Abstract: Premenstrual dysphoric disorder (PMDD), a subtype of premenstrual syndrome (PMS), involves physical and emotional symptoms that impact patients’ daily lives and productivity. A reliable, side-effect-free clinical intervention is needed. Shuyu capsule is an effective traditional Chinese medicine preparation for PMDD used in the clinics, but its therapeutic mechanism remains unclear. Previous research has suggested that the γ-aminobutyric acidergic (GABAergic) system in the periaqueductal gray (PAG) may play a role in treating PMDD with traditional Chinese medicine, but there is a lack of functional verification. This study aims to reveal the potential mechanism of the Shuyu capsule in treating PMDD. The study employed an experimental design using female C57BL/6J and Vgat-Cre mice to assess the effects of Shuyu capsules on PMDD, with a focus on the GABAergic system in the dorsal PAG (dPAG). Assessments were conducted using the forced swimming test (FST) to gauge depression-like behaviors and western blot (WB) and immunofluorescence (IF) to measure the numbers of active GABAergic neurons and the γ-aminobutyric acid type A receptor (GABA_AR) δ subunit (GABRD) expression. Chemogenetic techniques and adeno-associated virus were specifically used to activate GABAergic neurons and knock down the expression of subunits, respectively, providing insights into the neurobiological mechanisms underpinning the therapeutic effects of Shuyu capsules in treating PMDD. After being stressed by FST, the immobility duration of PMDD mice in the late dioestrus (LD) phase decreased after the Shuyu capsule intervention, implying that it can improve the estrous cycle-dependent depression-like phenotype in PMDD mice. Additionally, the application of Shuyu capsule can downregulate the expression of GABRD and reverse the downtrend of activated GABAergic neurons in the dPAG of PMDD model mice. We also found that single-target manipulation was enough to improve the depression-like behavior of PMDD model mice. Transgenic mice with GABRD knockout were established, and their behaviors were tested, revealing changes in their exploratory behaviors, indicating that the GABRD may be closely related to anxiety disorders. Shuyu capsule plays an anti-PMDD role by activating GABAergic neurons and downregulating the expression of GABRD in the dPAG. This provides a theoretical basis for the clinical treatment of PMDD with traditional Chinese medicine and promotes the development of drugs for treating PMDD.

舒郁胶囊通过激活dPAG脑区γ-氨基丁酸能神经元和下调GABA_A受体δ亚基的表达改善经前烦躁症小鼠动情周期依赖性抑郁样行为

[1]BehbehaniMM, 1995. Functional characteristics of the midbrain periaqueductal gray. Prog Neurobiol, 46(6):575-605.

[2]BerlyneDE, 1966. Curiosity and exploration: animals spend much of their time seeking stimuli whose significance raises problems for psychology. Science, 153(3731):25-33.

[3]BrackKE, LovickTA, 2007. Neuronal excitability in the periaqueductal grey matter during the estrous cycle in female Wistar rats. Neuroscience, 144(1):325-335.

[4]CampionKN, SavilleKA, MorganMM, 2016. Relative contribution of the dorsal raphe nucleus and ventrolateral periaqueductal gray to morphine antinociception and tolerance in the rat. Eur J Neurosci, 44(9):2667-2672.

[5]CarverCM, ReddyDS, 2013. Neurosteroid interactions with synaptic and extrasynaptic GABA_A receptors: regulation of subunit plasticity, phasic and tonic inhibition, and neuronal network excitability. Psychopharmacology (Berl), 230(2):151-188.

[6]ChungG, ShimHG, KimCY, et al., 2020. Persistent activity of metabotropic glutamate receptor 5 in the periaqueductal gray constrains emergence of chronic neuropathic pain. Curr Biol, 30(23):4631-4642.e6.

[7]DevallAJ, LovickTA, 2010. Differential activation of the periaqueductal gray by mild anxiogenic stress at different stages of the estrous cycle in female rats. Neuropsychopharmacology, 35(5):1174-1185.

[8]DevallAJ, LiuZW, LovickTA, 2009. Hyperalgesia in the setting of anxiety: sex differences and effects of the oestrous cycle in Wistar rats. Psychoneuroendocrinology, 34(4):587-596.

[9]DevallAJ, SantosJM, FryJP, et al., 2015. Elevation of brain allopregnanolone rather than 5-HT release by short term, low dose fluoxetine treatment prevents the estrous cycle-linked increase in stress sensitivity in female rats. Eur Neuropsychopharmacol, 25(1):113-123.

[10]ErikaEC, López-RubalcavaC, 2022. Can animal models resemble a premenstrual dysphoric condition? Front Neuroendocrinol, 66:101007.

[11]FarrantM, NusserZ, 2005. Variations on an inhibitory theme: phasic and tonic activation of GABA_A receptors. Nat Rev Neurosci, 6(3):215-229.

