This research was supported by grants from National Natural Science Foundation of China (No. 81630104&#65307;No. 81473597), and the Fundamental Research Funds for the Central Universities (No. 2018-JYBZZ-XS005). The protocol and results demonstrated in this paper originates from the article Involvement of Normalized Glial Fibrillary Acidic Protein Expression in the Hippocampi in Antidepressant-Like Effects of Xiaoyaosan on Chronically Stressed Mice by Dr. Xiu-Fang Ding, et al.

This protocol was approved by the Animal Ethics Committee of Beijing University of Chinese Medicine and was in accordance with all guidelines for the Care and Use of Laboratory Animals of China.&#160;There was no accidental death situation during the experimental procedure, and no animals needed to be euthanized in this study.
1. Preparing Drugs for the Experiment
NOTE: The Chinese herbs were purchased and then authenticated by Dr. B. Liu of the Beijing University of Chinese Medicine. Xiaoyaosan contains the following eight traditional Chinese drugs in a ratio of 6:6:6:6:3:6:2:2: Radix Angelicae Sinensis, Radix Paeoniae Alba, Poria, Radix Bupleuri, Radix Glycyrrhizae, Rhizoma Atractylodis Macrocephalae, Herba Menthae and Rhizoma Zingiberis Recens,.
<ol>
	<li>Prepare Xiaoyaosan as previously described16. 
	Note: We extracted these compounds in the Chinese medicine preparation room of the China-Japan Friendship Hospital, and the extraction yield was 18.8%.</li>
	<li>Verify the quality of Xiaoyaosan by high-performance liquid chromatography-mass spectrometry analysis (LC-MS/MS)17.</li>
</ol>
2. Preparing Mice for the Experimental Procedure
NOTE: For the experimental procedure, 60 healthy male C57BL/6J mice aged 12 weeks were selected, with each mouse weighing between 18 and 22 g.
<ol>
	<li>Keep mice individually in cages in a typical Specific Pathogen Free (SPF) laboratory animal room (22 &#177; 1 &#176;C, 12 h/12 h dark/light cycle, relative humidity: 30&#8211;40%) for one week before the CUMS procedure.</li>
	<li>Randomly divide all mice into a control group, a model group, a Xiaoyaosan treatment group, or a fluoxetine treatment group according to their body weight. Each group has 15 mice.</li>
	<li>Before initiation of the experiment, number the mice in each group on their tail using a marking pen.</li>
	<li>Test all mice using a five-day sucrose preference test (refer to step 5) and an open field test (refer to step 6).</li>
</ol>
3. Induction of Depression in Mice
NOTE: To induce depression, the mice in the model group and two treatment groups underwent CUMS for 21 days18. The plan can be changed according to the practical situation, but the principle that the same stressor cannot be used continuously should be followed.
<ol>
	<li>Expose mice daily to any two of the following seven stressors for 21 consecutive days19. Our modeling plan is designed as follows:
	<ol>
		<li>On Monday, expose mice to food deprivation (empty the feed of each cage for 24 h, 8:00 a.m. to 8:00 a.m.) and water deprivation (remove the drinking bottle of each cage for 24 h, 8:00 a.m. to 8:00 a.m.).</li>
		<li>On Tuesday, expose mice to restraint stress (restrain mice in a small plastic tub for 3 h, 8:00 a.m. to 11:00 a.m.) and empty cages (remove the padding in each cage for 11 h, 8:00 a.m. to 19:00 p.m.).</li>
		<li>On Wednesday, expose mice to wet and soiled cages (pour water into each cage and keep wet for 24 h, 8:00 a.m. to 8:00 a.m.) and crowded cages (put five mice in one cage for 24 h, 8:00 a.m. to 8:00 a.m.).</li>
		<li>On Thursday, expose mice to restraint stress and ice-cold swimming20,21 (force each mouse to swim in a clear glass aquarium with ice water for 5 min).</li>
		<li>On Friday, expose mice to wet and soiled cages and food deprivation.</li>
		<li>On Saturday, expose mice to restraint stress and crowded cages.</li>
		<li>On Sunday, expose mice to empty cages and ice-cold swimming.</li>
	</ol>
	</li>
	<li>Record the coat state and body weight weekly until the end of modeling (day 21). Calculate the total score of the coat state as the sum of scores from seven different body parts: head, neck, dorsal coat, ventral coat, forepaws, tail, and hind paws. A score of one indicates a well-groomed coat, and a score of zero indicates an unkempt coat22.</li>
	<li>Perform a five-day sucrose preference test (refer to step 5) and an open field test (refer to step 6) at the end of experiment.</li>
</ol>
4. Administration of Xiaoyaosan
NOTE: The dose of Xiaoyaosan (0.25 g/kg/day) was selected on the basis of its satisfactory efficacy as previously described9.
<ol>
	<li>During the experiment, use distilled water to prepare the Xiaoyaosan and fluoxetine suspensions, and make sure to use immediately.</li>
	<li>Give 0.5 mL of distilled water to the mice in the control group and model group by intragastric administration every day before modeling. Meanwhile, give Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day) to the mice in the two treatment groups.</li>
</ol>
5. Sucrose Preference Test
NOTE: No water or food deprivation was applied before the test. The protocol was performed as previously reported23.
<ol>
	<li>On the first two days, house the mice individually with two bottles filled with tap water.</li>
	<li>On the next two days, give the mice two bottles filled with 1% sucrose solution.</li>
	<li>On the last day, give the mice two bottles for 24 h, one with tap water and the other with 1% sucrose solution. To eliminate a side preference of the mice in drinking behavior, switch the position of the two bottles after 12 h.</li>
	<li>Record the volume (mL) of both the consumed sucrose solution and tap water. Calculate the sucrose preference as follows: sucrose preference (%) = sucrose consumption/total liquid consumption &#215; 100%.</li>
</ol>
6. Open Field Test
<ol>
	<li>Perform the open field test in a dimly lit and quiet room with a four-sided wooden black lusterless box. Divide the &#64258;oor into 25 equal squares by blue lines.</li>
	<li>Clean the open-field box with 5% alcohol prior to the access of each mouse. Then, place the mouse in the center of the open field gently.</li>
	<li>Suspend a high definition digital camera approximately 200 cm right above the field, and record the behavior of each mouse for 5 min.</li>
	<li>Evaluate the mice behavioral video by the video-tracking system software and record the data of the total running distance and number of entries into the center zone.</li>
	<li>Analyze the data of the total distance moved and number of entries into the center zone by statistical software.</li>
</ol>

