【作者】 何勇；

【导师】 谢鹏；

【作者基本信息】 重庆医科大学 ， 神经病学， 2023， 博士

【摘要】 研究背景抑郁症是一种慢性反复发作的神经精神障碍疾病,患者常出现持续的心境低落、快感缺失等症状,甚至伴有自杀行为倾向。抑郁症的发病机制极其复杂,并且现有的抗抑郁治疗效果十分有限。抑郁症发病涉及多个脑区,包括海马,前额叶,杏仁核,伏隔核,前扣带回,外侧僵核,腹侧被盖区等以及它们之间互相连接的神经环路。现有研究发现了众多与抑郁症相关的神经环路,但是这些神经环路依然还不够完善。一方面,因为不同的神经环路可能同时参与调控抑郁和焦虑,或者只参与调控其中一种行为表型。另外一方面,有些神经环路对疾病行为表现为正向调节,有些则为负向调节,还有些则为双向动态调节。因此,从某种程度上来说抑郁症也可以被看作是一种神经环路疾病。丘脑室旁核(paraventricular nucleus of thalamus,PVT)是位于中线丘脑背侧部的一个小核团,主要接受来自脑干,下丘脑,前额叶等脑区神经元的输入。丘脑室旁核是机体感受躯体和精神应激的前哨核团之一,无论是奖赏还是惩罚刺激均能激活PVT。同时,PVT有大量的谷氨酸能纤维投射到伏隔核(nucleus accumbens,NAc)和杏仁核(amygdala)等脑区,广泛参与成瘾和奖赏,应激和情绪,摄食等一系列行为的调控。抑郁症除了具有典型的行为绝望和快感缺失,同时多伴有睡眠障碍。而PVT是一个非常重要的参与调控睡眠和觉醒的核团。那么PVT是否参与了抑郁症的调控以及具体神经环路机制如何,这些问题仍然是不明确的,直接证据也比较缺乏。另外,对于抑郁症的研究,我们不能单独地从神经环路去解析,需要同时融合分子机制才能更全面的认识。研究目的本研究旨在探索PVT在抑郁样行为中的作用及其潜在的神经机制。利用病毒示踪和化学遗传操纵,同时借助分子生物学手段探索PVT在慢性束缚应激诱导小鼠抑郁样行为中的作用及其神经环路机制。研究方法1.首先对小鼠施加四种急性负面刺激,利用c-Fos染色探究急性负面刺激对PVT神经元活动的影响。构建21天慢性束缚应激抑郁小鼠模型,采用高架十字迷宫实验,旷场实验,悬尾实验,强迫游泳实验和糖水偏好实验评估小鼠焦虑样和抑郁样行为表型。同时通过PCR和免疫荧光染色验证PVT脑区反应神经元活动的基因:c-Fos和Fos B。2.借助化学遗传手段,在健康野生型小鼠PVT定位注射h M3D/h M4D病毒,通过激活/抑制PVT兴奋性神经元来观测行为学变化以探究其是否影响小鼠焦虑样和抑郁样行为的基线水平。利用ta Caspase3-Tobacco Etch Virus(ta Casp3-TEVp)工具病毒,特异性杀死PVT兴奋性神经元后评估小鼠行为表型以探究PVT在焦虑样和抑郁样行为中的作用。利用化学遗传激活/抑制策略,PVT定位注射h M3D/h M4D病毒后,叠加21天慢性束缚刺激/3天阈下束缚刺激以探索PVT是否参与介导了束缚应激诱导的焦虑样和抑郁样行为。3.采用顺行标记和逆行标记工具病毒示踪的策略,结合荧光成像以找到并详细了解PVT的主要输入和输出神经环路联系。构建慢性束缚应激抑郁模型,利用化学遗传Cre-DIO病毒标记系统,激活/抑制PVT→NAc通路并评估行为学变化以探究该通路是否介导了慢性束缚应激诱导的焦虑样和抑郁样行为。构建慢性束缚应激模型,化学遗传激活PVT→Ce A环路并检测小鼠相关行为表型以探究该环路是否参与调节慢性束缚应激诱导的焦虑样和抑郁样行为。建立慢性束缚应激抑郁模型,化学遗传激活m PFC→PVT→NAc环路并检测小鼠的焦虑样和抑郁样行为。4.借助RNA-sequence技术挖掘PVT中潜在的差异表达基因,并利用PCR,蛋白免疫印迹和免疫荧光实验对差异表达基因进行深入验证。构建慢性束缚应激小鼠模型,连续腹腔注射抗抑郁药丙咪嗪21天,评估小鼠行为以研究丙咪嗪是否能够逆转慢性束缚应激诱导行为缺陷和差异基因的表达。利用免疫荧光染色探索化学遗传激活和抑制PVT兴奋性神经元对PVT脑区差异表达基因的影响。借助导管埋植给药和慢性束缚应激造模,研究抑制差异表达基因在PVT的表达是否影响相关行为表型。研究结果1.四类急性负面刺激均能显著激活a PVT和p PVT神经元活动。慢性束缚应激可诱导小鼠出现焦虑样和抑郁样行为表型,并且21天慢性束缚刺激抑制p PVT兴奋性神经元的活动。2.化学遗传激活PVT兴奋神经元可提高健康野生型小鼠抗焦虑和抗抑郁水平,而抑制PVT兴奋性神经元则导致小鼠出现明显的焦虑样和抑郁样行为。特异性损毁PVT兴奋性神经元诱导小鼠出现抑郁样行为但并不影响小鼠焦虑样行为。化学遗传激活PVT兴奋性神经元可逆转慢性束缚诱导的焦虑样和抑郁样行为,而抑制PVT兴奋性神经元增加小鼠焦虑样和抑郁样行为的应激易感性。3.通过顺行示踪病毒标记,发现PVT主要输出的脑区为:伏隔核(NAc)和中央杏仁核(Ce A);而逆行标记则发现PVT主要接受前额叶(m PFC)的输入。化学遗传激活/抑制PVT→NAc通路可改善慢性束缚应激诱导的焦虑样和抑郁样行为/增加焦虑样和抑郁样行为的束缚应激易感性。但化学遗传激活PVT→Ce A环路并不影响慢性束缚应激诱导的行为缺陷。另外,化学遗传激活m PFC→PVT→NAc环路可一定程度上缓解慢性束缚应激诱发的焦虑样和抑郁样行为缺陷。4.慢性束缚应激导致PVT多种基因表达发生改变,主要集中在反应神经元活动的c-Fos和Fos B,以及多种肽能递质基因,尤其是以神经肽Y(NPY)在束缚小鼠中表达增高最为显著。慢性丙咪嗪处理缓解慢性束缚应激诱导的焦虑样和抑郁样行为,并且增加PVT脑区中的神经元活动和减少NPY蛋白表达水平。化学遗传激活/抑制PVT兴奋性神经元可减少/增加NPY蛋白表达水平。PVT区域埋管给药(NPY抑制剂-BIBP3226)能部分改善慢性束缚应激诱导的焦虑样和抑郁样行为。结论结合慢性束缚应激造模和化学遗传操纵,我们发现丘脑室旁核在抑郁症的发生发展过程中扮演重要的调控作用。同时,慢性束缚应激导致行为缺陷和NPY表达异常升高可被抗抑郁药/化学遗传激活/药理抑制剂所逆转,提示NPY参与了PVT调控抑郁症的过程。更多还原

【Abstract】 BackgroundDepression is a chronic and recurrent neuropsychiatric disorder,in which patients often have persistent symptoms such as low mood and anhedonia,even accompanied by suicidal behavior.The pathogenesis of depression is extremely complex,and the effectiveness of existing antidepressant treatment is limited.The pathogenesis of depression involves multiple brain regions,including the hippocampus,prefrontal cortex,amygdala,nucleus accumbens,anterior cingulate cortex,lateral habenula nucleus,ventral tegmental area,and their interconnecting neural circuits.Existing studies have identified a number of neural circuits related to depression,but these neural circuits are still not well established.On the one hand,because different neural circuits may be involved in the regulation of both depression and anxiety or even other diseases,or only one of the behavioral phenotype.On the other hand,some neural circuits are positively regulated,others are negatively regulated,and still others are dynamically regulated in both directions.So to some extent,depression can also be regarded as a neurological circuit disease.The paraventricular nucleus of thalamus(PVT)is a small nucleus located in the