shizuoka, Japan) weighing 25-30 g were housed at a room temperature of 23 ± 1 °C with a 12 h light-dark cycle (light on 7:00 a.m. to 7:00 p.m.). Food and water were available ad libitum. All experiments with animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of the International University of Health and Welfare.
Stress exposure
Mice were exposed to adaptable or
Comparison of 5-HT1A receptor or KIF13A protein expression in total extract of the hippocampus of mice exposed to adaptable or unadaptable stress
Western blot analysis demonstrated that there were no significant changes in the expression of 5-HT1A receptor in the total extract of hippocampus of mice that had been exposed to adaptable (Fig. 1A) or unadaptable (Fig. 1B) restraint stress. Similarly, there were no significant changes in the expression of KIF13A in both groups (Fig. 1C and 1D).
Comparison of 5-HT1A receptor or KIF13A protein expression in cytoplasmic or membrane extract of the hippocampus of mice exposed to adaptable or unadaptable stress
In the western blot analysis, abundant bands reactive to antibodies against the cytoplasmic marker GAPDH and the membrane marker Na+/K+-ATPase were
Discussion
In this study, we demonstrated that expression of 5-HT1A receptor was increased in the membrane fraction, but not the cytoplasmic fraction, of the hippocampus in mice that were exposed to repeated adaptable restraint stress, i.e., 1 h once a day for 14 days, as previously described [9]. In the CA1 and CA3 pyramidal cell layers and the dentate gyrus (DG) granule cell layer of the hippocampus, 5-HT1A receptors are widely expressed [15]. A recent study indicated that 5-HT1A receptors in mature DG
CRediT authorship contribution statement
Hiroko Miyagishi: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Visualization, Funding acquisition. Minoru Tsuji: Conceptualization, Methodology, Writing - original draft, Visualization. Kazuya Miyagawa: Investigation. Kazuhiro Kurokawa: Investigation. Atsumi Mochida-Saito: Investigation. Kohei Takahashi: Investigation. Kumiko Ishige: Writing - review & editing. Hiroshi Takeda: Conceptualization, Writing - review & editing, Funding acquisition,
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number JP17K15759.
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