<ol>
	<li>Chrousos, G. P., Gold, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concepts+of+stress+and+stress+system+disorders.+Overview+of+physical+and+behavioral+homeostasis.">The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis.</a> JAMA. 267 (9), 1244-1252 (1992).</li><li>Yao, Y., Huang, H. Y., Yang, Y. X., Guo, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cinnamic+aldehyde+treatment+alleviates+chronic+unexpected+stress-induced+depressive-like+behaviors+via+targeting+cyclooxygenase-2+in+mid-aged+rats.">Cinnamic aldehyde treatment alleviates chronic unexpected stress-induced depressive-like behaviors via targeting cyclooxygenase-2 in mid-aged rats.</a> Journal of Ethnopharmacology. 162, 97-103 (2015).</li><li>Murray, C. J., Lopez, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+health+statistics:+a+compendium+of+incidence+prevalence+and+mortality+estimates+for+over+200+conditions.">Global health statistics: a compendium of incidence prevalence and mortality estimates for over 200 conditions.</a> World Health Organization Harvard School of Public Health World Bank. , ii (1996).</li><li>Sackeim, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+definition+and+meaning+of+treatment-resistant+depression.">The definition and meaning of treatment-resistant depression.</a> Journal of Clinical Psychiatry. 62 (16), 10-17 (2001).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+compounds+of+soothing+liver,+invigorating+spleen,+tonifying+kidney+on+the+praxiology+and+immunological+function+of+chronic+immobilization+stressed+rats.">The effect of compounds of soothing liver, invigorating spleen, tonifying kidney on the praxiology and immunological function of chronic immobilization stressed rats.</a> Acta Laboratorium Animalis Scientia Sinica. 11, 33-37 (2003).</li><li>Meng, Z. Z., Chen, J. X., Jiang, Y. M., Zhang, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Xiaoyaosan+Decoction+on+Learning+and+Memory+Deficit+in+Rats+Induced+by+Chronic+Immobilization+Stress.">Effect of Xiaoyaosan Decoction on Learning and Memory Deficit in Rats Induced by Chronic Immobilization Stress.</a> Evidence-Based Complementary and Alternative. 2013 (17), 297154 (2013).</li><li>Zhao, H., Wan, X., Chen, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mini+review+of+traditional+Chinese+medicine+for+the+treatment+of+depression+in+China.">A mini review of traditional Chinese medicine for the treatment of depression in China.</a> American Journal of Chinese Medicine. 37 (02), 207-213 (2009).</li><li>Gao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabonomic+study+on+chronic+unpredictable+mild+stress+and+intervention+effects+of+Xiaoyaosan+in+rats+using+gas+chromatography+coupled+with+mass+spectrometry.">Metabonomic study on chronic unpredictable mild stress and intervention effects of Xiaoyaosan in rats using gas chromatography coupled with mass spectrometry.</a> Journal of Ethnopharmacology. 137 (1), 690-699 (2011).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNP+signal+pathway+up-regulated+in+rectum+of+depressed+rats+and+the+interventional+effect+of+Xiaoyaosan.">CNP signal pathway up-regulated in rectum of depressed rats and the interventional effect of Xiaoyaosan.</a> World Journal of Gastroenterology. 21 (5), 1518 (2015).</li><li>Li, X. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xiaoyaosan+exerts+anxiolytic-like+effects+by+down-regulating+the+TNF-&#945;/JAK2-STAT3+pathway+in+the+rat+hippocampus.">Xiaoyaosan exerts anxiolytic-like effects by down-regulating the TNF-&#945;/JAK2-STAT3 pathway in the rat hippocampus.</a> Scientific Reports. 7 (1), 353 (2017).</li><li>Jun-Sheng, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+the+endogenous+metabolites+in+depressed+patients+treated+with+TCM+formula+Xiaoyaosan+using+urinary+(1)H+NMR-based+metabolomics.">Dynamic analysis of the endogenous metabolites in depressed patients treated with TCM formula Xiaoyaosan using urinary (1)H NMR-based metabolomics.