dorsal part of the midline thalamus,which receives input from neurons in brain stem,hypothalamus,prefrontal cortex and other brain regions.The paraventricular nucleus of the thalamus is one of the sentinel nuclei for the body to feel physical and mental stress.Both reward and punishment stimuli can activate the PVT.At the same time,PVT has a large number of glutamatergic fibers projecting to brain regions such as nucleus accumbens(NAc)and amygdala,which are widely involved in the regulation of a series of behaviors such as addiction and reward,stress and emotion,and feeding.In addition to the typical behavioral despair and anhedonia,depression is often accompanied by sleep disorders.The PVT is a very important nucleus involved in the regulation of sleep and arousal.However,whether PVT is involved in the regulation of depression and the specific neural circuit mechanism are still unclear,and direct evidence is also lacking.In addition,for the study of depression,we cannot analyze it from the neural circuit or molecular level alone,and we need to integrate both to have a more comprehensive understanding of depression.ObjectivesThe purpose of this study was to explore the role of PVT in depression-like behavior and its potential neural mechanism.The role of PVT in depression-like behavior induced by chronic binding stress in mice and its neural circuitry mechanism were explored by using viral tracer and chemogenetic manipulation as well as molecular biology.Methods1.First,four kinds of acute negative stimuli were applied to the mice,and c-Fos staining was used to explore the effect of acute negative stimuli on PVT neuronal activity.A 21-day chronic restraint stress depression mouse model was established.Elevated plus maze test,open field test,tail suspension test,forced swimming test and sucrose preference test were used to evaluate the anxiety-like and depression-like behaviors of the mice.At the same time,PCR and immunofluorescence staining were used to verify the genes that reflect neuronal activity in the brain area of PVT:c-Fos and Fos B..2.Using the chemogenetic method,h M3D/h M4 D virus was injected into the PVT of healthy wild-type mice,and the behavioral changes were observed by activating/inhibiting PVT excitatory neurons to explore whether it affected the baseline level of anxiety-like and depression-like behaviors in mice.The ta Casp3-TEVp tool virus was used to evaluate the behavioral phenotype of mice after specifically killing PVT excitatory neurons to explore the role of PVT in anxiety-like and depression-like behaviors.Using a chemogenetic activation/inhibition strategy,PVT-localization injection of h M3D/h M4 D virus was combined with 21 days chronic restraint stimulation /3 days sub-threshold restraint stress to explore whether PVT is involved in the regulation of restraint stress-induced anxiety-like and depression-like behaviors in mice.3.The strategies of anterograde and retrograde labeling tool virus tracing,combined with fluorescence imaging,were used to find and understand the main input and output neural circuit connections of PVT.A chronic restraint stress-induced depression model was constructed.Chemogenetic Cre-DIO virus system was used to activate/inhibit PVT→NAc pathway and evaluate behavioral changes to explore whether this pathway mediated anxiety-like and depression-like behaviors induced by chronic restraint stress.A chronic restraint stress model was established,and the PVT→Ce A loop was chemically activated and the related behavioral tests were detected to explore whether this loop was involved in the regulation of anxiety-like and depression-like behaviors induced by chronic restraint stress.The chronic restraint stress depression model was established,and the m PFC→PVT→NAc loop was chemically activated to