[12]FirstMB, 2013. Diagnostic and statistical manual of mental disorders, 5th edition, and clinical utility. J Nerv Ment Dis, 201(9):727-729.

[13]GaoXX, SunP, QiaoMQ, et al., 2014. Shu-Yu capsule, a traditional Chinese medicine formulation, attenuates premenstrual syndrome depression induced by chronic stress constraint. Mol Med Rep, 10(6):2942-2948.

[14]GengXW, WangXY, LiuK, et al., 2024. ShuYu capsule alleviates emotional and physical symptoms of premenstrual dysphoric disorder: impact on ALLO decline and GABA_A receptor δ subunit in the PAG area. Phytomedicine, 130:155549.

[15]GirdlerSS, StranevaPA, LightKC, et al., 2001. Allopregnanolone levels and reactivity to mental stress in premenstrual dysphoric disorder. Biol Psychiatry, 49(9):788-797.

[16]GoldsteinJM, JerramM, PoldrackR, et al., 2005. Hormonal cycle modulates arousal circuitry in women using functional magnetic resonance imaging. J Neurosci, 25(40):9309-9316.

[17]GriffithsJ, LovickT, 2005. Withdrawal from progesterone increases expression of α4, β1, and δ GABA_A receptor subunits in neurons in the periaqueductal gray matter in female Wistar rats. J Comp Neurol, 486:89-97.

[18]GulinelloM, GongQH, SmithSS, 2002. Progesterone withdrawal increases the α4 subunit of the GABA_A receptor in male rats in association with anxiety and altered pharmacology—a comparison with female rats. Neuropharmacology, 43(4):701-714.

[19]HoltRA, BatesonAN, MartinIL, 1996. Chronic treatment with diazepam or abecarnil differentially affects the expression of GABA_A receptor subunit mRNAs in the rat cortex. Neuropharmacology, 35(9-10):1457-1463.

[20]HsiungA, PohJH, HuettelSA, et al., 2023. Curiosity evolves as information unfolds. Proc Natl Acad Sci USA, 120(43):e2301974120.

[21]HuZF, MuYM, HuangL, et al., 2022. A visual circuit related to the periaqueductal gray area for the antinociceptive effects of bright light treatment. Neuron, 110(10):1712-1727.e7.

[22]IsmailiE, WalshS, O'BrienPMS, et al., 2016. Fourth consensus of the international society for premenstrual disorders (ISPMD): auditable standards for diagnosis and management of premenstrual disorder. Arch Womens Ment Health, 19(6):953-958.

[23]JensenML, TimmermannDB, JohansenTH, et al., 2002. The β subunit determines the ion selectivity of the GABA_A receptor. J Biol Chem, 277(44):41438-41447.

[24]KadianS, O'BrienS, 2012. Classification of premenstrual disorders as proposed by the international society for premenstrual disorders. Menopause Int, 18(2):43-47.

[25]KlatzkinRR, MorrowAL, LightKC, et al., 2006. Associations of histories of depression and PMDD diagnosis with allopregnanolone concentrations following the oral administration of micronized progesterone. Psychoneuroendocrinology, 31(10):1208-1219.

[26]KohW, LeeCJ, 2024. Diagnostic and therapeutic potential of tonic gamma-aminobutyric acid from reactive astrocytes in brain diseases. Clin Transl Med, 14(4):e1642.

[27]KohW, KwakH, CheongE, et al., 2023. GABA tone regulation and its cognitive functions in the brain. Nat Rev Neurosci, 24(9):523-539.

[28]KyzarEJ, PhamM, RothA, et al., 2012. Alterations in grooming activity and syntax in heterozygous SERT and BDNF knockout mice: the utility of behavior-recognition tools to characterize mutant mouse phenotypes. Brain Res Bull, 89(5-6):168-176.

[29]LevyDR, HunterN, LinS, et al., 2023. Mouse spontaneous behavior reflects individual variation rather than estrous state. Curr Biol, 33(7):1358-1364.e4.

[30]LiF, FengJ, GaoD, et al., 2016. Shuyu capsules relieve premenstrual syndrome depression by reducing 5-HT_3AR and 5-HT_3BR expression in the rat brain. Neural Plast, 2016:7950781.

[31]LovickTA, GriffithsJL, DunnSM, et al., 2005. Changes in GABA_A receptor subunit expression in the midbrain during the oestrous cycle in Wistar rats. Neuroscience, 131(2):397-405.

[32]Lowery-GiontaEG, DibertoJ, MazzoneCM, et al., 2018. GABA neurons of the ventral periaqueductal gray area modulate behaviors associated with anxiety and conditioned fear. Brain Struct Funct, 223(8):3787-3799.