</a> Journal of Ethnopharmacology. 158, 1-10 (2014).</li><li>Dai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+study+on+the+anti-depression+effect+of+xiaoyaosan+on+rat+model+of+chronic+unpredictable+mild+stress.">Metabolomics study on the anti-depression effect of xiaoyaosan on rat model of chronic unpredictable mild stress.</a> Journal of Ethnopharmacology. 128 (2), 482-489 (2010).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antidepressant-like+effects+of+the+fractions+of+Xiaoyaosan+on+rat+model+of+chronic+unpredictable+mild+stress.">Antidepressant-like effects of the fractions of Xiaoyaosan on rat model of chronic unpredictable mild stress.</a> Journal of Ethnopharmacology. 137 (1), 236-244 (2011).</li><li>Wang, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Costs+and+Benefits+of+Enhanced+Depression+Care+to+Employers.">The Costs and Benefits of Enhanced Depression Care to Employers.</a> Archives of General Psychiatry. 63 (12), 1345-1353 (2006).</li><li>Pirraglia, P. A., Rosen, A. B., Hermann, R. C., Olchanski, N. V., Neumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-utility+analysis+studies+of+depression+management:+a+systematic+review.">Cost-utility analysis studies of depression management: a systematic review.</a> American Journal of Psychiatry. 161 (12), 2155-2162 (2004).</li><li>Wang, S. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xiaoyaosan+Decoction+Regulates+Changes+in+Neuropeptide+Y+and+Leptin+Receptor+in+the+Rat+Arcuate+Nucleus+after+Chronic+Immobilization+Stress.">Xiaoyaosan Decoction Regulates Changes in Neuropeptide Y and Leptin Receptor in the Rat Arcuate Nucleus after Chronic Immobilization Stress.</a> Evidence-based Complementary and Alternative Medicine. 2012 (4), 381278 (2012).</li><li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xiao+Yao+San+Improves+Depressive-Like+Behavior+in+Rats+through+Modulation+of+&#946;-Arrestin+2-Mediated+Pathways+in+Hippocampus.">Xiao Yao San Improves Depressive-Like Behavior in Rats through Modulation of &#946;-Arrestin 2-Mediated Pathways in Hippocampus.</a> Evidence-based Complementary and Alternative. 2014, (2014).</li><li>Ding, X. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+Normalized+Glial+Fibrillary+Acidic+Protein+Expression+in+the+Hippocampi+in+Antidepressant-Like+Effects+of+Xiaoyaosan+on+Chronically+Stressed+Mice.">Involvement of Normalized Glial Fibrillary Acidic Protein Expression in the Hippocampi in Antidepressant-Like Effects of Xiaoyaosan on Chronically Stressed Mice.</a> Evidence-Based Complementary and Alternative Medicine. 2017, 1960584 (2017).</li><li>Ding, X. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+glutamate/glutamine+cycle+and+glutamate+transporter+GLT-1+in+antidepressant-like+effects+of+Xiao+Yao+san+on+chronically+stressed+mice.">Involvement of the glutamate/glutamine cycle and glutamate transporter GLT-1 in antidepressant-like effects of Xiao Yao san on chronically stressed mice.</a> BMC Complementary &amp; Alternative Medicine. 17 (1), 326 (2017).</li><li>Murua, V. S., Gomez, R. A., Andrea, M. E., Molina, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shuttle-box+deficits+induced+by+chronic+variable+stress:+reversal+by+imipramine+administration.">Shuttle-box deficits induced by chronic variable stress: reversal by imipramine administration.</a> Pharmacol Biochem Behav. 38 (1), 125 (1991).</li><li>Duan, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+of+5-fluorouracil+and+cyclophosphamide+in+depression+rats.">Pharmacokinetics of 5-fluorouracil and cyclophosphamide in depression rats.</a> European Review for Medical &amp; Pharmacological Sciences. 16 (4), 427-436 (2012).