detect anxise-and depression-like behaviors in mice.4.RNA-sequence technology was used to explore the potential differentially expressed genes in PVT,and PCR,Western blot and immunofluorescence experiments were used to further verify the differentially expressed genes.A chronic restraint stress mouse model was established,and antidepressant imipramine was injected intraperitoneally for 21 days.The behavior of the mice was evaluated to study whether imipramine could reverse the behavioral defects and differential gene expression induced by chronic restraint stress.Immunofluorescence staining was used to explore the effects of chemogenetic activation and inhibition of PVT-excitatory neurons on differentially expressed genes in PVT.Cannula implantation and chronic restraint stress were used to establish a model to study whether inhibiting the expression of differentially expressed genes in PVT affected related behavioral phenotypes.Results1.All four types of acute negative stimuli significantly activated a PVT and p PVT neuronal activity.Chronic restraint stress can induce anxiety-like and depression-like behaviors in mice,and 21-day chronic restraint stress inhibits the activity of excitatory neurons in p PVT.2.Chemogenetic activation of PVT-excitatory neurons can improve the anti-anxiety and anti-depression levels in healthy wild-type mice,while inhibition of PVT-excitatory neurons leads to significant anxiety-like and depression-like behaviors in mice.Specific depletion of PVT excitatory neurons induces depression-like behaviors but does not affect anxiety-like behaviors in mice.Chemogenetic activation of PVT excitatory neurons can reverse the anxiety-like and depression-like behaviors induced by chronic restraint,while inhibition of PVT excitatory neurons increases the stress susceptibility of anxiety-like and depression-like behaviors in mice.3.Through anterograde virus labeling,it was found that the main output connections of PVT were nucleus accumbens(NAc)and central amygdala(Ce A);In contrast,retrograde labeling revealed that the PVT received input from the prefrontal cortex(m PFC).Chemogenetic activation/inhibition of PVT→NAc pathway can improve chronic restraint stress-induced anxiety-like and depression-like behaviors/increase the susceptibility to restraint stress-induced anxiety-like and depression-like behaviors.However,chemogenetic activation of PVT→Ce A loop does not affect the anxiety-like and depression-like behaviors induced by chronic restraint stress.In addition,activation of m PFC→PVT→NAc loop can alleviate the anxiety-like and depression-like behavioral deficits induced by chronic restraint stress to a certain extent.4.Chronic restraint stress causes changes in the expression of many genes in PVT,mainly in c-Fos and Fos B,and many peptide transmitter genes,especially neuropeptide Y(NPY).Chronic imipramine treatment alleviated chronic restraint stress-induced anxiety-like and depression-like behaviors,increased neuronal activity and decreased NPY protein expression of PVT.Chemogenetic activation/inhibition of PVT excitatory neurons can reduce/increase the protein expression level of NPY.Cannula administration of NPY inhibitor BIBP3226 in PVT can partially improve the anxiety-like and depression-like behaviors induced by chronic restraint stress.ConclusionCombined with chronic restraint stress modeling and chemogenetic manipulation,we found that the paraventricular nucleus of thalamus plays an important regulatory role in the development of depression.At the same time,chronic restraint stress induced behavioral deficits and abnormal increase of NPY expression can be reversed by antidepressant or chemogenetic activation or pharmacological inhibitors,suggesting that NPY is involved in the process of PVT-regulated depression.更多还原