[33]MaguireJ, ModyI, 2008. GABA_AR plasticity during pregnancy: relevance to postpartum depression. Neuron, 59(2):207-213.

[34]MarvánML, SantanaS, Chávez ChávezL, et al., 1997. Inescapable shocks accentuate fluctuations of forced swimming immobility in different phases of the rat estrous cycle. Arch Med Res, 28(3):369-372.

[35]ModyI, PearceRA, 2004. Diversity of inhibitory neurotransmission through GABA_A receptors. Trends Neurosci, 27(9):569-575.

[36]MöhlerH, 2006. GABA_A receptor diversity and pharmacology. Cell Tissue Res, 326(2):505-516.

[37]NobleRE, 2005. Depression in women. Metabolism, 54(5):49-52.

[38]OsborneL, YenokyanG, SanyalA, et al., 2019. 37. The role of allopregnanolone in perinatal mood and anxiety disorders. Biol Psychiatry, 85(10):S15-S16.

[39]PinnaG, UzunovaV, MatsumotoK, et al., 2000. Brain allopregnanolone regulates the potency of the GABA_A receptor agonist muscimol. Neuropharmacology, 39(3):440-448.

[40]PuiaG, MienvilleJM, MatsumotoK, et al., 2003. On the putative physiological role of allopregnanolone on GABA_A receptor function. Neuropharmacology, 44(1):49-55.

[41]ReichlingDB, BasbaumAI, 1990. Contribution of brainstem GABAergic circuitry to descending antinociceptive controls: I. GABA-immunoreactive projection neurons in the periaqueductal gray and nucleus raphe magnus. J Comp Neurol, 302(2):370-377.

[42]ReidRL, SoaresCN, 2018. Premenstrual dysphoric disorder: contemporary diagnosis and management. J Obstet Gynaecol Can, 40(2):215-223.

[43]RodgersRJ, DalviA, 1997. Anxiety, defence and the elevated plus-maze. Neurosci Biobehav Rev, 21(6):801-810.

[44]RoughanJV, Wright-WilliamsSL, FlecknellPA, 2009. Automated analysis of postoperative behaviour: assessment of HomeCageScan as a novel method to rapidly identify pain and analgesic effects in mice. Lab Anim, 43(1):17-26.

[45]RudolphS, GuoC, PashkovskiSL, et al., 2020. Cerebellum-specific deletion of the GABA_A receptor δ subunit leads to sex-specific disruption of behavior. Cell Rep, 33(5):108338.

[46]SchneiderT, PopikP, 2007. Increased depressive-like traits in an animal model of premenstrual irritability. Horm Behav, 51(1):142-148.

[47]SekhonS, GuptaV, 2024. Mood disorder. In: Statpearls. StatPearls Publishing, Treasure Island (FL).

[48]SmithAJ, OxleyB, MalpasS, et al., 2004. Compounds exhibiting selective efficacy for different β subunits of human recombinant γ-aminobutyric acid_A receptors. J Pharmacol Exp Ther, 311(2):601-609.

[49]SmithSS, RudermanY, FryeC, et al., 2006. Steroid withdrawal in the mouse results in anxiogenic effects of 3α,5β-THP: a possible model of premenstrual dysphoric disorder. Psychopharmacology (Berl), 186(3):323-333.

[50]SpindeleggerCJ, PapageorgiouK, GrohmannR, et al., 2015. Cardiovascular adverse reactions during antidepressant treatment: a drug surveillance report of German-speaking countries between 1993 and 2010. Int J Neuropsychopharmacol, 18(4):pyu080.

[51]Sundström-PoromaaI, ComascoE, SumnerR, et al., 2020. Progesterone ‒ friend or foe? Front Neuroendocrinol, 59:100856.

[52]WalzN, MühlbergerA, PauliP, 2016. A human open field test reveals thigmotaxis related to agoraphobic fear. Biol Psychiatry, 80(5):390-397.

[53]WangXL, GaoJ, WangXY, et al., 2018. Treatment with Shuyu capsule increases 5-HT1AR level and activation of cAMP-PKA-CREB pathway in hippocampal neurons treated with serum from a rat model of depression. Mol Med Rep, 17(3):3575-3582.

[54]WeiS, GengXW, LiZF, et al., 2020. A forced swim-based rat model of premenstrual depression: effects of hormonal changes and drug intervention. Aging (Albany NY), 12(23):24357-24370.

[55]Yankelevitch-YahavR, FrankoM, HulyA, et al., 2015. The forced swim test as a model of depressive-like behavior. J Vis Exp, (97):52587.

[56]ZhangH, QiaoMQ, GaoDM, et al., 2023. Shuyu capsule alleviates premenstrual depression via allopregnanolone metabolic pathway targeting GABA (A) receptors δ subunit in the hippocampus. Asian J Psychiatr, 90:103800.