</li><li>Cao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-derived+ATP+modulates+depressive-like+behaviors.">Astrocyte-derived ATP modulates depressive-like behaviors.</a> Science Foundation in China. 19 (1), 56 (2013).</li><li>Kirshenbaum, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+neuronal+Na+,+K++-ATPase+activity+in+Atp1a3+heterozygous+mice+increases+susceptibility+to+depression-like+endophenotypes+by+chronic+variable+stress.">Decreased neuronal Na+, K+ -ATPase activity in Atp1a3 heterozygous mice increases susceptibility to depression-like endophenotypes by chronic variable stress.</a> Genes Brain &amp; Behavior. 10 (5), 542-550 (2011).</li><li>Liu, X. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+metabonomic+study+using+a+CUMS+rat+model+of+depression.">Urinary metabonomic study using a CUMS rat model of depression.</a> Magnetic Resonance in Chemistry. 50 (3), 187-192 (2012).</li><li>Zhu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpredictable+chronic+mild+stress+induces+anxiety+and+depression-like+behaviors+and+inactivates+AMP-activated+protein+kinase+in+mice.">Unpredictable chronic mild stress induces anxiety and depression-like behaviors and inactivates AMP-activated protein kinase in mice.</a> Brain Research. 1576, (2014).</li><li>Yang, C., Wang, G., Wang, H., Liu, Z., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+alterations+in+rat+hippocampus+following+chronic+unpredictable+mild+stress+and+re-exposure+to+acute+and+chronic+unpredictable+mild+stress.">Cytoskeletal alterations in rat hippocampus following chronic unpredictable mild stress and re-exposure to acute and chronic unpredictable mild stress.</a> Behavioural Brain Research. 205 (2), 518-524 (2009).</li><li>Ding, X. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xiao+Yao+San+Improves+Depressive-Like+Behaviors+in+Rats+with+Chronic+Immobilization+Stress+through+Modulation+of+Locus+Coeruleus-Norepinephrine+System.">Xiao Yao San Improves Depressive-Like Behaviors in Rats with Chronic Immobilization Stress through Modulation of Locus Coeruleus-Norepinephrine System.</a> Evidence-Based Complementary and Alternative. , 605914 (2014).</li><li>Wu, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antidepressant-Like+Effects+of+Fractions+Prepared+from+Danzhi-Xiaoyao-San+Decoction+in+Rats+with+Chronic+Unpredictable+Mild+Stress:+Effects+on+Hypothalamic-Pituitary-Adrenal+Axis,+Arginine+Vasopressin,+and+Neurotransmitters.">Antidepressant-Like Effects of Fractions Prepared from Danzhi-Xiaoyao-San Decoction in Rats with Chronic Unpredictable Mild Stress: Effects on Hypothalamic-Pituitary-Adrenal Axis, Arginine Vasopressin, and Neurotransmitters.</a> Evidence-Based Complementary and Alternative Medicine. 2016 (3), 1-11 (2016).</li><li>Stanford, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Open+Field+Test:+reinventing+the+wheel.">The Open Field Test: reinventing the wheel.</a> Journal of Psychopharmacology. 21 (2), 134 (2007).</li><li>Zeldetz, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Method+for+Inducing+a+Depression-Like+Behavior+in+Rats.">A New Method for Inducing a Depression-Like Behavior in Rats.</a> Journal of Visualized Experiments. (132), e57137 (2018).</li><li>Willner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+mild+stress+(CMS)+revisited:+consistency+and+behavioural-neurobiological+concordance+in+the+effects+of+CMS.">Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological concordance in the effects of CMS.</a> Neuropsychobiology. 52 (2), 90-110 (2005).</li><li>Wood, G. E., Norris, E. H., Waters, E., Stoldt, J. T., Mcewen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+immobilization+stress+alters+aspects+of+emotionality+and+associative+learning+in+the+rat.">Chronic immobilization stress alters aspects of emotionality and associative learning in the rat.</a> Behavioral Neuroscience. 122 (2), 282-292 (2008).</li></ol>

The authors have nothing to disclose.

The CUMS model used in this study is a common method to establish the stress diathesis model of depression24. Most manifestations of CUMS can be restored by antidepressant drugs, which indicated that this model method has a strong predictive validity25. By this method, animals receive chronic unpredictable stressful stimuli and develop core symptoms of major depression, including decreased place preference conditioning, anhedonia, and impaired emotion-like behaviors<sup cla...

MDD is a chronic, highly recurrent mental disease that presents with persistent low mood, depression, low self-esteem, mental retardation, loss of interest, sleep disorders, profoundly pessimistic or suicidal tendencies; it has caused great harm to human health and social stabilization1. It has become one of the world&#39;s primary cause of disability, and it may be one of the main leading causes of disease burden by 2030 according to the World Health Organization2,3. Currently, antidepressant medications and psychotherapy are considered to be the first choice to treat MDD. Nevertheless, an enormous number of patients diagnosed with MDD do not respond to continuous medication or therapy, and they are listed as having treatment-resistant depression4.
Xiaoyaosan is a classic Chinese compound5 that has been widely used to treat psychological diseases for thousands of years since the Song dynasty in China6. It is composed of 8 Chinese herbs: Radix Bupleuri, Radix Paeoniae Alba, Radix Angelicae Sinensis, Poria, Rhizoma Atractylodis Macrocephalae, Radix Glycyrrhizae, Rhizoma Zingiberis Recens, and Herba Menthae. Xiaoyaosan contains multiple constituents, has multiple targets, and utilizes multiple pathways7. Xiaoyaosan has been commonly shown to be a safe and effective formula for treating various diseases related to mental disorders, and modern studies have also focused on the anti-depression mechanism of this compound8,9,10. The pathogenesis of MDD is yet unclear, even though there are multifarious hypotheses, including the monoamine hypothesis, the neurotransmitter receptor hypothesis, the neuroendocrine function change hypothesis, the immune system abnormality hypothesis, etc. Several studies have shown that the antidepressant effect of Xiaoyaosan has a connection with the material bases of the above hypotheses11,12, and its therapeutic action manifests as an improvement to depressive-like behaviors13.
Moreover, with the prevalence of depression, modern society has suffered from reductions in the work force, early retirements, and expensive treatments14. Expensive treatment needs strict medical attention, which means a range of increased medical costs and precise anticipation of precise undesirable outcomes as well as side effects15. The Chinese herb Xiaoyaosan has accessible components, unique effects, and slight side effects. Its application can play a role in reducing the burden of modern medical treatment. Therefore, to provide a more theoretical basis for the application of Xiaoyaosan, an alternative clinical therapy should be offered for the treatment of depression, the incidence of which increases year by year, and should improve human health at the same time. This paper describes a protocol involving the use of Xiaoyaosan to prevent the depressive-like behaviors by establishing the depressive model of the mouse. The open field test and sucrose preference test were used to verify the antidepressant effects of Xiaoyaosan.

<table>
	<tbody>
		<tr>
			<td>Chinese Herbs</td>
			<td>Tongrentang (Bozhou, AnHui) Decoction Pieces Limited Company</td>
			<td>-</td>
			<td>The drugs were extracted by the Chinese medicine preparation room of the China-Japan Friendship Hospital, distilled water was used to prepare the suspension of Xiaoyaosan, and the dose was 0.25 g/kg/d.</td>
		</tr>
		<tr>
			<td>Fluoxetine</td>
			<td>PATHEON FRANCE</td>
			<td>5545A</td>
			<td>The distilled water was used to prepare the suspension of fluoxetine, and the dose 2.6 mg/kg/d.</td>
		</tr>
		<tr>
			<td>C57BL/6j mice</td>
			<td>Beijing Vital River of the Charles River Company</td>
			<td>SCXK 2011-0004</td>
			<td>60 healthy male C57BL/6J mice aged 12 weeks were purchased from Beijing Vital River of the Charles River Company.</td>
		</tr>
		<tr>
			<td>Mouse cage</td>
			<td>Styling Biotechnology</td>
			<td>34853016</td>
			<td>318&#215;202&#215;135 mm</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>-</td>
			<td>-</td>
			<td>The water was used througout the experiment.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>-</td>
			<td>The sucrose was used in the sucrose preference test to prepare 1% sucrose solution.</td>
		</tr>
		<tr>
			<td>ACS-2EAS Electronic Balance</td>
			<td>Beijing Hengzhongheng Electronic Technology Co., Ltd.</td>
			<td>-</td>
			<td>It was used to weigh the avoirdupois of mice at different stages.</td>
		</tr>
		<tr>
			<td>Wooden Black Lusterless Box</td>
			<td>-</td>
			<td>-</td>
			<td>(40 cm x 40 cm x 15 cm), divided into 25 equal squares by blue lines.</td>
		</tr>
		<tr>
			<td>WV-CP470 Camera</td>
			<td>Panasonic, Japan</td>
			<td>-</td>
			<td>A HD digital camera which was used for recording the whole process of the open field test.</td>
		</tr>
		<tr>
			<td>Etho Vision 3.0 Software</td>
			<td>Noldus, Wageningen, Holland</td>
			<td>-</td>
			<td>A video-tracking system which was used to evaluate the locomotor function and emotionality of mice.</td>
		</tr>
		<tr>
			<td>SPSS Statistics 21.0 Software</td>
			<td>IBM</td>
			<td>-</td>
			<td>SPSS 21.0 software was used to analyze the data, one-way ANOVA or non-parametric test was used for general data based on normality test and homogeneity test for variance, and LSD method was adopted for the comparisons between groups.</td>
		</tr>
		<tr>
			<td>Graphpad Prism 6.0 Software</td>
			<td>Graphpad Software,Inc.USA</td>
			<td>-</td>
			<td>Graphpad Prism 6.0 software was used to draw statistical graphs.</td>
		</tr>
	</tbody>
</table>

evaluating the anti-depression effect of xiaoyaosan on chronically-stressed mice

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall improvement of patients with major depressive disorder (MDD). Therefore, the purpose of this study was to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and then evaluate the anti-depression effect of Xiaoyaosan. Mice were randomly divided into four groups: a control group, a model group, a treatment group with Xiaoyaosan, and a treatment group with fluoxetine. All mice were individually kept in cages, and depression was induced in the mice by exposing them to several designed manipulations of CUMS for 21 days, as described in the protocol. Mice in the control group and model group received 0.5 mL of distilled water, while mice in the treatment groups received either Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day). The drugs used in the study were given intragastrically daily during the entire three weeks. To estimate the depressive-like behaviors, a series of parameters including the coat state, body weight, open field test score, and sucrose preference test score were recorded. Data analysis showed that behaviors of model mice were significantly changed compared to behaviors of mice in the control group, which were improved by the treatment of Xiaoyaosan and fluoxetine. The current findings demonstrated the anti-depression effects of Xiaoyaosan on the behaviors of CUMS-induced mice and revealed that compounds from the Xiaoyaosan prescription may be worthwhile for treating depression, considering their beneficial effects on depressive-like behaviors.

Note that the results have been previously published by Dr. Xiu-Fang Ding, et al.18
Data were expressed as the mean &#177; standard error of the mean (SEM). A one-way ANOVA or a non-parametric test was used for general data based on the normality test and the test for homogeneity of variance. The LSD test was used for the comparisons between groups. In addition, the repeated measurement process o...

Watch this Scientific Journal Video about Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice at JoVE.com

Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice

Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall ...

evaluating-anti-depression-effect-xiaoyaosan-on-chronically-stressed

Research

Education

JoVE Journal

JoVE Core

Introduction to Psychology

Pharmacology

Behavior

Unit 1

States of Consciousness

Pharmacotherapy of Depression and Anxiety Disorders

SCIENTISTS IN ACTION

9.1K Views.  Beijing University of Chinese Medicine. Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.

Video: Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Eye Tracking, Cortisol, and a Sleep vs. Wake Consolidation Delay: Combining Methods to Uncover an Interactive Effect of Sleep and Cortisol on Memory

Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these two factors may interact to influence consolidation. Here we present a protocol to examine the interactive influence of cortisol and sleep on memory consolidation, by combining three methods: eye tracking, salivary cortisol analysis, and behavioral memory testing across sleep and wake delays. To assess resting cortisol levels, participants gave a saliva sample before viewing negative and neutral objects within scenes. To measure overt attention, participants&#8217; eye gaze was tracked during encoding. To manipulate whether sleep occurred during the consolidation window, participants either encoded scenes in the evening, slept overnight, and took a recognition test the next morning, or encoded scenes in the morning and remained awake during a comparably long retention interval. Additional control groups were tested after a 20&#160;min&#160;delay in the morning or evening, to control for time-of-day effects. Together, results showed that there is a direct relation between resting cortisol at encoding and subsequent memory, only following a period of sleep. Through eye tracking, it was further determined that for negative stimuli, this beneficial effect of cortisol on subsequent memory may be due to cortisol strengthening the relation between where participants look during encoding and what they are later able to remember. Overall, results obtained by a combination of these methods uncovered an interactive effect of sleep and cortisol on memory consolidation.

We present a protocol used to discover an interactive effect between sleep and cortisol on memory consolidation, particularly for negative arousing images. Specifically, the experimental design utilizes eye tracking, salivary cortisol analysis, and behavioral memory testing &#8211; methods that can be used with both healthy and clinical participants.

Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these ...

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

Comparing the Frequency Effect Between the Lexical Decision and Naming Tasks in Chinese

In psycholinguistic research, the frequency effect can be one of the indicators for eligible experimental tasks that examine the nature of lexical access. Usually, only one of those tasks is chosen to examine lexical access in a study. Using two exemplar experiments, this paper introduces an approach to include both the lexical decision task and the naming task in a study. In the first experiment, the stimuli were Chinese characters with frequency and regularity manipulated. In the second experiment, the stimuli were switched to Chinese two-character words, in which the word frequency and the regularity of the leading character were manipulated. The logic of these two exemplar experiments was to explore some important issues such as the role of phonology on recognition by comparing the frequency effect between both the tasks. The results revealed different patterns of lexical access from those reported in the alphabetic systems. The results of Experiment 1 manifested a larger frequency effect in the naming task as compared to the LDT, when the stimuli were Chinese characters. And it is noteworthy that, in Experiment 1, when the stimuli were regular Chinese characters, the frequency effect observed in the naming task was roughly equivalent to that in the LDT. However, a smaller frequency effect was shown in the naming task as compared to the LDT, when the stimuli were switched to Chinese two-character words in Experiment 2. Taking advantage of the respective demands and characteristics in both tasks, researchers can obtain a more complete and precise picture of character/word recognition.

Researchers adopt both the lexical decision task and the naming task to investigate some important topics such as character/word recognition by comparing the frequency effect between these two tasks. This article introduces this approach through two exemplar experiments and elaborates on the underlying logic.

In psycholinguistic research, the frequency effect can be one of the indicators for eligible experimental tasks that examine the nature of lexical access. ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

Evaluating Skilled Prehension in Mice Using an Auto-Trainer

We describe a method to introduce na&#239;ve mice to a novel prehension (reach-to-grasp) task. Mice are housed singly in cages with a frontal slot that permits the mouse to reach out of its cage and retrieve food pellets. Minimal food restriction is employed to encourage the mice to perform the food retrieval from the slot. As the mice begin to associate coming to the slot for food, the pellets are manually pulled away to stimulate extension and pronation of their paw to grasp and retrieve the pellet through the frontal slot. When the mice begin to reach for the pellets as they arrive at the slot, the behavioral assay can be performed by measuring the rate at which they successfully grasp and retrieve the desired pellet. They are then introduced to an auto-trainer that automates both the process of providing food pellets for the mouse to grasp, and the recording of successful and failed reaching and grasping attempts. This allows for the collection of reaching data for multiple mice with minimal effort, to be used in experimental analysis as appropriate.

Method to assess the impact of training on motor skills is a useful tool. Unfortunately, most behavioral assessments can be labor intensive and/or expensive.We describe here a robotic method of assessing prehension (reach-to-grasp) skill in mice.

We describe a method to introduce na&#239;ve mice to a novel prehension (reach-to-grasp) task. Mice are housed singly in cages with a frontal slot that ...

Investigating the Effect of Visual Imagery and Learning Shape-Audio Regularities on Bouba and Kiki

This article presents a protocol for investigating the role of visual imagery in the bouba/kiki-effect, whether training in noticing the bouba/kiki shape-audio regularities affects the bouba/kiki-effect and the recognition of individual bouba and kiki shapes, and finally what mental images these regularities produce. To generate bouba/kiki shape-audio regularities, there were two types of shapes (filled; outlined) and two types of audio (word; non-word sound). Three groups of individuals participated in three experiments: Blind, blindfold, and vision. The experiments were conducted in fixed order across participants, with no break between them. In Experiment 1 (pre-test-post-test design with three repeated within-group measures) the participants were asked to pick out the shape they associated with the auditory bouba/kiki; in Experiment 2 (within-subject design), to name one shape and some audio (sometimes congruous; sometimes incongruous) as &#8216;bouba&#8217; or &#8216;kiki;&#8217; and in Experiment 3 (post-test only design), to draw the shape they associated with the auditory bouba/kiki. The results suggest that the blindfold-group draw upon visual imagery to solve new problems, but not long term; that training in noticing bouba/kiki shape-audio regularities affects the bouba/kiki-effect and the recognition of individual bouba and kiki shapes, but differently in each experimental group; and that all experimental groups create mental images of the most characteristic shape feature of bouba (curve) and kiki (angle). In fact, the effect of visual imagery is robust across tasks, but not long term; the effect of learning shape-audio regularities is robust long term, but not across tasks. The presented protocol is appropriate for investigating the effect of visual imagery and learning shape-audio regularities, when they occur and how robust they are; in specific individuals and groups of individuals. This protocol is unique in that it keeps under control both the visual imagery and the sensory information during training and testing.

The aim of the presented protocol is to investigate the role of visual imagery in the bouba/kiki-effect, whether training in noticing the bouba/kiki shape-audio regularities affects the bouba/kiki-effect and the recognition of individual bouba and kiki shapes, and finally what mental images these regularities produce.

This article presents a protocol for investigating the role of visual imagery in the bouba/kiki-effect, whether training in noticing the bouba/kiki ...

Assessment of Sexual Behavior of Male Mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

This article describes how to perform sexual behavior tests in male mice.

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...